Research Collection
Doctoral Thesis
Biological screening of cyanobacteria and phytochemical investigation of Nostoc commune and Tolypothrix byssoidea
Author(s): Jaki, Birgit
Publication Date: 2000
Permanent Link: https://doi.org/10.3929/ethz-a-003926384
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ETH Library Diss. ETH No. 13582
Biological Screening of Cyanobacteria and Phytochemicai
Investigation of Nostoc commune and Tolypothrix byssoidea
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH
for the degree of
Doctor of Natural Science
Presented by
Birgit Jaki
Pharmacist
Born May 4, 1970
Karlsruhe, Germany
accepted on recommendation of
Prof. Dr. Otto Sticher, examiner
Dr. Jörg Heilmann, co-examiner
Dr. Hans-Rudolf Bürgi, co-examiner
Zürich 2000 Acknowledgements
and This study was carried out at Division of Pharmacognosy Phytocbemistry,
Department of Applied BioSiences, Institute of Pharmaceutical Science, Swiss Federal
Institute of Technology (ETH) Zurich, Switzerland.
excellent I am most grateful to Prof. Sticker for being supervisor, providing working facilities and his interest throughout all phases of this work.
me to this his I wish to express my gratitude to Dr. Jimmy Orjala for introducing project, helpful and intensive discussions and his continuous motivation
Great thanks are due to Dr. Jörg Heilmaim for many stimulating discussions, his encouragement and for being co-examiner.
and T also wish to express my gratitude to Dr 11ans-Rudolf Bürgi for fruitful discussions being co-examiner.
of I am highly appreciated to Dr. Oliver Zerhe for introducing me to basic techniques
NMR spectroscopy and structure determination of peptides, as well as excellent discussions concerning structural problems.
Special thanks go to Dr. Anthonv Linden for perfonning X-ray crystallographic analysis and his fruitful collaboration.
I owe a special word of appreciation to Dr. Bernhard Vogler for performing LC-NMR experiments.
1 am especially indebted to Dr. Marianne Bosh and Frank Sunder for providing, identifying and cultivating the algal material. I also wish to thank Drs. Walter Amrein and Peter James as well as Oswald Greter and
Manfredo Quadroni for recording mass spectra and Dr. Engelbert Zass for performing literature searches.
Special thanks for support m informatics and patience are due to Daniel Förderer, Phillip
Stalder, andlvo Fähnle.
I wish to thank all my colleagues and staff at the Institute of Pharmaceutical Sciences for the pleasant working affair they afforded me. Special thanks are due to Eva Hoberg,
Carmen Volken, Tulla Spineili, Wolfgang Schiihly, and Michael Wasescha (the staff of the 'Chaos Laboratory') for their friendship and our not always phytochemicai discussions.
Finally, I want to express my warmest thanks to mv family and friends for their encouragement, support and patience during this study. Contents Page
list of Abbreviations 1
Summary 4
Zusammenfassung 5
Preface 7
Ï THEORETICAL PART
1 General Introduction
1.1 Introduction
I * Ai References
2 Aim of the Present Investigation 10
3 Introduction to the Cyanobacterial Biology 11
3.1 Taxonomy 11
3.1.1 Botanical Approach 12
3 1.2 Bacteriological Approach 13
3.2 Anatomy, Morphology, and Physiology 16
3.2.1 Cellular Organization 16
3.2.2 Surface Structures 16
3.2.3 Nuclear Apparatus 18
3.2.4 Cyloplasnuitic Inclusions and Reserve Products 18
3.2.5 Ileterocysts 21
3.2.6 Motility 21
3.2.7 Modes of Reproduction 22
3.2.8 Cyanobacteria in Svmbiotic Associations 23
3.2.8.1 Cyanelles and the Origin of Chloroplasts 23
3.2.8.2 Casual Associations 23 2.8.3 Associations between Fungi and Cyanobacteria
2.8.4 Associations between Liverworts and Cyanobacteria
.2.8.5 Association between a Fern and a Cyanobacterium
2.8.6 Associations between Higher Plants and Cyanobacteria
2.8.7 Associations between Invertebrates and Cyanobacteria
3 Metabolism
3.1 Nitrogen-Fixation
3.2 Photosynthesis
3.2.1 Photosynthetic Membrane System
3.2.2 Photosynthetic Metabolism
3.3 Heterotrophic Growth
4 Ecology
.4.1 Flabitats
4.2 Public Health
.4.2 Detection of Toxins and Cyanobacteria! Growth Control
4.3 The Use of Cyanobacteria as Bio fertilizer
5 Pharmaceutical and Pharmacological
Interest of Cyanobacteria
6 References
Secondary Metabolism
1 Introduction
1.1 Secondary Metabolism of Marine Cyanobacteria
1.2 Secondary Metabolism of Freshwater and
Terrestrial Cyanobacteria
2 Bioactive Compounds Produced by Cyanobacteria
2.1 Cyanobacterial Toxins 4.2.1.1 Cytotoxics 42
4.2.1.2 Biotoxins 42
4.2.1.2.1 Neurotoxins 42
4 2 1 2 '? Hepatotoxins 44
as a Source of Medicinal 47 *" jC* . ^W . Cyanobacteria Agents
4.2.2.1 Antimicrobial Compounds 47
4 22'^ Antiviral Compounds 50
with 52 I* Jl^ . ,w • Jj , Compounds Multidrug-Resislance
Reversing Activity
4.2.2.4 Cytotoxic Compounds 54
4.2,2.5 Enzyme Inhibitory Compounds 59
4.2.2.6 Cardioactive Compounds 67
4.2.2.7 Anti -In f] am m atoiy Compounds 69
43 References 70
5 The Genera Nostoc and Tolypothrix 77
5. / The Genus lYostoc 77
5.2 The Genus Tolypothrix 79
5.3 References 80
6 Collection and Cultivation 81
6.1 Collection 81
6.2 Isolation 81
6.2.1 Isolation by Liquid Enrichment 82
6.2.2 Direct Manual Isolation 82 63 Culturing 82
6.3.1 Stock-Cultures 82
6.3.2 Large-Scale-Cultures 83
6.3.3 Composition of the Culture Media 83
6.4 References 87
7 Methodology of Isolation Procedure 88
7.1 General Isolation Strategy 88
7.1.1 Isolation of Intracellular Compounds 88
7.1.2 Isolation of Extracellular Compounds 88
7.1.2.1 Solid Phase Extraction 89
7.2 Chromatographic Methods 90
7.2.1 Normal Phase Chromatography 90
7.2.2 Reverse Phase Chromatography 90
7.2.3 Thin Layer Chromatography (TEC) 90
7.2.4 Vacuum-Liquid Chromatography (VLC) 91
7.2.5 Open Column Chromatography 91
7.2.6 Gel Permeation Chromatography 93
7.2.7 High Performance Liquid Chromatography 93
73 References 95
8 Methodology of Structure Elucidation 96
8.1 Spectroscopic and Spectrometric Methods 96
8.1.1 Ultraviolet (EV) Spectroscopy 96 8.1.2 Infrared (IR) Spectroscopy 96
8.1.3 Mass Spectrometry (MS) 96
8.1.3.1 Electron Impact Mass Spectrometry (ELMS) 97
8.1.3.2 Fast-Atom Bombardment Mass Spectrometry (FAB-MS) 97
8.1.3.3 Matrix Laser Desorption/Ioni/ation 98
Mass Spectrometry (MALDEMS)
8.1.3.4 Electron Spray Ionization Mass Spectrometry (ESI-MS) 98
8.1.3.5 Chemical Ionization Mass Spectrometry (CI-MS) 98
8.1.3.6 Tandem-Mass Spectrometry (MS-MS) 99
8.1.4 Nuclear Magnetic Resonance (NMR) Spectroscopy 101
8.1.4.1 One-Dimensional NMR Spectroscopy 101
8.1.4.2 Two-Dimensional NMR Spectroscopy 102
8.1.4.2.1 Hornonuclear Experiments 102
8.1.4.2.2 Fleteronuclear Experiments 10:.1
8.1.4.3 High Performance Liquid Chromatography 103
Proton Nuclear Resonance On-Line Coupling (LC-NMR)
8.2 Physical Methods 107
8.2.1 X-ray Crystallography 107
8.2.2 Optical Rotation 107
8.3 Chemical Methods 107
8.4 References 108
9 Biological Investigation
9.1 Screening for Biological A ctive Metabolites
and Determination of Pure Compounds
9.1.1 Introduction 9.1.2 Brine Shrimp Lethality Test
9.1.3 Bioassay for Antibiotic Activity
9.1.3.1 Diffusion Method
9.1.3.2 Agar Overlay Method
9.1.3.3 MIC Determination of Pure Compounds
9.1.3.4 Assay for Molluscicidal Activity
9.1.3.5 Assay for Cytotoxic Activity
9.1.3.6 Assay for Antiplasmodial Acthity
9.2 References
10 Diterpenoids
10.1 Introduction
10.2 Biosynthesis
10.2.1 Acetate/Meval onatc Pathw ay
10.2.2 Cyclization
10.2.3 Triose Phosphate/Pymvate Pathway
103 References
11 Anthraquinones
11.1 Introduction
11.2 Biosynthesis
11.2.1 Shikimi Acid'Mevalonate Pathway
11.2.2 Polyketide Pathway
113 References
12 Cyclic Peptides 12.1 Introduction 135
12.2 Biosynthesis 135
12.2.1 Nonribosomal Biosynthesis of Unusual Peptides 135
(Thiotemplate Pathway)
123 References 139
II EXPERIMENTAL PART 141
13 Introduction 141
14 Extraction 142
14.1 Materialfor Biological Investigation 142
14.2 Materialfor Phytochemicai Investigation 142
15 Biological Screening 143
16 Phytochemicai Investigation of Nostoc commune (EAWAG 122 b) 147
16.1 Fractionation and Isolation 147
16.1.1 Fractionation o f Extract B 147
16.1.2 Fractionation of Extract C 147
16.2 Structures of the Isolates 15 0 163 Structure Determination 153
16.3.1 Structure Determination of NC-1 153
16.3.2 Structure Determination of NC-2 160
16.3.3 Stracrare Determination of NC-3 - NC-7 166
16.3.4 Structure Determination of NC-8 180
16.3.5 Structure Determination of NC-9 183
16.3.6 EC-NMR Experiments 187
16.4 Biological Testing 189
17 Phytochemicai Investigation of 190
Tolypothrix byssoidea (EAWAG 195)
17.1 Fractionation and Isolation 190
17.2 Structures of the Isolates 192
17.3 Structure Determination 194
17.3.1 General Structure Determination Strategy of Peptides 194
by means of NMR
I/.,3.1.1 Choice of the NMR Solvent 194
17.,3.1.2 Sequence Speci fic Resonance Assignment of Peptides 194
17.,3.2 Residue Masses of Neutral Amino Acids 195
17..3.3 Structure Determination of TB-1 and TB-2 196
17.,3.4 Determination of the Absolute Stereochemistry of the 221
Amino Acid Residues according to Marfey
17.,3.4.1 Introduction 221
17.,3.4.2 Acid Hydrolysis of EB-1 and TB-2 222
17. 3.4.3 HPLC Analysis of the Marfey Derivatives 222
17. 3.4.4 Preparation of the Acetyl Derivatives of Threonine 223 17.4 Biological Testing
17.5 References 227
18 Publication 1 228
19 Publication 2 242
20 Publication 3 249
21 Publication 4 267
22 Publication 5 278
23 Discussion 296
23.1 Selection of the Cyanobacterial Strains 2%
233 Cultivation 296
233 Biological Testing 297
23.4 Isolation 297
23.5 Structure Elucidation 298
23.6 Biological Active Secondary Metabolites 299
24. Conclusions 301
list of Publications
List of Poster Presentations List of Abbreviations
AcThr Acetyl-threonine
Arg Arginine
c Concentration
CD,OD Deuterated Methanol
CHCb, Chloroform
DCM Di ch lorom ethane
d Doublet
dd Double doublet
DEPT Distortionsless Enliancement by Polarisation Transfer
Dhha Dehydroh om oal anine
DQF-COSY Double Quantum Correlation Spectroscopy
CIMS Chemical Ionization Mass Spectrometry
EIMS Electron Impact Mass Spectrometry
ESIMS Electron Spray Mass Spectrometry
EtOAc Ethyl Acetate
eV Electron Volt
FABMS Fast Atom Bombardment Mass Spectrometry
HIV Human Immunodeficiency Virus
HMBC Heteronuclear Multiple Bond Correlation
HMQC Heteronuclear Multiple Quantum Correlation
H20 Water
HPLC High Performance (Pressure) Liquid Chromatography
Hz Flertz
He Isoleucine
IR Infrared Spectroscopy
KBr Potassium Bromide
LC-NMR High Perfomiance Liquid Chromatography Proton Nuclear Magnetic
Resonance On-Line Coupling
Leucine
m Multiplet
1 MeCN Acetonitrile
Me2CO Acetone
MeOH Methanol
MeOFEd4 Deuterated Methanol
MALDIMS Matrix Laser Desorption/Ionisation Mass Spectrometiy
Met Methionine fig Microgram
MHz Megahertz
M.p. Melting Point
MS-MS Mass Mass Spectrometry (Tandem Vlass Spectrometry) m.w. Molecular Weight
NMR Nuclear Magnetic Resonance
MET lodonitrotetrazolium chloride nOe Nuclear Overhauser Effect
NOESY Nuclear Overhauser Enhancement Spectroscopy
NP Norma! Phase
Phe Phenylalanine ppm Parts per million
Pro Proline q Quartet
ROESY Rotating-Frame Overhauser Spectroscopy
RP Reversed Phase s Singlet
Si Gel Silica Gel
Thr fhrconine t lYiplet
TLC Thin Layer Chromatography
TOCSY Eotal Correlation Spectroscopy
Tyr Tyrosine
UV Ultra Violet Spectroscopy
Val Valine
VLC Vacuum Liquid Chromatography
3 C
|«4 f ^"y VA l-1'
1 Summary
The extracts of 43 different cyanobacterial strains were investigated during a biological
screening for their antibacterial, antifungal, cytotoxic, andmolluscicidal activity. Thirty-
six of these strains were selected from the Culture Collection of Algae of the Swiss
Federal Institute of Water Resources and Water Pollution Control (EAWAG), while
seven further isolated strains originated from three different field collections.
Additionally, the three field-collected samples (one Nostoc commune strain and two mixed samples) were added to the same biological screening program.
Based on the results of the biological screening two cyanobacterial strains, Nostoc commune (EAWAG 122b) and Tolypothrix byssoidea (EAWAG 195) were chosen for large-scale cultivation.
Using different chromatographic methods, such as VLC and HPEC, a bioguided fractionation of the culture medium of both strains and of the cell material of the strain
Nostoc commune led to the isolation of ele\en compounds that were unknown in
literature.
Seven diterpenoids, isolated from the culture medium of the strain Nostoc commune comprise two different unprecedented diterpenoid skeletons. Additionally, a new anthraquinone with an unusual substitution pattern and an in dan derivative, that is only reported as synthetic product were obtained from the cell mass.
The investigation of the culture medium of the strain Tolypothrix byssoidea resulted in the isolation of two novel cyclic tridecapeptides, both comprising the unusual amino acid didehydrohomoalanine (Dhha).
All isolates show a biological activity in at least one of the test systems applied.
The structures of the isolates were established by spectroscopic and chemical methods,
UV, IR and NMR MS and MS-MS as well as including , spectroscopy, spectrometiy, by single-crystal X-ray analysis.
EC-NMR experiments were done to detect the extracellular compounds also in the cell material.
The presented results support the general notation that cyanobacteria are a promising source to yield chemical and pharmaceutical interesting compounds.
4 Zusammenfassung
Die Extrakte von insgesamt dreiundvierzig verschiedenen Cyanobakterienstämmen
wurden im Rahmen eines biologischen Screenings bezüglich ihrer antibakteriellen,
antifungalen, zytotoxischen und molluskiziden Wirkung untersucht. Sechsunddreißig
dieser Stamme wurden aus der Algenkultursammhmg der Eidgenössischen Anstalt für
Wasserversorgung, Abwasserreinigung und Gewässerschutz (EAWAG), ausgewählt,
während sieben weitere isolierte Stämme aus drei verschiedenen Wildsammlungen
resultierten. Zusätzlich wurden die drei wild gesammelten Proben (ein Nostoc commune
Stamm und zwei gemischte Proben) direkt in das biologische Screening eingeschleust.
Anhand der Resultate des biologischen Screenings wurden zwei Cyanobakterienstänime,
Nostoc commune (EAWAG 122b) und Tolypothrix byssoidea (EAWAG 195), für eine
Kultivierung im großen Maßstab ausgewählt.
Die bioaktivitätsgeleitete Fraktionierung der Kulturmedien beider Stämme und des
Zellmaterials des Stammes Nostoc commune führte mit Hilfe verschiedener
Chromatograph is eher Methoden zur Isolierung von elf in der Literatur bisher nicht beschriebeneu Substanzen.
Sieben Diterpene, die aus dem Kulturmedium des Stammes Nostoc commune isoliert wurden, weisen zwei neue diterpenoide Grundkörper auf. Darüber hinaus wurden aus der
Zeilmasse des selben Stammes ein neues Anthrachinon mit bisher nicht bekanntem
Substitutionsmuster und ein Indan Derivat, das bisher nur synthetisch gewonnen werden konnte, isoliert.
Die LIntersuchung des Kulturmediums des Stammes Tolypothrix byssoidea führte zur
Isolierung von zwei neuartigen zyklischen ITidecapeptiden, die beide die nicht-natürliche
Aminosäure Dehydrohomoalanin (Dliha) enthalten.
Alle isolierten Reinsubstanzen zeigen eine biologische Aktivität in mindestens einem der eingesetzten Testsysteme.
Die Strukturaufklärung der isolierten Substanzen erfolgte mittels spektroskopischer, spektrometrischer und chemischer Methoden, einschließlich UV-, IR- und NMR-
Spektroskopie, MS- und MS-MS-Spektrometrie, sowie durch Einkristall-
Röntgenstrukturanalyse. LC-NMR Untersuchungen wurden durchgeführt um
5 extrazellulär gewonnene Substanzen auch im Zellmaterial des Stammes Nostoc commune nachzuweisen.
Die hier beschriebenen Ergebnisse unterstützen die allgemeine Ansicht, daß
Cyanobakterien eine vielversprechende Quelle für die Gewinnung chemisch und pharmazeutisch interessanter Substanzen sind.
6 Preface
This thesis is composed of two parts. The Theoretical Part comprises general aspects concerning an introduction to the biology and the secondary metabolism of cyanobacteria. The Experimental Part describes a biological screening of several cyanobacterial strains as well as the isolation and structure elucidation of secondaiy metabolites isolated from two of these strains, Nostoc commune and Tolypothrix byssoidea. 1 General Introduction
1.1 Introduction
Cyanobacteria (blue-green algae) are a remarkable group of simple photosynthetic
microorganisms. In evolutionary terms they represent a link between bacteria and green plants.
Their cellular organization, known as prokaryotic, is characterized by the lack of membrane-
bound organelles such as true nucleus, a cliloroplast or a mitochondrion, and resembles that
found in bacteria. Hence the genetic material, the photosynthetic apparatus and the respiratory-
system are not segregated by means of internal membranes from the rest of the cell. Their
principle mode of nutrition, oxygen-evolving photosynthesis, however, is similar to that which
operates in all other nucleate or eukaryotic algae and in green plants.
Cyanobacteria apparently have a long histoiy on earth. Their fossils were recently identified in
sediments from the Early Precambrian period, over three billion years old. At that time they were
probably the chief primary producers of organic matter and the first organisms to release
elementary oxygen, 0?, into the primitive atmosphere, which was until then free from 02. Thus
cyanobacteria were most probably responsible for a major evolutionary transformation leading to
the development of aerobic metabolism and to the subsequent rise of higher plant and animal
forms It is (1). suggested that certain of the photosynthetic cyanobacterial cells were taken up
permanently by other microbes, lost the ability to function and became chloroplasts (2).
Cyanobacteria are widespread in marine and freshwater aquatic environments. Most
cyanobacteria are planctonic organisms that grow best in warm, calcareous, still, eutrophic or
hypertrophic waters. They are most noticeable, and probably most harmful, when they rise to the
surface of inshore waters and form a bloom (3).
Toxic cyanobacterial waterblooms are found worldwide in eutrophic lakes, ponds, drinking water
reservoirs and coastal waters, where they cause animal poisonings and pose risk to human health (4).
Toxins produced by cyanobacteria present a serious health problem in water supplies, both for
livestock and for human health. This is a worldwide problem due to algal blooms (5). In the
course of the toxic determining constitutents in cyanobacteria, a diverse array of secondary metabolites with interesting chemical and biological features was encountered. These metabolites
8 have been important biomedically as leads to new pharmaceutical compounds, herbicides and pesticides (6).
1.2 References
1. Fay, P.: The Blue-Greens (Cyanophyta-Cyanobacteria). Edward Arnold Ltd, London. 160:
4-18(1983)
2. Carmichael, W. W.: The Toxins of Cyanobacteria. Sei. Amer. 270: 64-72 (1994)
3. Hunter, P. R.: Cyanobacterial Toxins and Human Health../. Appl. Microbiol. Symp. Suppl.
84:35-40(1998)
4. Namikoshi, M., Rinehart, K. L.: Bioacbve Compounds Produced By Cyanobacteria. J.
Ind. Microbiol. & Biotechnol. 17: 373-384 (1996)
5. Rinehart, K. L., Namikoshi, M., Choi, B. W.: Structure and Biosynthesis of Toxins from
Blue-Green Algae (Cyanobacteria). J Appl. Phvcol. 6: 159-176 (1994)
6. Shin, H. J., Murakami, M., Matsuda, H., Yamaguchi, K.: Microviridins D-E, Serine
Protease Inhibitors from the Cyanobacterium Oseillaforia agardhii (NIES-204).
Tetrahedron 52: 8159-8168 (1996)
9 2 Aim of the Present Investigation
This study is part of a project between the Swiss Federal Institute of Technology (ETH), Zurich
(Switzerland) and the Swiss Federal Institute for Water Resources and Water Pollution Control
(EAWAG), Dübendorf (Switzerland), concerning the biological and phytochemicai investigation of cyanobacteria.
Since cyanobacteria are known to provide chemical interesting as well as unusual and biologically active secondaiy metabolites it is of growing interest to examine these organisms in detail.
The first part of the project was the cultivation and the biological screening of a choice of 43 different stock cultured cyanobacterial strains originating from different habitats (roadside and waterfalls). Additionally three field-collected samples were passed in this screening program.
The aim of this part of the project was to find the most active and chemical interesting cyanobacterial strains for further investigation and to compare the biological activities of stock- cultured and field-collected strains.
From these initial experiments two strains, Nostoc commune (EAWAG 122b) and Tolypothrix byssoidea (EAWAG 195) were chosen for large-scale cultivation.
The aim of this part of the work was to isolate the biological active compounds from the cyanobacterial cell material and the culture media by means of bioactivity guided fractionation and to determine their chemical structures and their biological activities.
10 3 Introduction to the Cyanobacteria! Biology
3.1 Taxonomy
The blue-green algae are a rather circumscribed class of pigmented protophyta. The class contains about 150 genera and about 2000 species ( 1 ). Cyanophyceans (Cyanobacteriales) are the most diverse and widespread of the phototrophic prokaryotes. Unlike purple and green photobacteria, they have a photosynthetic apparatus similar m structure and function to that of the eukaiyotic chloroplast. The fundamental difference in the cellular organization of cyanophyceans and other algae however, led to the taxonomically treatment of blue-green algae as a separate class of division (2), (3). Their capacity to perform oxygenic photosynthesis and exhibiting an algal-like morphology make the taxonomic treatment of the cyanophyceans by the rules of the
International Code of Botanical Nomenclature (1972) relevant. The species concept in cyanoprokaryotes is under intense discussion. The bacterial features, which are typically possessed by blue-green algae, make also a classification based upon the principles of the
International Code of Nomenclature of Bacteria (1975) suitable. Thus, two possible methodologies of biological classification are appropriate. A recent synthetic approach provides a promising alternative (4), (5), The cyanophyceans are accordingly placed within the group eubacteria in the phylogenetic taxonomy, distinct and apart from the archaebacteria and eucaryotes (see Figure 2.1). (6). The two nomenclature systems for classification of blue-green algae are now, in conformity with this, applied in parallel (7). Consequently many names have been proposed for this group of microorganisms and have been used synonymously at the liberty of the authors: Myxophyceae (Wallroth, 1833). Phycochromophyceae (Rabenhoerst, 1863),
Cyanophyceae (Sachs, 1874), or Schizophyceae (Cohn, 1879) are the most well known examples.
The name Cyanophyceae has survived the longest and is still widely used by phycologists and botanists today. In view of the prokaiyotic (thus bacterial) cellular properties of blue-green algae the name Cyanobacteria, which was first proposed by Stanier (1974) is the most common nowadays (8). A system based on morphological relationships can be depicted from Figure 3.2 (9).
11 3.1.1 Botanical Approach
Chronologically, the botanical approach was the first for the cyanobacterial taxonomy. As for
other algae, the classical taxonomy of the cyanobacteria is based on morphological features and their nomenclature is ml cd by the Botanical Code. This means that each new species has to be
described in Latin, and that its reference is a herbarium specimen. For the simple filamentous
(Oscillatoriaceae) and the heterocystous species (Nostocaccae and Stigonemataceae), the starting
points for valid publication of names are the monographs written by Gomont (10) and Bornet and
Flahaut (11), respectively. Since the last century, numerous new species have been described. Tn
Geitler's determination key (12) published in 1932, about 1300 species and 145 genera were recognized. This key was devised for Germany, Austria and Switzerland but it was used till over the world and is still the basis of numerous taxonomic works. Among botanical taxonomists, there is a suspicion that too many species have been described over the years. Many are based on
a single character difference, such as the presence or absence of sheath or slight deviations in cell
dimension or form (13, 14). The problem of morphological variability has prompted Drouet (15) to revise the taxonomy profoundly. His basic idea was that there exist ecophenes, which were
organisms sharing the same genotype but expressing distinct morphologies under the influence of
environmental factors. But these results do not correspond with cyanobacterial diversity in nature
and in cultures. He reduced the number of species down to 62 by selecting certain morphological
features that he believed to be invariant with the environment. Classical taxonomists were quite
critical of this approach (4, 16). Later, DNA-DNA hybridization showed that some taxa placed by
Drouet in the same species were genotypically different (17, 18).
Recently, Anagnostidis and Komarek (4, 13, 14, 19, 20) published anew and deeply recognized taxonomic revision. The authors made an extensive review of the literature and tried to integrate
all the biochemical, ultrastructural molecular characters (21). The investigative process of the
genotype diversity is still in its early stage. Only a very small percentage of taxonomic units are
in culture.
12 3.1.2 Bacteriological Approach
The classical botanical taxonomy was confronted in the seventies with the rather different bacteriological approach. Hereby, the reference for each species becomes a pure culture instead of a herbarium specimen. The physiological and genotypic characters were determined with axenic cultures (22). The characters employed included the pigment composition, fatty acid analysis, heterotrophic growth, nitrogenase activity, DNA base composition, and genome length
(23, 24, 25, 26). The basis of bacteriological taxonomy of the cyanobacteria was published by
Rippka et al. (26). This taxonomic system which relies largely on the morphology, allows the identification of the strains of the Pasteur Culture Collection at the generic level (21 ).
According to the proposal of Gibbons and Murray (27), cyanobacteria are members of the kingdom Procaryotae and are included in the division Gracilicutes (bacteria with gram negative cell wall); they are assignable to the class Photobacteria which, in its subclass Oxyphotobacteria, embraces them as the order Cyanobacteriales.
13 EUKARYOTA EUBACTERIA
Plant chloroplasts Stramenopiies Alveolates
Red algae
Agrobacterium Slime moulds
Entamoebae
Heterolobosea Plant mitochondria Kinetoplastids
Euglenoids
Enterobacteria crosporidians
Trichomonads
ARCHAEBACTERIA
Sulfolobus
Thermoplasma
Methanobacteria Halobacteria
Figure 3.1 'Tree of Life' adapted to Woese (6)
14 Tricfiodesmium ^pharazcmenon Gteoinchta Pseuefosnabasna Lyngbya Cytodrospermum Rävölana Phormk&r« Nosîœ Toîypoah« Âffâbasoa Nosîœhopsîs Gardnerula Hydtoeoteym Noduiaria Scytonema Mastigocfatft» Sîlgonema
Dicboîhrix Fisc h«relia Schizolhrw Kyrtuthnx L Masîigocoieus J
Gsmtm fcfer&cftaetas ßrachytnchsa Hapalosiphon Microcoieus
J
Osciitotoriales Noftacafes Stîgonematates T
HORMOGOHEAE Nyefla Aphanoîtece C^to« Hydîococcus Co^haenum ***** Chaemosiphon Dôfmoca/pa Aphanocapsa Gieothece Hormathonema 1 Gomptosphaena ChroococcidaDpsssPfeurocapsa Gteœapsa L 1 Sîœhossphon _ Johannesbaptîstia L Myxosarctna Mefismopedta Chroococcus £f!îDphysaîts Sspfiononema L Clasîsdium Synecnocoocus Eucapsia Xenococcus CMorogloea Cyanophanon SynechocystÄ J
I . ^
Chroococcales Cbsemosiphenaies Pi#urocap««îes
COCCOGONEÂE
CYÄMOPHYTA 3.2 Anatomy, Morphology, and Physiology
3.2.1 Cellular Organization
Cyanobacteria comprise microscopic organisms that could be included under two broad categories of morphological organization, namely unicellular or colonial (— coccoid) forms and filamentous forms. A considerable degree of modification and diversification of the two basic types of morphological forms results in a variety of thalli that ranges from a simple unicellular organism to a branched filamentous member.
The coccoid members consist of simple unicells that may be ovoid, spherical or cylindrical, occurring as free cells or cells aggregated into a mucilaginous matrix. Some unicellular forms have, besides the colonial sheath, a firm sheath for each individual cell grouping together to form a colony. Ordered arrangements of cells in a definite shaped colony (plate like or cubical) and irregularly arranged colonies form another set of coccoid members. Polarity of the cells, production of exospores (exocytes) or endospores (baeocytes), pseudoparenchymatous and pseudofilamentous nature of the thai lus differentiates yet another group of coccoid cyanobacteria.
Thus, the system of unicellular and colonial cvanoprokaryotes is based on the combination of supraspecilic criteria like unicellular or colonial mode, form of cells, polarity of cells and colonies, and type of cell division (binary or multiple fission).
The filamentous forms are separated on the basis of the presence or absence of specially differentiated cells called heterocysts into non-heterocysts (see 3.2.5) or heterocystous forms.
Heterocystous filamentous forms may be of uniform width or showing base apex differentiation, and there are forms that exhibit various types of true branching and false branching. Those exhibiting true branching are the heterotrichous forms with main and lateral branches, the highest level of differentiation seen in cyanobacteria (28).
3.2.2 Surface Structures
The cells of the most cyanobacteria measure from 2 to 5 jjm in diameter. They show a typical prokaiyotic organization, profoundly different from the more complex eukaiyotic structure of
16 plant and animal cells. It is nevertheless possible to make a rough distinction between two main regions of the cytoplasm; the peripheral and the central regions. The central (or nucleopiasmic) region is lightly granulated due to the presence of ribosomes, and contains the extremely folded thin thread of the prokaiyotic type of circular chromosome. Thin sheets of the photosynthetic membrane system, incorporating the photosynthetic pigments traverse the peripheral (or chromatoplasmic) region. A membrane, the pi asm al em ma, which is fortified by a multilayered cell wall, encloses the cell. External to the wall, the cell may be surrounded by a gelatinous sheath or a firmer envelope (29).
The gram-negative cell wall is uniform in all cyanoprokaryotes. It lies between the cytoplasmatic membrane and the mucilaginous sheath, and all together constitutes the envelope. The cell wall consists of a complex, usually multilayered structure of three obligatory and additional facultative layers (30). The innermost layer of the cell wall, the electron-opaque or peptidoglycan layer overlays the cytoplasmatic membrane. In ultrathin sections, the peptidoglycan layer is separated from the cytoplasmatic membrane by an electron transparent space. The peptidoglycan layer of most cyanobacteria is thicker than in most gram-negative bacteria, and the width varies between
1 and 10 nm and can go to 200 nm in Oscillatoria princeps (31). The peptidoglycan layer which form a polymeric fibrous mesh is primarily responsible for the mechanical strength of the wall; h confers the shape of the cell and protects it against osmotic damage (29).
Regularly arranged discontinuities have been observed in peptidoglycan layers of many cyanobacteria. Pores with average 13 nm in diameter are located in single circumferential rows on either side of every cross wall and are formed from the inside outward (32). Pit-like pores which average 70 nm diameter are uniformly distributed over the cell surface with an average ccnter-to-center spacing of 200 nm (33). It is suggested that the pores allow diffusion of sheath precursors and permit close contact with and steady ALP supply to the postulated contractile fibrils of the outer membrane (34).
The outer membrane of the cell wall, the lipoprotein layer, appears as a double-track structure of ultrathin sections (diameter 7-10 nm). The outer layer probably controls the transport of solutes
(29).
The space between the two layers, termed the periplasmatic space, appears to have a similar content of lipopolysaccharides and degradative enzymes as in Gram-negative bacteria (29).
Numerous cyanobacteria possess a polysaccharide envelope outside the outer membrane, called sheath, glycocalyx, capsule, mucilage, or slime. Many sheaths contain fibrils, which are 1-2 pm
17 long, made of polysaccharides and embedded in the amorphous matrix. These sheaths are
composed of polysaccharides consisting of glucose, hexuronic acids, xylose, ribosc, galactose, rhamnose, arabinose, and polypeptides. Thickness and consistency of the gelatinous sheath
depend on environmental conditions. One principal function of the sheath appears to be the protection of desiccation and therefore cyanobacteria are able to survive for long periods of
drought. Additionally, polysaccharides of the sheath may absorb essential nutrient from the
environment (35).
3.2.3 Nuclear Apparatus
The DNA fibrils are organized in a complex helical and folded configuration and are distributed uniformly throughout the centroplasm. The size of the genome varies widely in cyanobacteria, having a molecular weight between 1.6 x 109 and 8,6 x 109 daltons. Most unicellular forms possess genomes of about 1.6 x 109 to 2.7 x 109 daltons, similar in size and shape to that found
in other prokaiyotic microorganisms. Cells of the pleurocapsalean and filamentous cyanobacteria however contain larger genomes.
Ribosomes in cyanobacteria are seen diffusely distributed throughout the cytoplasm and particular concentrated in the nucleoplasm^ region. Their size, about 10-15 nm in diameter, and their sedimentation properties, 70 S (Svedberg units), are typically prokaiyotic (29).
3.2.4 Cytoplasmatic Inclusions and Reserve Products
The cyanobacterial cytoplasm contains a series of inclusions, most of which function as reserves
of carbon and energy, and which accumulate under extreme nutrient and stressful conditions (36).
• Glycogen granules, which are minute (about 30 x 65 nm in size), ovoid or rod-shaped
structures deposited primarily in the cytoplasm between the thylakoids, serve as a carbon or
energy source.
® Lipid globules, which are spherical, -variable in size (up to 30 - 90 nm in diameter), and most
abundant near the cell surface, possibly represent lipid stores for use in membrane synthesis.
• Cyanophycin granules are relatively large in size (up to 500 nm in diameter), and are also
deposited mainly at the cell periphery. They consist of a unique polypeptide (multi-L-arginyl
18 poly-L-aspartic acid) composed only of two amino acids, arginine and aspartic acid. The
arginyl residues are attached at each free carboxyl group of the polyaspartate core. They are
produced, unlike other polypeptides, by a ribosome-independent mechanism, and have
function in nitrogen turnover.
in • Polyphosphate bodies, which are fairly large structures, appear highly electron-dense even
thin sections, and serve as phosphate store.
- 300 run in • Carboxysomes (or polyhedral bodies) are semicrystallme structures about 200
diameter. They contain a reserve form of the primary photosynthetic enzyme, ribulose-1,5-
bisphosphate carboxylase, which, in its active form, catalyzes photosynthetic C02 fixation
into ribulose-1 „5-biphosphate.
Several other inclusions (like microtubules, microfilaments, wall bodies, crystalline and trilamellar bodies, and also granular and fibrillar ciystals) have been observed in various
cyanobacterial cells but so far less is known about their function (29).
Furthermore, aerotopes (group of gas \ esicles)) are found m cells of many planctonic species.
'They are important in regulating buoyancy and responsible for vertical migration (37).
A schematically system of a cyanobacterial cell is shown in Figure 2.3 (38).
19 jS^îe veer/
20 3.2.5 Heterocysts
Heterocysts are differentiated cells that are specialized in fixation of N2 in an aerobic environment (see 2.3.1). In the light, Photosystem I generates ATP, without photosynthetic production of 02. Instead, NH4~ moves into heterocysts from vegetative cells. In return, fixed nitrogen moves from heterocysts to vegetative cells (39).
3.2.6 Motility
Most cyanobacteria show gliding, flexi onal, or rotary movement, though they do not possess flagella or cilia at any stage. The mechanism causing the movement is not understood, though various hypotheses have been suggested which involve excretion of mucilaginous material, propagation of rhythmic waves of contraction in the cells, or mechanisms associated with osmosis or surface tension. Many of the filamentous forms, which show the most rapid movements, also show the presence of pores through the cell wall through which strands of mucilaginous substances might be secreted (40).
For a prokaryote or eukaryote gliding is a self-propulsion across a solid or semi-solid material without the aid of any visible organ (flag ell urn) or apparent change in the shape of the organism.
However, Visible' is defined by the limits of the light microscope and by the inability of the electron microscope to utilize living material. Gliding is a relatively slow movement (its speed varies between about 1 and 10 urn s"1), and because of this inherent sluggishness, gliding is scarcely important as a means of dispersing the species over a large area. It may, however, help the organism to adjust its position in the environment, by gliding forward and backward, and to settle in area where conditions are favorable for growth. This kind of movement depends on temperature, light intensity and quality. pH value and chemical composition of the environment
(41, 42). Furthermore, light can detenuine the direction of movement by inducing both phototaxis
(negative and positive) and Photophobie reactions (43).
21 3.2.7 Modes of Reproduction
Most unicellular cyanobacteria reproduce themselve by three types of binary fission (simple binary fission, asymmetrical binary fission, and irregular binary fission). Spherical cells are budded off at one end (the apical pole) of the ovoid or elongated sessile vegetative cell, which displays a distinct, basal to apical, polarity.
There arc various modes of reproduction in the filamentous forms, and some involve the formation and germination of specific reproductive structures.
• The simplest and in some genera the commonest way of reproduction is by random trichomc
fragmentai ion. The trichomc fragments may vary m size but even a single detached cell may
be capable under favorable conditions to reproduce the characteristic filamentous morphology
of a particular species.
• Filament fragmentation is often an ordered process associated with the production of distinct
reproductive trichome segments called hormogones (or hormogonia). These are short 5 to 15
celled segments which separate by the rounding off of their end cells within the trichomc
envelope. They exhibit active gliding motion (see 2.2.6) upon their liberation, and can
develop into a new trichome (44). In some cases specialized cells (necridia), which become
biconcave in shape due to lysis and dehydration permit fragmentation of the trichome and
subsequent liberation of the hormogonia (45).
• Exocyte and baeocyte division are characteristic of the two orders Chamaesiphonaceae and
Dermocapcllaceac. Exocytes develop by successive cleavage from the apical pole of an
elongated vegetative cell. Baeocytes are formed by multiple fission of a large spherical
vegetative cell and are subsequently released by rupture of the parental wall and form new
vegetative cells (45).
• Many of the heterocystous cyanobacteria are also capable to produce akinetes or spores,
under adverse environmental conditions, which dewelop through the transformation of
vegetative cells. Akinetes are reversible differentiated cells, which arc growing by stroring
food reserves (cyanophycin granules, glycogen, and lipids). RNA, as well as carotenoid
pigments. Photosynthetic capacity however decreases or ceases completely. Akinetes tolerate
drying, freezing, and many years of storage m anaerobic sediments. After this resting period,
which is not absolutely necessary, the akinete may germinate, resulting in a vegetative
1") trichome (44). Usually the akinete is larger, its cell wall is thicker and its cytoplasm more
granular than the vegetative cell (45).
3.2.8 Cyanobacteria in Symbiotic Associations
Cyanobacteria occur in many symbiotic relationships such as in lichens, in the roots of cycads and with certain bryophytcs and pteridophytes. In some of these relationships the cyanobacteria are thought to contribute most or all of the reduced carbon and nitrogen compounds required for growth by the host cell (40).
3.2.8.1 Cyanelles and the Origin of Chloroplasts
Several eukaryotic protists contain green chloroplast-like structures in their cytoplasm. It is suggested that these organelles, called cyanelles. may have originated as independent cyanobacterial cells which invaded the host and became established as intracellular symbionts, which were modified to function as organelles of the host cell. 'There are many similarities between chloroplasts and cyanobacterial cells. There is considerable morphological, enzymatic, and chemical evidence in favor of this possibility, although such an evolutionary development of chloroplasts from cyanobacteria would entail many alterations in structure and chemical composition of the endosymbiont (40).
3.2.8.2 Casual Associations
Many bacteria arc embedded in the often extensive mucilaginous sheath surrounding cyanobacteria. These bacteria are adapted to the microenvironment of the mucilage envelope, and thrive on the extracellular organic products released by the cyanobacteria. Bacterial assimilation of organic substrates results in the production of COi. which could immediately be available to the cyanobacterium and re-assimilated in photosynthesis (49). Also cyanobacteria are often in the mucilage of other algae (e.g. Phormidium muacola).
23 3.2.8.3 Associations between Fungi and Cyanobacteria
The symbiotic association of some fungal partners (e. g. Coccomyxa) and cyanobacteria (e. g.
Nostoc) forms a special morphological entity. It is suggested that some fungi are able to modify the permeability properties of the cyanobacterial plasma membrane by chemical action. Increased permeability enhances the movement of metabolites, sugars and ammonia, from the cyanobacterial cells to the fungus. The fungus furnishes water and mineral supply, optimal light and oxygen conditions to the cyanobacterium (49).
3.2.8.4 Associations between Liverworts and Cyanobacteria
Cyanobacteria form symbiotic associations not only with heterotrophs but also with photosynthetic organisms. Common associations can be found between Nostoc and the bryophytes Blasia, Anthoceros and Cavicularia. The cyanobacterial endosymbiont occupies mucilage-filled intercellular cavities withm the liverwort thalli. The invading Nostoc filaments enter through small mucilaginous pores. Once installed inside the liverwort cavity, the Nostoc colony becomes metabolically specialized for the function of N2-fixation and depends almost completely on the host for a source of carbon and energy. Fixed carbon, probably as sucrose, is liberated by the host cells and translocated to the endosymbiont (49).
3.2.8.5 Association between a Fern and a Cyanobacterium
The only known example of a symbiosis between a fern and a cyanobacterium is the association between the floating water fern A/olla and the cyanobacterium Anabaena. The fern as host provides the N,-fixing cyanobacterium with photosynthetic products, which allows N2-fixation to continue even at night (49).
3.2.8.6 Associations between Higher Plants and Cyanobacteria
Two of groups seed-bearing plants, the gymnosperm group of cycads and the angiosperm genus
Gunnera, harbor hetereocystous cyanobacteria.
24 In the root nodules of cycades, nostocacean filaments occupy the mucilage-filled intercellular
spaces in the distinctly green middle zone of the root cortex. Following invasion, the cells
bordering this intercellular space produce tubular outgrowth and secret at least part of the mucilaginous matrix. Although the fonnation of root nodules is independent of the presence of
Nostoc, only those, which arc invaded by the cyanobacteria. persist (49).
3.2.8.7 Associations between Invertebrates and C\ anobactcria
Many different bacterial and cyanobacterial symbionts arc found in marine sponges. The chemical relationship between secondary metabolites from cyanobacteria and sponges, tunicates and other aquatic organisms, has led to the speculation that the secondary metabolites of marine invertebrates may have originated from symbiotic cyanobacteria (50).
Aggregates of unicellular cyanobacteria occur in vacuolated cells of the sponges. The endosymbiont is able to fix N2 within the sponge system. This may be particular beneficial to sponges in tropical seas, which are generally short of Nb (49),
33 Metabolism
3.3.1 Nitrogen-Fixation
Cyanobacteria can assimilate nitrogen from nitrate, nitrite, ammonia, hydroxylamin, urea, casein, amino acids, uric acid, and NT. There is considerable variation among species to which forms of
can be nitrogen assimilated, but all cyanobacteria apparently can grow with nitrate as a sole nitrogen source (40).
The ability to reduce the nitrogen molecule to ammonia is restricted to prokaryotes, where it is
scarce but rarely spread widely through the systematic groups. Among these organisms, a prominent role is occupied by the cyanobacteria. They fix N: both m the free-living state and in symbiosis with a wide range of partners (51 ).
25 The machinery for fixing N2 has been shown to be present in all the currently recognized taxonomic groups of cyanobacteria.
The enzyme complex nitrogenase catalyzes the ATP-dependent reduction of N2 to two molecules of ammonium. This reaction requires the transference of six electrons to N2.
The reduction of N, to NII( catalyzed by nitrogenase is a highly endergonic reaction that requires metabolic energy in the form of A'fP. The biological reduction of N, by nitrogenase always occurs concomitantly with the reduction of protons to II,, a wasteful reaction costing both energy and rcductant that decreases the efficiency of the N. fixation process (39).
The N2-fixation reaction is one of the fundamental biological processes, essential for the maintenance of the nitrogen status of the whole biosphere.
Nitrogenase is extremely sensitive to free O-, and can function only under anaerobic conditions.
Direct exposure of the enzyme to air results in the inactivation and even the destruction of the component proteins (52). This consists the development of heterocysts. These arc specialized cells that, under conditions of aerobiosis and combincd-N deprivation, differentiate from vegetative cells at semi-regular intervals in the filament and bear a series of modifications devoted to the protection of the NVfixation apparatus from O, (39). For the unicellular and filamentous strains which do not differentiate heterocysts, the mechanism by which the nitrogenase complex is protected is less well known (36).
The 02 sensitivity and the fact that the enzyme system is present only in prokaryotes suggest that the ability to fix N2 has originally evolved during the early anoxygenic period of earth history.
There is reasonable geochemical and fossil evidence in support of this suggestion. With the development, initially in ancient cyanobacteria, of O-,-evolving photosynthesis, the atmosphere has become gradually more oxygenic until the partial pressure of O, in the atmosphere reached its present level (52). The fixation of N, is particularly important m the colonization of recently exposed land surfaces and in accumulating a reservoir of reduced carbon and nitrogen compounds which is essential for the establishment of metazoans. cukaryotic algae, and higher plants. Such colonization is important in areas denuded of life by vulcanic activity or excessive radiation, as well as in desert areas, sand dunes, and on land exposed by receding glaciers (40).
26 3.3.2 Photosynthesis
3.3.2.1 Photosynthetic Membrane System
The peripheral region of the cytoplasm contains the photosynthetic apparatus of cyanobacteria. In unicellular forms this commonly consists of a few membrane layers which extend in concentric
sheets beneath the cell membrane and surround the central nucleoplastic region. The more elaborate membrane structure seen in the cells of filamentous species is apparently formed by means of expansion and invagination of the membranes. The basic structure of the membranes resembles a flattened sac and is known as the tlyvlakoid. The apprcssed membranes of the thylakoid may occasionally or partially separate displaying the inner cavity of the thylacoid sac.
The lipid bilayer of the thylacoid membrane incorporates the lipophilic photosynthetic pigments, chlorophyll a and various carotenoids. It also incorporates the components of the electron transport chain (like cytochromes, plastocyanm, and ferredoxin). Chlorophyll a, which is the only chlorophyll species in cyanobacteria. is present m three different forms which can be distinguished on the basis of their absorption characteristics (maximum light absorption, Xmax at
670, 680 and 700 nm, respectively). Among the carotenoids, which absorb in the range between
480 and 520 nm wavelength, ß-carotene appears to be unnersal present in all cyanobacteria while the presence and abundance of different xanthophylls (echienonc, zeaxanthin, oscillaxanthin, myxoxanthophyll) varies according to species.
An important part of the photosynthetic pigment complement of cyanobacteria is located in granular supramolecular complexes, called phycobihsomes. They are water-soluble proteins, which consist of the two blue biliproteins c-phycocyamn (Xmn 620 nm), c-allophycocyanin (Xmax
550 - 570 nm), and the red c-phycoerythrin (X11U1X 650-670 nm) as chromophoric groups. The relative quantities of these pigments may vary according to species and the spectral composition of light, and will determine the colored appearance of these organisms (29). These accessory pigments are the primary light harvesting pigments of photosystem II. They possess a remarkable efficiency of nearly 100 % in light harvesting and energ\ transfer (36).
27 3.3.2.2 Photosynthetic Metabolism
The main characteristics of photosynthesis, originally elucidated in green algae and higher plant
chloroplasts, also apply to cyanobacteria. There are some important aspects of photosynthesis, which are, however, particular to cyanobacteria. One contains the spectral characteristics of light absorption in cyanobacteria, which are inherently different from those of other photosynthetic organisms. High rates of photosynthetic activity are measured in cyanobacteria not only in the spectral region between 665-680 nm wavelength, were light absorption of chlorophyll is greatest, but also around 620 nm or 560 nm, where phycocyanin and phycocrythrin, respectively, absorb light effectively. It was shown that light absorbed by the phycobiliproteins is used by cyanobacteria as efficiently as light absorbed by chlorophyll. Excitation energy is transmitted from bilyprotein pigments to chlorophyll with great efficiency. Energy transfer proceeds as a rule from a pigment with an absorption maximum at a shorter wavelength of light to a pigment with greatest light absorption at a longer wavelength of light. The sequence is: phycocrythrin v> phycocyanin => allophycocyanin =» chlorophyll a (Si).
Efficient energy transfer enables cyanobacteria to prosper under particular light regimes, like those encountered by aquatic forms deep below the water surface (52).
Another thing is, that cyanobacterial strains can grow under anaerobic conditions or switch to a bacteria-like photosynthesis in which H2S is comerted to sulfur (54).
28 Ferredoxin
NADP M !
ADP
r cytochromes a
piâsîoquînofie ATP
cytochrome c e~ Tx. plastocyanin
PftOföS¥St«m
H,0 'P68CP ^_ Photo« system If ^^"^ HO»
UCHT>660nm
Phycobilisome
LIGHT < 660 nm
Figure 3.4. fight reactions in phtosynthesis of cyanobacteria (53)
29 3.3.3 Heterotrophic Growth
Some cyanobacteria will grow not only in the light with C0? but also under conditions where growth is dependent on exogenous organic compounds. Such heterotrophic growth can take two different forms: photoheterotrophy, which is growth in the light on an organic compound in the absence of CO-, fixation, and chemoheterotrophy, which is growth on an organic substrate in complete darkness. In the latter case, the organic compound provides the organism with a source of carbon and energy while in photoheterotrophy the organic compound is used only as a source of carbon, while light is supplying the energy needs of the cell (55).
3.4 Ecology
3.4.1 Habitats
The cyanobacteria are widely distributed in practically all types of habitats in temperate, tropical, and polar regions. Most commonly, they occur in fresh water, marine or brackish water, however, they can also grow in hot springs, saline lakes, frigid lakes in the Antarctic, oil field ponds, desert areas, and on bare rocks (46).
Many cyanobacteria grow attached on the surface of hygropetric rocks and stones (epilithic forms), on submerged plants (epiphytic forais) or on the bottom sediments (epipelic forms) of lakes. Planktonic cyanobacteria are rather uncommon in rivers or streams, except during periods of reduced flow. But attached forms, well adapted to strong currents, may grow abundantly receiving a continuous supply of nutrients. Many cyanobacteria arc epiphytic on larger algae and on other river plants. Thermophilic cyanobacteria were found to grow at temperatures as high as
73 °C. They are most abundant in the alkaline hot springs and absent from hot acid springs. The hot source water contains most of the essential nutrients and permits a steady growth throughout the year.
Cyanobacteria are also extremely hard in regard to survive the generally deleterious effects of low temperatures. The cells flourish on exposed land surfaces and on ice in the Antarctic, which presents severe climatic conditions. 'The temperatures are not only as low as -88 °C, but cells are exposed to severe desiccation conditions imposed by low atmospheric humidity. They are also
30 subjected to high light intensities in the summer months, followed by many months of darkness
(40). Cyanobacteria are of widespread distribution in and around the oceans and form an important element of the vegetation of some particular marine habitats, like the iiitertidial zone of temperate and tropical seas and in estuarinc areas (47).
In the marine environment cyanobacteria occur as symbionts in the surface tissues of invertebrates (e. g. sponges), or as epiphytes upon larger algae or sea grasses. Endolithic cyanobacteria are found in living corals, in fossil limestone, in dead corals, and in the shells of living and dead mollusks (48). Although cyanobacteria are more apparent in aquatic habitats, they can often be seen on the surface of moist soil and growing beneath the soil surface. In the temperate region cyanobacteria are especially common in calcareous and alkaline soils (47).
3.4.2 Public Health
Extensive growth of cyanobacteria can create severe problems in the maintenance of water supplies and in meeting the ever-increasing demand for potable water. Cyanobacteria produce a diverse range of secondary metabolites including hepatotoxins. neurotoxins, and cytotoxins.
Much research on these toxins focus on freshwater where cyanobacterial toxins are responsible for, or contribute to, acute and chronic human and animal health problems and the impairment of water supply- and water-based-activities. Cyanobacterial blooms in brackish- and marine waters can also produce toxins. Their impact in cstuanne and marine environments is unclear, although shellfish harvesting and fisheries are adversely affected.
Cyanobacterial bloom toxicity appears to be a global phenomenon. Awareness of cyanobacterial blooms and scums, and of health hazards which they can present, is long established and has been heightened by a history of associated animal death and outbreaks of human illness. Human health incidents associated with, or attributed to cyanobacterial toxins include gastroenteritis outbreaks and atypical pulmonary consolidation. These incidents were recorded after single- or relatively short-term exposures to cyanobacterial blooms, scums and toxins. A major human poisoning episode, involving the death of at least 55 people, and attributed to exposure to microcystins, occurred in 1996 in Brazil. The deaths occurred after haemodialysis treatment at a clinic in
Caruaru, Brazil, with victims presenting li\er damage consistent with microcystin poisoning and the presence of microcystins in clinical and post-mortem specimens (57).
31 The need to investigate the consequences of long-term exposure to cyanobacterial toxins is apparent since they can be present in drinking water in the absence of effective water treatment and long-term or at least multiple exposures may occur during recreational water activities and bathing or showering. Additionally, some toxins (e. g. microcystins) arc potent tumor-promoters in animal studies. Thus, studies are in progress in the Peoples' Republic of China and in Riga in regions where a higher incidence of human primarily liver cancer occurs among populations using surface water containing microcystins for drinking, than in neighboring areas where ground water, apparently free from microcystins is used for drinking. World Health Organization (WHO)
Guidelines for human drinking water quality have not yet included Guideline Values (GV) for cyanobacterial toxins. However, based on exposure assessments of studies of the effects of microcystin-LR in animals and risk assessment to humans via drinking water, a GV of I ug/L has been proposed.
Proactive monitoring and toxin analysis programs show that cyanobacterial toxins commonly occur in waters with cyanobacterial blooms worldwide and that a precautionary principle in cyanobacterial bloom management and water treatment is appropriate with respect to cyanobacterial toxins (56, 57),
3.4.3 Detection of Toxins and Cyanobacterial Growth Control
To control toxic cyanobacterial blooms it is important to be able to determine quantity and quality of toxins as well as to understand the circumstances under which they occur.
Methods of detection for the cyanotoxins use bioassav. chemical assay, and immunoassay techniques. The mouse bioassay has been the typical first test for toxicity in screening water bloom material and laboratory cultures or cell extracts. Its advantages are that it is easy to use assuming that laboratory test mice are readily available. The method is inexpensive and can detect within a few hours, the qualitative and quantitative characteristics of the toxin present.
From the signs of poisoning it is possible to distinguish hepatotoxins from neurotoxins and even the different types of neurotoxins. The disadvantages of the mouse bioassay arc its inability to detect low level amounts of toxins, especially in drinking water, and the inability to distinguish between homologues of different toxins. In addition it is desirable to minimize the use of laboratory test animals, especially mammals (58).
32 This has led to the investigation of other bioassays, including invertebrate, bacterial and mammalian cultured cells (59). Chemical detection assays for the cyanotoxins have also been developed. These include high performance liquid chromatography, high performance thin layer chromatography, thin layer chromatography, gas chromatography, and fast-atom bombardment mass spectrometry. None of these techniques has been developed into a standard method.
The most promising method of detection for these toxins would seem to be some type of immunoassay. To date only the peptide toxins have been used to develop immunoassay techniques. These techniques have also been successful to detect low level toxin amounts in drinking water and animal tissue (58).
Chemicals are widely used to prevent the growth of cyanobacteria, and the commonest treatment is the application of copper sulfate (at a concentration of 0.5 - 1.0 mg/L). Although initially these low concentrations are not toxic to fish or other aquatic animals, repeated administration may increase the concentration of copper m the water to a toxic level. A number of other algicides arc used effectively in water supplies, like phenolic compounds, amide derivatives, quaternary ammonium compounds and quinone derivatives. Nevertheless, the hazards of using toxic chemicals in the natural environment arc well documented, and it would seem to be advisable to replace such practices by other less hazardous methods. The introduction of natural grazers and pathogens of cyanobacteria can achieve biological control. Some invertebrates and snails arc known to graze on cyanobacteria and diatoms.
Microorganisms (fungi, bacteria and viruses) appear to play an important part in regulation the growth of cyanobacteria in freshwaters. Thus, some bacterial pathogens, belonging to the group
Myxobacterialcs, can effect rapid lysis of a wide range of unicellular and filamentous cyanobacteria. Another approach to regulate the growth may be the removal of nutrients essential for the growth of cyanobacteria in water reservoirs (60).
Destratification by aeration of water reservoirs to maintain thorough mixing of the water body has been shown in some cases to decrease the incidence of cyanobacterial blooms (61).
33 3.4.4 The Use of Cyanobacteria as Biofertilizer
Organic fertilizers are of great value for agriculture. Natural manner, in contrast to chemical
fertilizers, is a continuous source of nutrients, which arc slowly released in accordance with
requirements of plant growth.
In order to use their beneficial effects, cyanobacteria are grown on a large scale in India, the
Philippines and China, and disseminated in paddy fields (60).
Nitrogen is usually the limiting factor to produce high rice yields. The idea of utilizing algal
biofertilizer as an alternative or supplementary source of nitrogen for rice is a potential biological
system in low cost rice production technology. The resulting nitrogen fertility by algal
application has permitted moderate but constant producth ity in fields where no nitrogen fertilizer
is applied. The cyanobacteria used as biofertilizers consist of Nostoc, Anabaena, Tolypothrix,
Plectonemma, Aphanothece, Oscillatoria, Cylindrospermum, Aulosira and Scytonemma.
Application of dried cyanobacteria at the rate of 10 kg per hectare is recommended to the
farmers. A composite start culture consisting of more than one species or genera is more
preferable. Further, it is always preferable to have the local florae of cyanobacteria. as they
acclimatize to the local surroundings much more easily than the introduced ones. Fast growing
species, which can tolerate certain amount of extremities, are more suitable to slow growing and
highly susceptible ones (62).
3.5 Pharmaceutical and Pharmacological Interest of Cyanobacteria
Natural products are an important source of new pharmaceuticals and pharmaceutical lead
compounds (63). The investigation of chemicals produced by plants and microorganisms has
resulted in the discovery of numerous organic compounds, many of which have found
applications as pigments, fragrances, insecticides, pharmaceuticals, or biomedical tools. Early
studies which focused on the terrestrial plants and microorganisms, proved extremely fruitful,
yielding many useful organic compounds, including for example, as many as 25% of the
currently used anticancer drugs, with another 25% coining from synthetic derivatives of natural products (64).
34 The cyanobacteria in general have produced a number of interesting secondary metabolites, including peptides, alkaloids, nucleosides and lactones. These organisms may arguable be the class of algae with the highest potential as a source of commercially valuable natural products of pharmaceutical interest (65). Cyanobacteria have been recognized in the last several years as a
source of novel cytotoxic, antineoplastic, antiviral, antialgal, growth stimulatory, immuno¬ modulatory, antibacterial and antifungal metabolites as well as specific enzyme inhibitors (66).
Chapter 4 provides an overview of the most interesting and potent secondaiy metabolites reported from cyanobacteria.
3.6 References
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7. Skulberg, O. M.. Carmichael, W. W., Codd. G. A., Skulberg, R., Taxonomy of Toxic
Cyanophyceae (Cyanobacteria), in Algal Toxins in Seafood and Drinking Water. London,
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8. Rippka, R., Recognition and Identification of Cyanobacteria, /// Methods in Enzymology.
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Cycinobaklerien. Krumbern, W. E. Oldenburg, Universität Oldenburg: 107-112 (1979)
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35 11. Bornct, E., C, F.: Révision des Nostocacces hètèrocystées. Ann. Sei. Nat. 7: 323-381/343-
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14. Komarek, E, Anagnostidis, K.: Modern Approach to the Classification System of
Cyanophytes. 4-Nostocales. Arch. Hydrobwl. (Suppl. 823) 56: 247-345 (1989)
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Relationship between some Blue-Green Algae (Cyanophyceae). Acta. Bot. Nederl. 26:
327-342 (1975)
18. Stam, W. T.: Relationships between a Number of Filamentous Blue-Green Algal Strains
(Cyanophyceae) Revealed by DNA-DNA Hybridization. Ach. Ilydrobiol (Suppl. 56) 25:
351-374(1980)
19. Anagnostidis, K., Komarek, J.: Modern Approach to the Classification System of
Cyanophytes. 3-Oscillatoriales. Arch. Hvdrobiol. (Suppl. 80) 50/53: 327-472 (1988)
20. Anagnostidis, K., Komarek. E: Modern Approach to the Classification System of
Cyanophytes. 5-Stigonematales. Arch. Ilydrobiol. (Suppl. 86) 59: 1-73 (1990)
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Molecular Biology of Cyanobacteria. Dordrecht, The Netherlands, Kluwcr Academic
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Green Algae) under the Rules of the International Code of Nomenclature of Bacteria. Int.
J. Svst. Bacteriol. 28: 335-336 (1978)
23. Herdnian, M., ct ah: Deoxyribonucleic Acid Base Composition of Cyanobacteria. J. of.
Gen. Microbiol. HE 63-71 (1979a)
24. Herdman, M., Janvier, VI., Rippka. 11., Stanier, R. Y.: Genome Size of Cyanobacteria. J.
of. Gen. Microbiol. 111: 73-85 (1979b)
36 25. Kcnyon, C. N., Rippka, R., Stanier, R. Y.: Fatty Acid Composition and Physiological
Properties of some Filamentous Blue-Green Algae. Arch. Mikrobiol. 83 : 216-236 (1972)
26. Rippka, R., Deruelles, R., Watcrbury, J. B., Herdman, M., Stanier, R. Y.: Generic
Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. J. Gen.
Microbiol. Ill: 1-61 (1979)
27. Gibbons, N. E.. Murray, R. G. E.: Bacteriological Taxonomy. Int. J. Syst. Bacteriol. 28: J -
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28. Anand, N., Morphology, Classification, and Taxonomic Studies, in Indian Phycological
Review. Dehra Dun, India, Bishen Singh Mahendra Pal Singh (1993)
29. Fay, P.: The Blue-Greens (Cyanophyta-Cyanobactena). Edward Arnold Ltd, London. 160:
4-18(1983)
30. Bold, H, C, Wynne, M. E: Introduction to the Algae: Structure and Reproduction.
Prentice-Hall: 34-35 (1985)
31. Halfen, L. N., Castenholz, R. W.: Gliding Motility in the Blue-Green Alga Oscillatoria
princcps. J. Phycol. 7: 133-145 (1971)
32. Frank, IT. Lefort, M., Martin, IE IE: Elektronenoptischc und Chemische Untersuchungen
an den Wänden der Blaualge Phormidium unanatum, Z. Naturf. 7: 262-268 (1962)
33. Shukovsky, E. S., Halfen. L. N.: Cellular Differentiation of Terminal Regions of
Oscillâtoria princcps. J. Phycol. 12: 336-342 (1976)
34. Halfen, L. N., Castenholz, R. W.: Energy Expenditure for Gliding Motility in a Blue-
Green Alga. J. Phycol. 7: 258-260 (1971)
35. Castenholz, R. W., Watcrbury. J. B.: Bergey's Manual of Systematic Bacteriology.
Williams and Wilkins, Baltimore. 3: 1710-1711 (1989)
36. Tandeau de Vlarsac. N., Houmard, E: Adaption of Cyanobacteria to Environmental
Stimuli: New Steps Towards Molecular Mechanism. FEMS Microbiol. Rev. 104: 119
(1993)
37. Walsby, A. E.: The Prokaryotes, a Handbook on Habitats, Isolation, and Identification of
Bacteria. Springer-Verlag, Berlin: 224-230 (1981)
38. Wartenberg, A.: Systematik der niederen Pflanzen, fhieme, Stuttgart: 62 (1979)
39. Flores. E., Herrero, A., Assimilatory Nitrogen Metabolism and its Regulation, in The
Molecular Biology of Cyanobacteria. Dordrecht, Boston, London, Kluwer Academic
Publishers (1994)
37 40. Holm-Hansen, O.: Ecology, Physiology, and Biochemistry of Blue-Green Algae. Ann.
Rev. Microbiol. 22: 47-70 (1968)
41. Fay, P.: The Blue-Greens (Cyanophyta-Cyanobacteria). Edward Arnold Ltd, London. 160:
47-59(1983)
42. Castenholz, R. W.: Motility and Taxes, in The Biology of Cyanobacteria. Oxford,
London, Edinburgh, Boston, Melbourne, Blackwell Scientific Publications (1982)
43. Hader, D.-P.: The Cyanobacteria. Else\ 1er, Amsterdam: 437-440 (1987)
44. Fay, P.: The Blue-Greens (Cyanophyta-Cyanobacteria). Edward Arnold Ltd, London. 160:
19-29(1983)
45. Nichols, J. M., Adams. D. G., Akinetes. in The Biology of Cyanobacteria. Oxford,
London, Edinburgh, Boston, Melbourne. Blackwell Scientific Publications (1982)
46. Ahluwalia, A. S.: Habitats and Distribution, in Cyanophyta (Cyanobacteria). Dehra Dun,
India, Bishen Singh Mahendra Pal Singh ( 1993)
47. Fay, P.: 'The Blue-Greens (Cyanophyta-Cyanobacteria). Edward Arnold Ltd, London. 160:
(1983)
48. Kazmierczak, E, Kempe, S.: Modern Cyanobacterial Analogues of Paleozoic
Stromatoporoids. Science 259: 1244-1248 (1990)
49. Fay, P.: Ehe Blue-Greens (Cyanophyta-Cyanobacteria). Edward Arnold Ltd, London. 160:
60-68(1983)
50. Fusetani, N., Matsunaga, S.: Bioactive Sponge Peptides. Chem. Rev. 93: 1793-1806
(1993)
51. Bothe, H., Nitrogen Fixation, in The Biology of Cyanobacteria. Oxford, London,
Edinburgh, Boston, Melbourne. Blackwell Scientific Publications. (1982)
52. Fay, P.: Ehe Blue-Greens (Cyanophyta-Cyanobacteria). Edward Arnold Ltd, London. 160:
30-46(1983)
53. Fay, P.: The Blue-Greens (Cyanophyta-Cyanobacteria). Edward Arnold Ltd, London. 160:
32(1983)
54. Cohen, Y., Jorgenscn. B. B., Revsbach, N. P., Poplawsky, R.: Photosynthtic Metabolism
of Cyanobacteria. Appl. Environm. Microbiol. 51: 398 (1986)
55. Smith, A. E, Modes of Cyanobacterial Carbon Metabolism, in The Biology of
Cyanobacteria. Oxford. London, Edinburgh, Boston, Melbourne. Blachwell Scientific
Publications (1982)
38 56. Codd, G. A., Ward, C. E, Beatti, K. A., Bell, S. G., Widening Perceptions of the
Occurence and Significance of Cyanobacterial Toxins, in The Phototrophic Prokaryotes.
New York, Kuwer Academic/ Plenum Publishers (1999)
57. Codd. G. A., Cyanobacterial Blooms and Toxins in Fresh-, Brackish-, and Marine Waters,
in Harmful Algae., Xunta de Galicia and Intergovernmental Océanographie Commission
of UNESCO (1998)
58. Carmichael, W. W.: Cyanobacteria Secondaiy Metabolites - lire Cyanotoxins. J. Appl.
Bacteriol. 72: 445-459 (1992)
59. Codd, G. A., Brooks, W. P.. Priestly. 1. M., Poon, G. K., Bell, S. G.: Production,
Detection, and Quantification of Cyanobacterial Toxins. Toxicity Assessment 4: 499-511
(1989)
60. Fay, P.: The Blue-Greens (Cyanophyta-Cyanobacteria). Edward Arnold Ltd, London. 160:
77-84(1983)
61. Pastorak, R. A., Ginn, E. C, Eorenzen, M. W.: Restoration of Lakes and Inland Waters.
U. S. EPA-440/5-81-010: 75-80 (1980)
62. Kannaiyan, S., Algal Biofertilizer 'Technology for Rice Crop, in Cyanophyta
(Cyanobacteria). Dehra Dun, India. Bishen Singh Mahendra Pal Singh. (1993)
63. Schwartz, R. E., ct ah: Pharmaceuticals from Cultured Algae. J. Ind. Microbiol. 5: 113-
124(1990)
64. Davidson, B. S.: Mew Dimensions in Natural Product Research: Cultured Marine
Microorga.msms.Current Oppinion in Biotechnology 6: 284-291 (1995)
65. Cannell, R. .1. P.: Algae as a Source of Biologically Active Products. Pesticide Science 39:
147-153(1993)
66. Behrens, P. W.: New Drugs from Natural Sources. Microalgae as a Source oj Bioactive
Products. IBC Technical Services, London: 166-175 (1992)
39 4 Secondary Metabolism of Cyanobacteria
4.1 Introduction
Most plants and microorganisms, from the simplest unicellular algae to complex angiosperms,
metabolites. Thousands of produce a variety of compounds collectively referred to as secoudary these compounds have been chemically characterized and classified on the basis of their
metabolites have useful functional groups and chemical properties. Some of these
pharmacological properties, for example, the antibiotics, penicillin and erythromycin and drags
such as morphine and vinblastin (1).
Since secondaiy metabolites serve no basic obvious metabolic function and vary in concentration
and structure even in closely related species, they were once considered metabolic waste products
(2). However, most of these compounds are structurally very complex and therefore
metabolically expensive. In addition they have a rapid turnover rate in the cell. Such properties
are hardly those of waste products (3).
Advances in plant physiology and biochemistry have shown that many secondary metabolites
facilitate interactions between plants and their environment. Examples of these are the plant
hormones, auxins, and gibberellins, which govern plant growth and development in response to
environmental parameters such as light and temperature. The synthesis of other secondary
metabolites, the phytoalexins, is induced by parasitic invasion and may enable plants to fight off
infections (4). It has also been suggested that many secondary metabolites are allopathic
substances; i. e., compounds produced by one organism which inhibit the growth and
development of competing species. Although difficult to prove in an ecological setting, this is
apparently the rational behind bacterial and fungal production of antibiotics. It has also been
shown that similar substances produced by higher plants can be used as 'natural herbicides' (5).
While the secondary metabolism of terrestrial species has received the greatest attention, aquatic
organisms, particularly those in the marine environment, are also known to produce a complex
array of unusual metabolites (6). Although the function of most of these compounds is unknown,
some have pharmacological uses such as the antihelmintic agent, kainic acid from the red algae,
Digenca simplex, or possible antitumor activity as demonstrated by a number of halogenated
metabolites from marine algae (7). Aquatic algae and bacteria are also known to synthesize
40 compounds with functions similar to their terrestrial counterparts, such as hormones and antibiotics. Among these, the secondary metabolism of the cyanobacteria has received relatively little attention. However, like other marine algae, these prokaiyotic algae produce a wide variety of secondaiy metabolites (8).
4.1.1 Secondary Metabolism of Marine Cyanobacteria
The biosynthesis of halogenated secondary metabolites in the marine environment is much more widespread than in both terrestrial and freshwater ecosystems. Biological halogenation requires the involvement of specific peroxidases. Peroxidases utilize hydrogen peroxide in a variety of reactions, but perhaps the most unusual one is that of biological halogenation by the enzymes
Gl¬ chloroperoxidase and bromoperoxidasc. These enzymes are responsible for the oxidation of and Br- to give an enzyme-bound electrophilic halogenating agent. This halogcnating agent was
shown to react with a variety of nucleophiles to give halogenated reaction products, which are exclusively produced by marine organisms (9).
Several of these halogenated natural products are cytotoxic, and some of them resemble potential medicaments, insecticides or pesticides (1).
4.1.2 Secondary Metabolism of Freshwater and Terrestrial Cyanobacteria
In contrast to the marine algae, the secondary metabolism of freshwater and terrestrial
cyanobacteria has been relatively ignored. Although these organisms might be expected to be as metabolically versatile as their marine counterparts, research on freshwater cyanobacteria has
been directed to those species, which are known to synthesize toxms. Since many cyanobacteria
give rises to blooms in drinking water supplies, problems related to their growth and metabolism
have become a public health concern ( 10), (see Chapter 3.4.2).
41 4.2 Bioactive Compounds Produced by Cyanobacteria
4.2.1 Cyano b acteri al Toxins
Toxins are secondaiy compounds that have harmful effect on other tissues, cells or organisms.
They are grouped into two main categories, the cytotoxins and the biotoxins, based on the type of bioassay used to screen for their activity. The cytotoxins are detected on the basis of mammalian cell lines, especially tumor cell lines, whereas the biotoxms arc assayed with small animals, like mice or aquatic invertebrates (10).
4.2.1.1 Cytotoxins
Ehe discovery of cytotoxins was largely due to the search for new pharmaceutical and agrochemical compounds including enzymes, antibiotics, and anticancer agents (see 4.2.2-4.2.7).
4.2.1.2 Biotoxins
Biotoxins constitute a major source of natural product toxins that arc found in surface supplies of freshwater. Species and strains in all of the common planctonic cyanobacterial genera including
Anabaena, Aphanizomenon, Microcystis, Nodulana, Xostoc. and Oscillatoria produce biotoxins.
Tests on water blooms are frequently positive for the two common groups of cyanobacterial biotoxins - the alkaloid neurotoxins and the cyclic peptide hepatotoxins (10).
4.2.1.2.1 Neurotoxins
Species and strains of Anabaena, Aphanizomenon, Oscillatoria, and Trichodesmium produce neurotoxins (10). Anatoxin-a was the first toxin from a freshwater cyanobacterium that was chemically and functionally defined. It is the secondaiy atmn, 2-acetyl-9-aza-bicyclo[4.2.l]non-
2~ene (11). This alkaloid neurotoxin is a potent postsynaptic cholinergic nicotinic agonist, which causes a depolarizing neuromuscular blockade. Signs of toxicosis in field cases of wild and domestic animals include staggering, muscle fasciculation, gasping, convulsions. Death is
42 probably due to respiratory arrest and occurs within minutes or a few hours, depending on
species, dosage or prior food consumption. Clinical signs of toxicosis in mice, rats, and calves
dosed in the laboratory follow a progression of muscle fasciculation, decreased movement,
collapse, exaggerated abdominal breathing, cyanosis, convulsions, and death. The LDS0
intraperitoneal (i.p.) for purified toxin is about 200 U-gTcg body weight in mice, with a survival time of minutes. The oral dose necessary that produces acute lethality is much higher (hundreds of mg/kg body weight for the dry weight cell material). Nevertheless, toxicity is still so high that animals need to ingest only a few milliliters to a few liters of the toxic surface water bloom to receive a lethal bolus. No chemical antidote exists for anatoxin-a intoxication (12).
Most reports of anatoxin-a occurrence are associated with Anabaena flos-aquae, Anabaena spiroides, Anabaena cicinalis, and Oscillatona (12). Oscillatoria rnbescens was shown to produce a methylene homologue of anatoxin-a termed homoanatoxin-a (13).
Figure 4.1 Anatoxm-a: R = CH,
Ilomoanatoxm-a: R = CPECTf
The production of neurotoxin by Aphanizaomenon flos-aquae was first demonstrated by Sawyer et al. (1968) (14). These neurotoxins were later shown to be saxitoxin (= Aphantoxin II) and neosaxitoxin (-- Aphantoxin I) (LDW i.p. mouse equals about 10 pg/kg). the two primary toxins of red tide paralytic shellfish poisoning (15). 'These toxms arc fast-acting neurotoxins that inhibit nerve conduction by blocking sodium channels without affecting permeability to potassium, the transmembrane resting potential, or membrane resistance (16).
43 H?NOO
hbN
Figure 4.2 Saxitoxin: R = II
Neosaxitoxin: R -- Oil
Another neurotoxin with some different signs of neurotoxicosis, in particular, market salivation
this from in laboratory mice, was isolated from Anabaena flos-aquae. In order to differentiate signs observed with anatoxin-a the toxin was designated anatoxin-a(s) (s = salivation) (17). It is a unique N-hydroxyguanidine methyl phosphate ester. Anatoxin-a(s) is the only naturally occurring organophosphate. It was shown to be a potent irreversible inhibitor of Cholinesterase. Its toxicity was determined as LDS0 20 pg/kg in mice. Cholinergic reactions, such as viscous salivation, are observed prior to death by respiratory arrest. Atropine appears to be a possible antagonist of anatoxin-a(s) (18).
Figure 4.3 Anatoxin-a(s)
4.2.1.2.2 Hepatotoxins
Acute hepatotoxicosis involving the hepatotoxins (fixer toxins) is the most commonly encountered toxicosis involving cyanobacteria. These toxins are produced by strains of species within the genera Microcystis, Anabaena. Nodulana, Oscdlaloria, and Nostoc. In addition,
44 chemically undefined hepatotoxins have been studied in Cylindrospermopsis, Aphanizomenon,
Gfoeotrichia, and Coelosphaerium. Clinical signs of hepatotoxicosis have been observed in field poisonings involving cattle, sheep, horses, pigs, ducks, and other wild and domestic animals.
Most laboratory studies have involved the use of mice, rats, guiuca pigs, rabbits and pigs.
Collectively the signs of poisoning in these animals include weakness, anorexia, pallor of mucous membranes, vomiting, cold extremities and diarrhea. Death occurs within a few hours to a few days after initial exposure and may be preceded by coma, muscle tremors, and forced expiration of air. Death from these hepatotoxins is most likely the result of intrahepatic haemorrhage and hypovolemic shock (12).
The best known toxins responsible for the hepatotoxicitx arc the microcystins. They arc obtained not only from Microcystis, but also from Anabaena, Nostoc, and Oscillatoria, which also form water blooms (19).
The microcystins arc cyclic heptapeptides like the representative microcystin-LR. The unique structural feature in microcystins is the C20 amino acid, (2S,3S,8S,9S)-3~amino-9-methoxy-
2,6,8-trimethyl-10-phenyldeca-4E,6E-dienoic acid (Adda), xvhich plays an important role in their toxicity. Ehe suffix LR identifies the two xariable amino acids at positions 2 and 4, i. e., Leu and
Arg. More than 50 structural x^anations in microcystins have been found. The general structure of microcystins is cyclo-(-D-Ala-X-D-MeAsp-Z-Adda-D-Glu-Mdha), in which MeAsp is erythro-ß- methylaspartic acid and Mdha symbolizes N-methyldehydroalaninc. X and Z arc the commonly variable amino acids (positions 2 and 4), but structural modifications have been detected in all seven amino acid units (19).
Microcystins are strong inhibitors of protein phosphatases 1 and 2a, similarly to okadaic acid.
These phosphatases are involxred in cellular regulation and therefore are probably the key enzymes that reverse the action of protein kinase C. The f D value for most microcystins is 0.07 mg per kg of body weight in mice (19). Additionally, they are considered as potent tumor promoters.
Cyclosporin-A, rifampicin. and Silymarin are the most successful antagonists of microcystins when given before or coadministered with the toxin (20).
45 aA^' o
D ei \lhio McAsp
Figure 4.4 Microcystm LR
The structurally related nodularins haxe been isolated from the brackish water-dwelling cyanobacterium Nodularia spumigena. Nodularins are cyclic pcntapeptides and contain the Adda unit or its derivative. Nodularin is also a strong inhibitor of protein phosphatases 1 and 2a and has been reported to be a strong tumor promotor (19).
I) GIu
Mdln
o rooH
D sn thio MeA^p
Figure 4.5 Nodularin
A structurally unrelated cyanobacterial hepatotoxin, cylindrospermopsin, has been isolated from
Cylindrospermopsis raciborskii and Umczakia nutans. Structurally it is an unusual alkaloid, possessing a tricyclic guamdine moiety together with a hydroxymethyluracil part. In contrast to
46 microcystins and nodularins, it does not inhibit proteinphosphatases 1 and 2a. It has an acute (24 h) LD,0 of 2.1 mg/kg (i. p.) in mice (19).
QH b a 5 OaSC^ _._... . n )3SV
OCT t
Figure 4.6 Cylindrospermopsin
4.2.2 Cyanobacteria as a Source of Medicinal Agents
Natural products are an important source of new pharmaceuticals and pharmaceutical lead compounds. The overwhelming majority of active compounds have been derived from streptomycetes and fungi, although additional significant sources include other bacteria and plants (21). Cyanobacteria are rapidly proving to be an extremely important source of biologically active metabolites with potential benefits against human disease (22).
4.2.2.1 Antimicrobial Compounds
Antimicrobially active compounds of cyanobacteria arc of great interest because the discovery of new antibiotics is necessary due to the resistance of some microbes to common antibiotics.
Kawaguchipeptins A and B are cyclic undecapeptides with antibacterial activity from the cultured cyanobacterium Microcystis aeruginosa. Both of them inhibit the growth of the gram- positive bacterium Staphylococcus aureus at a concentration of lug/niL (MIC) (23).
47 Figure 4.7 Kawaguchipeptin A
Figure 4.8 Kawaguchipeptin B
Hapalindoles are antibacterial and antifungal alkaloids from the cyanobacterium Eïapalosiphon fontinalis. Hapalindole A is an unusual chlorine- and isonitrile containing indole alkaloid. Several similar active indoles, hapalindoles B-V, have been isolated from the same cyanobacterial strain.
Some of them possess an isothiocyanate instead of the isonitrile function (24).
48 Figure 4.9 Hapalindole A
Efierridin A and B are the first secondary metabolites from the cyanobacterium Phormidium eclocarpi with activity against Plasmodium falciparum. The IC50 of the mixture of both compounds against chloroquine sensitive clones and chloroquinc resistant clones were determined to be 5.2 ug/mL and 3.7 jig mL respectively (25).
OCH3
Figure 4.10 Hierridin A: R - C17I1„
HicrridinBiR-C^H,,
The laxaphycins are a large family of cyclic undeca- and dodecapeptides, the major representative of each class being laxaphycin A and laxaphycin B, respectively, that are responsible for the antifungal activity of the crude extract of Anabaena laxa. The antifungal effect exhibited by these peptides is unusual because the peptides act synergist!cally with each other to inhibit growth. In order to achieve maximium biological potency, a member of each class of peptide must be present. The mode of action, howex-er, is not novel and does not involve a
specific receptor. Lysis of cells occurs in non-specific manner (26). The laxaphycins closely
49 resemble, both structurally and biologically, a group of cyclic peptides known as the
hormothamnins (27) that have been isolated from the marine cyanobacterium Hormothamnion
enteromorphoides. Laxaphycin A differs from hormothamnin in the geometry of the double bond
in the didehydrobutyrinyl unit.
Figure 4.11 Eaxaphycine A Figure 4.12 Laxaphycine B
4.2.2.2 Antiviral Compounds
As the part of U.S. National Cancer Institute (NCI)'s effort to discover and develop new classes of compounds which inhibit the mfectixity and or cytopathic effects of the human immunodeficiency virus (HIV), an extensive collection of extracts from terrestrial plants, marine organisms, and selected microbial sources has been screened for m vitro anti-FIIV activity.
Numerous HIV-inhibitory compounds xxith diverse xiral and cellular targets have been isolated and identified from natural product extracts, which were active m the NCI screen. These include novel metabolites, which inhibit reverse transcriptase, block HIV-induccd cell fusion, disrupt protein kinase-C mediated processes, and bind to the cellular receptor CD4 (28).
50 A novel anti-HIV protein, cyanoviridin (CV-N), was isolated from the cultured cyanobacterium
Nostoc ellipsosponan. CV-N consists of a single 101 amino acid chain which exhibits significant internal sequence duplication, but no significant homology to previously described proteins or to the transcription products of known nucleotide sequences. The protein was produced
recombinantly in Escherichia coli by expression of a synthetic DNA coding sequence corresponding to the amino acid sequence deduced initially for natural CV-N. It potently inhibits the in vitro cytopathicity of diverse clinical isolates and laboratory strains of HIV type 1, HIV type 2 and simian immunodeficiency virus. CV-N also effectively prevented cell-to-cell fusion and transmission of HIV from infected cells to uninfected host cells. Pretreatment of HIV virions with CV-N irreversibly neutralized virus infectixity. CV-N was nontoxic to host cells. The virucidal effects of CV-N result, at least in part, from its association with the viral envelope glycoprotein gpl20; CV-N apparently interferes with interactions between viral gpl20 and cell surface receptors which are required for successful virus fusion and entry into the cell (28).
A group of novel diacylated sulfoglycobpids and aeylatcd diglycolipids were isolated from
Scytonema sp. and Oscillatoria raoi. Initial screening for the inhibitory activity of the DNA polymerase function of HlV-1 reverse transcriptase (RI) revealed that some of them arc potent inhibitors. At a final concentration of 10 u.M. these compounds totally abolished the initial enzymatic activity. It is suggested that the inhibitory activity of these compounds in tissue results from their potential to abolish the HIV-1 RT-associated DNA polymerase activity (29).
OR,
- Figure 4.13 Sulfoglycobpids: R, linolcoyl. R, == palmitoyl, R, - palmitoyl
R, linolcoyl, R, = palmitoyl, R-, = H
- R, palmitoyl IE = palmitoyl, R, - II
R, = oleoxl. R-, - nalnutovl, R, - II
51 Calcium Spirulan (Ca-SP), an inhibitor of enveloped virus replication was isolated from Spirulina platensis. This polysacharide was composed of rhamnose, ribose, mannose, fructose, galactose, xylose, glucose, glucuronic acid, galacturonic acid, sulfate and calcium. It was found to inhibit the replication of several enveloped viruses, including Herpes simplex vims type 1 (EDS0 0.92 jig/mL), human cytomegalovirus (ETV„ 8.3 pgrniL). measles virus (EDS0 17.0 ug/mL), mumps virus (ED,0 23.0 p:g/mL), influenza A vims (ETV0 9.4 pg niL), and HIV-1 (ED50 2.3 pg/mL). It was revealed that Ca-SP selectively inhibited the penetration of virus into host cells (30).
Bauerine A-C are ß-carbolines from Dichothrix baucrinana that demonstrate •mti-Herpes simplex-2 activity (]C50 2-3 mg niL) as well as cytotoxicity (IC50 3, 5, and 0.03 pg/mL).
Structurally they are related to the h arm an alkaloids, which are known from higher plants (3 I).
,/^X
Figure 4.14 Bäuerin A: R =H. Bauerin C
Bäuerin B: R - CTE
4.2.2.3 Compounds with Multidrug-Resistance Reversing Activity
Tumor cells that survive initial chemotherapy often recover with increased resistance to both the original therapeutic agent and other seemingly unrelated drugs, resulting in the ultimate failure of chemotherapy. An overexpression or activation of P-glycoprotein, a transmembrane protein, which acts as an AEP-depcndent drug efflux pump, frequently causes the phenomenon termed multiple-drug resistance (MDR). Enhanced efflux results in a reduction of intracellular drug accumulation xvith a concomitant decrease in cytotoxicity. Thus, agents that are capable of overcoming P-glycoprotein-mediated multidrug resistance have potential use in the treatment of cancer patients undergoing chemotherapy. Over the past decade, several drugs have been discovered that reverse P-glycoprotein mediated MDR. Verapamil is one of the first MDR reversing agents to be discovered and studied in the clinic (32).
found Elapalosin, a cyclic depsipeptide from the cyanobacterium Hapalosiphon welwitschii was to have better MDR reversing activity than verapamil. SKVLBl cells accumulate 0.7 pmol of
of taxol in a control experiment. Verapamil cause dose-dependent increases in the accumulation
taxol in the cells reached 270% of the control at 20 tiM. In the case of hapalosin taxol
accumulation reached 440% of control at 20 uM (32).
Figure 4.15 Hapalosin
Dendroamide A, a new anti-MDR active bistratamide-type cyclic hexapeptides from the
terrestrial cyanobacterium Stigonema dendroideum is more potent than verapamil in its ability to
increase the accumulation of vinblastine (33).
Figure 4.16 Dendroamide A
A group of anti-MDR active porphinoids, the tolyporphins A-K, xvas isolated from the
cyanobacteriuni Tolypothrix nodosa (34).
53 OH
Figure 4.17 Tolyporphin K
4.2.2.4 Cytotoxic Compounds
Since there is a great interest in finding nexv antitumor compounds, natural products arc mainly evaluated for their cytotoxic properties. The cyanobacteria have been identified as one of the most promising group of organisms from which to isolate new anticancer-type natural products (35).
Tantazoles and mirabazoles are modified heterocyclic peptides that comprise one of four classes of cytotoxins in terrestrial Scvtonema mirabilc. Most of them are tumor-selective cytotoxins.
Tantazole B and didchydromirabazole A are also solid tumor selective (36, 37).
UN N-^^A
Figure 4,18 Tantazole B and Didehvdroimrabazoie A
54 Westiellamide is a weakly cytotoxic, modified cyclic hexapeptide from Westiellopsis prolifica.
Its structure is identical with that of cyclooxazoline from the ascidian Lissoclinum bislratum. It provides circumstantial evidence for algal symbionts and plays a role in the biosynthesis of closely related cyclic peptides found in marine tunicates. Similar cyclic peptides, like dolastatin, found in marine mollusks undoubtedly have a cyanobacterial origin (38).
\—»o
Figure 4.19 Westiellamide
The ciyptophycins comprise the largest class of cyanobacterial depsipeptides to date. All of the cyclic cryptophycins consist of a 6-hydroxy acid unit (A), an a-amino acid unit (B), a ß-amino acid unit (C), and an «-hydroxy unit (D), connected together in a cyclic ABCD sequence.
Cryptophycin-1 was first isolated from Nostoc sp. as an antifungal agent (39). However, it appears to be too toxic to be of practical use as an antifungal agent. But cryptophycin-1 was discovered to be a new microtubule-dcpolymerizing agent showing excellent activity against a broad spectrum of solid tumors implanted in mice, including drag-resistant and multiple drag- resistant ones. One compound from the cryptophxcm line, cryptophycin-52 is planned for human clinical trials as an anticancer drug soon (21, 40).
och3
Figure 4.20 Cryptophycin 1
55 Curacin A, an antimitotic, antiproliverative and brine shrimp toxic thiazoline containing lipid, was isolated from Lyngbya majuseula. It is assessed as an antitubulin agent. Further investigations revealed that curacin A interacts at the colchicine class binding domain on tubulin, similarly to podophyllotoxin. The mammalian cell antiproliferative activity was determined with an IC,0 of 6.8 ng/mE in the Chinese hamster AuxBl cell line. Antimitotic activity in three cell lines xvith IC<;0 values ranging from 7 to 200 nM (41 ).
Figure 4.21 Curacin A
The macrolide compounds scytophycine A-E were isolated from the terrestrial cyanobacterium
Scytonema pseudohofmanni. Scytophycin A and B demonstrated potent cytotoxic activity. Both are also antifungal agents. Scytophycins are structurally related with the antifungal swinholid A, isolated from the sponge Theonella swinhoei. Thus, the cyanobacterial origin of swinholid A is suggested (42, 43).
Tolytoxin, another compound, xvhich is structurally related to the scytophycins, is a niacrolidc lactone from the terrestrial cyanobacterium Tolypothrix conglutinata var. coloraia. It shows murine and human solid tumor selective cytotoxicity. It is suggested that the cytotoxicity is due to the disraption of microfilament organization. These results in the loss of the ability to form the contractile ring needed for cytokinesis in eukaryotic cells. Additionally tolytoxin is a broad- spectrum fiingicidc with MICs in the range of 0.25 - 8 nM, being more potent than nystatin (44).
56 Figure 4.22 Scytophycin A
Figure 4.23 Scytophycin B: R, H, R, = H
'Tolytoxin R, - OH, R, - CEE
The phytochemicai investigation of another Tolypothrix species, Tolypothrix byssoidea, leads to two cytotoxic pyrrolopyrimidiiie nucleosides, tubercidin and toyocamycin. They have previously been isolated from Streptomyces tubercidicus. Both of them show significant inhibitory activity
against KB cells (MIC values of 0.07 and 0.56 ug'mL for tubcrcidin and its glucosidc,
respectively, and 0.06 and 0.3 Lig/iiiE for toyocamyeme and its glucosidc). Additionally,
tubcrcidin exhibits a potent inhibitory effect against NITE3T3 cells and in vivo against P-388
lymphocytic leukemia in mice. Tubercidin acts by disrupting the nucleic acid structure and
therefore inhibits DNA, RNA and protein synthesis (45, 46).
57 CH2OH NHo CH2OH
OH OH
OH OH
Figure 4.24 Cytotoxic Nucleosides
Tubcrcidin- Deazaadcnosinc-
5'-a-D-ghicopyranoside: R - II 5-a-D~glucopyranoside
Toyacamycin-
5'-a-D-glucopyranoside: R - CN
The dolastatins arc a scries of remarkable cytotoxic compounds isolated from the Indian seahare
Dolabella auricularia (47). The most important of these is dolastatin 10, which is in phase I trials as an anticancer agent. Symplostatin I, a solid tumor selective cytotoxic analogue of dolastatin
10, has been isolated from the marine cyanobacterium Symploca hydnoides. Symplostatin 1 exhibited a cytotoxicity 1C50 value of 0.3 ng-'mL against KB cells, as compared to ^ 0.1 ng/mL for dolastatin 10 (48).
Ehe cytotoxic peptolide dolastatin 12 and the closely related analogue lyngbyastatin 1 were isolated from collections of the cyanobacterium Lvngbva majuscula and assemblages oîEvngbya majuscula and Schizothrix calieola. These discox eries support again the proposal that many compounds isolated from seahares are of cyanobacterial origin. Lyngbyastatin 1 and dolastatin 12 were isolated as inseparable mixtures with their C-15 epimers. Complexes of the epimeric mixture of lyngbyastatin 1 have MIC values of 0.1 jig'mE against KB and of 0.5 pg/mE against
LoVo cells. Complexes of the epimeric mixture of dolastatin 12 possess MIC values < 0.05 p,g/mE against KB and 0.08 |ig/mL against LoVo cells (49).
58 Figure 4.25 Dolastatin 10: R = H
Symplostatin i : R - CI I
- Fi eure 4.26 Dolastatin 12: R -- OCH,, R' CH„
R" - (CEE)2CHCEI2
epimeric at C-15
wnebvastatin R =- H, R' - CIL,,
R" - (GifECHCEI,
epimeric at C-15
4.2.2.5 Enzyme Inhibitory Compounds
Cardiovascular diseases such as stroke and coronary artery occlusion continue to be major causes of morbidity and mortality, and it is likely that improxred pharmacological control of blood clot formation xvill remain a major goal. A complex proteolytic cascade that leads to the formation of fibrin triggers the process of blood coagulation. Serine proteases, such as thrombin, trypsin, and chymotrypsin are the key enzymes. Their inhibitors may be good targets and useful
59 chemotherapeutic agents for these diseases (50). Most of them were isolated from the freshwater cyanobacterium Oscillatoria agardhii.
the Micropeptins are a class of cyclic depsipeptides containing Ahp (3-amino-6-hydroxy-2- piperidone) unit, which have been frequently found in the constituents of freshwater-blooming
A and B cyanobacteria. These peptides are known to be protease inhibitors, such as micropeptins
(51), micropeptin 90 (52), and micropeptins 478-A and 478-B (53) from Microcystis aeruginosa,
which inhibit plasmin and trypsin. Oscillapeptin (54) from Oscillatoria agardhii, nostopeptins
(55) from Nostoc minuium, and micropeptin 103 (56) from Microcystis viridis are inhibitors of
clastase and cbymotrypsin. Micropeptins 88-A to 88-F, isolated from Microcystis aeruginosa,
also inhibit chymotrypsin (57).
Figure 4.27 Micropeptin 88-A
Aeruginosins are linear peptides and glycopeptides with serine protease inhibitory activity.
the Aeraginosins 102-A and B (58) as xvell as 103-A (59), three linear peptides are isolated from
cyanobacterium Microcystis viridis. Aeruginosin 102-A inhibits thrombin with an IC50 of 0.04
jlg/mL, trypsin and plasmin with an ICsn of 0.2 and 0.3 lig/'mL, respectively. Aeruginosin 102-B
inhibits thrombin, trypsin and plasmin with an \C,n of 0.1. El, and 0.8 pg/mE, respectively, while
aeruginosin 103-A inhibits thrombin, trypsin, and plasmin with an 1CS0 value of 9.0, 51.0, and
68.0 jig/niE, respectively. The trypsin and thrombin inhibitors aeraginosins 205-A and B are
linear glycopeptides from the cyanobacterium Oscillatoria agardhii: both inhibit thrombin with
IC50 values of 1.5 and 0.17 pg-'niE, respectively (60).
60 argal (argininealdehyd)
I I OH V NH
./ 'NH;
H03SO
Figure 4.28 Aeruginosin 102-A: L-argal
Aeruginosin 102-B: D-argal
O NH E N NHa
HO'
Figure 4.29 Aeruginosin 103-A
61 plas (phenyllactic acid 2-O-sulfate)
HO3SO HN
hleu (3-hydroxyleucine) , ^ q-- 4hq ,Q
Ov
OH
Figure 4.30 Aeruginosin 205-A: L-plas, (2R, 3S)-Hlcu
Aeruginosin 205-B: D-plas, (2S. 3R)-Hleu
A thrombin inhibitor, the cyclic heptapeptide agardhipeptin A, was additionally derived from
Oscillatora agardhii. It inhibits plasmin xvith an IC\0 of 65 fig/mE. The structurally related compound agardhipeptin B, isolated from the same cyanobacterial strain has no inhibitory effects
(61).
Figure 4.31 Agardhipeptin A
Oscillatoria agardhii is also the source for the chymotiypsin inhibitor oscillatorin, a cyclic decapeptide containing (3a-cis)-1.2,3,3a.8.8a-hexahydro-3a-(3-methyl-2~2butenyl-)pyrrolof2,3-
62 b]indol-2~carboxylic acid (oscillatoric acid, Ose) (62). The fundamental skeleton of flustramine A
and B, two isolates from the marine bryozoa Flustra foliacea resembled closely that of oscillatoric acid (63). Therefore it is suggested that these alkaloids may be produced by the
symbiotic cyanobacteria in marine bryozoa.
HoN
Figure 4.32 Oscillatorin
Figure 4.33 Flustramine A Flustramine B
Other serine protease inhibitors from Oscillatoria agardhii arc the microviridins, which are cyclic or dicyclic peptides. They shoxv strong actixity against elastase and chymotiypsin. Microviridins
D and E inhibit elastase potently, with IC,-0 values between of 0.6 and 5.8 pg/mE, respectively.
Microviridn D and E are also chymotiypsin inhibitors with an TC,0 value of 1.2 and 1.1 fig/mL, respectively (64).
63 ~S P: K Ä
>=0 HN
OH HO- /~\ Vs1
Figure 4.34 Microviridin D
A90720A, is a serine protease inhibitor, which is active against bovine thrombin (TC,0 no
bovine =~ ng/niL), trypsin (IC,P 10 and human - ng/niL) plasmin (ICE0 30 ng/niL). It is a cyclic
with a sulfated acid depsipeptide, glyceric moiety in the sidcchain. A90720A was isolated from the terrestrial cyanobacterium Microchaete loktakensis (65).
64 OH
Figure 4.35 A90720
Radiosumin (66) and dehydroradiosumin (67) are dipeptides with trypsin inhibitory effects, isolated from the freshwater cyanobacteria Anabaena cvhndrica and Plectonema radiosum, respectively. Both of them are potent trypsin inhibitors, dehydroradiosumin demonstrates also weak plasmin inhibitory activity. Dehydroradiosumin inhibits trypsin with an IC0 of 0.1 pg/mL and plasmin with an I(\0 of 90 Ug'mL,
,*v 'A
o 4"
N COO H
Figure 4.36 Dehvdroradiosumrn
Nostopeptins A and B are cyclic depsipeptides containing Ahp (3-amino-6-hydroxy-2- piperidone) with elastase and chymotiypsin inhibitory effects, which were isolated from the freshwater cyanobacterium Nostoc munition.
As elastase is suggested to be involved in pulmonary emphysema, rheumatoid arthritis, adult respiratory distress syndrome, and other inflammatory states, its inhibitors might be useful therapeutic agents for these diseases.
65 11.0 and also (K%0 1.4 Nostopeptin A and B inhibit elastase (TC50 1.3 and ug/mE) chymotrypsin
and 1.6 pg/mE).
Figure 4.37 Nostopeptin A: R - (CEEECEE,
Nostopeptin B: R - Cil-,
66 Circinamide, isolated from the cyanobacterium Anabaena circinalis (68), is a papain inhibitor,
containing an unusual amino acid (3-epoxy succinic acid) and a polyamine (homospermidine).
Spermidine is a natural occurring polyamine and is present in ptilomycalin A (69) and
crambcscidins (70) as a structural unit of the major metabolites of sponges, while
homospemiidine is a very rare triamine. Circinamide is related to E-64, a potent cysteine protease
inhibitor isolated from cultures of Aspergillus faponicus. (68) Based on the prototype E-64,
loxistatin was designated as a clinically usable drug for the treatment of muscular dystrophy (71).
o o
Figure 4.38 Circinamide
4.2.2.6 Cardioactive Compounds
A high percentage of hydrophilic extracts of cyanobacteria exhibit cardiotonic activity in isolated
mouse atria. Tolypophycins A and B. two water soluble substances of unknown structure,
account for the inotropic activity of Tolypothrix bvssoidea. The inotropic activity of the terrestrial
cyanobacterium Anabaena sp. is due to a different, less polar substance, an unusual chlorine
containing cyclic decapeptide named puwainaphycin 0 (EDM) - 0.2 (ig/mE). The closely related puwainaphycins A. B, D and E, however, were inactixe (72).
67 OH
Figure 4.39 Puwainaphycin C
Scytonemin A from a Scytonema sp. possesses potent calcium antagonistic properties. Calcium antagonistic effects were noted at 5 ugfiiiL on atria and calcium blocking effects were noted at 20 flg/mL on rat portal vein (73).
o
O, T'A o OH
NH
NH O
HN HO J^ O N 'HOH HO/;,>M„
,NH O
K ""OH o H(\ °Y HN^- NH
F i mire 4.40 S cvton emin
68 isolated from the Microginin, an angiotensin-converting enzyme inhibitory pentapeptide, was freshwater cyanobacterium Microcystis aeruginosa. Angiotensin-converting enzyme inhibitors have been developed as antihypertensive agents. Microginin inhibits the angiotensin-converting enzyme with an IC50 of 7.0 pig/mL (74).
OH y"-MA
Figure 4.41 Microginin
4.2.2.7 Anti-inflammatory Compounds
The anti-inflammatory diterpenoid tolypodiol, the first reported occurrence of a diterpenoid
nodosa. compound in cyanobacteria, was isolated from the terrestrial cyanobacterium Tolypothrix
of 30 It shows strong anti-inflammatory activity in the mouse car edema assay, with an ED50
obtained for the jig/ear. The EDS0 value of tolypodiol compares very favorably with those
standards hydrocortisone (20 ug/car) and manoahdc (100 jig/ear) in the same assay (75).
OCH-,
OH
Fiuurc 4.42 Tolvpodiol
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50. Bonjouklian, R.. Smitka, T. A.. Hunt, A. IE, Occolowitz, J. L., Permi, T. E, Doolin, L.,
Stevenson, S., Knauss. E.. Wijayaratne, R., Szcwczyk, S., Patterson, G. M. E.: A90720A,
a Serine Protease Inhibitor Isolated from a Terrestrial Blue-Green Algae Microchaete
loktakensis. Tetrahedron 52: 395-404 ( 1996)
51. Okino, T.. Murakami, M.. Haraguchi, R., Weckesser. E: Micropeptins A and B, Plasmin
and Trypsin Inhibitors from the Blue-Green Algae Microcystis aeruginosa. Tetrahedron
Lett. 34: 8131-8134(1993)
52. Ishida, K., Munekata, H., Matsuda, H., Yamaguchi. K.: Micropeptin 90, a Plasmin and
Trypsin Inhibitor from the Blue-Green Algae Microcystis aeruginosa. Tetrahedron Lett.
36:3535-3538(1995)
53. Ishida, K., Matsuda, H., Murakami, M., Yamaguchi, K.: Micropeptins 478-A and -B,
Plasmin Inhibitors from the Cyanobacterium Microcystis aeruginosa. J. Nat. Prod. 60 :
184-187(1997)
54. Sano, T., Kaya, K.: Oscillapeptin G. a Tyrosinase Inhibitor from Toxic Oscillatoria
agardhii. J. Nat. Prod. 59: 90-92 (1996)
55. Okino, T., Sun, Q., Matsuda, H., Murakami. VE, Yamaguchi, K.: Nostopeptins A and B,
Elastase Inhibitors from the Cyanobacterium Nostoc minutum. J. Nat. Prod. 60: 158-161
(1997)
56. Murakami. M., Kodani. S., Ishida, K., Matsuda. H.. Yamaguchi. K.: Micropeptin 103, a
Chymotiypsin Inhibitor from the Cyanobacterium Microcystis viridis. Tetrahedron belt.
38: 3035-3038(1997)
57. Ishida, K., Matsuda, IE, Murakami, M.: Micropeptins 88-A to 88-F, Chymotrypsin
Inhibitors from the Cyanobacterium Microcystis aeruginosa (N1ES-88). Tetrahedron 54: 5545-5556(1998)
74 58. Matsuda, EL, Okino, T., Murakami, M., Yamaguchi, K.: Aeraginosins 102-A and B, New
Thrombin Inhibitors from the Cyanobacterium Microcystis viridis (N1ES-102).
Tetrahedron 52: 14501-14506 (1996)
59. Kodani, S., Ishida, K., Murakami, M.: Aeruginosin 103-A, a Thrombin Inhibitor from the
Cyanobacterium Microcystis viridis. J, Nat. Prod. 61: 1046-1048 (1998)
60. Shin, H. J., Matsuda, IE, Murakami, M., Yamaguchi, K.: Aeraginosins 205-A and -B,
Serine Protease Inhibitory Glycopeptides from the Cyanobacterium Oscillatoria agardhii.
J Org. Chem. 62: 1810-1813 (1997)
61. Shin, H. J., Matsuda, H.. Murakami, M., Yamaguchi, K.: Agardhipeptins A and B, Two
New Cyclic Hcpta- and Octapeptides, from the Cyanobacterium Oscillatoria agardhii.
Tetrahedron 52: 13129-13136 (1996)
62. Sano, 'T.. Kaya, K.: Oscillatorin, A Chymotrypsin Inhibitor from Toxic Oscillatoria
agardhii. Tetrahedron Letters 37: 6873-6876 (1996)
63. Hoist, P. B., Anthoni, U., Christophersen. C. Nielsen. P. EL: Two Alkaloids, Flustramine
E and Debromoflustramine B. from the Marine Bryozoan Flustra foliacea. J. Nat. Prod.
57:997-1000(1994)
64. Shin, IE E, Murakami, M., Matsuda, IE, Yamaguchi, K.: Microviridins D-F, Serine
Protease Inhibitors from the Cyanobacterium Oscillatoria agardhii. Tetrahedron 52:
8159-8168(1996)
65. Bonjouklian, R., et al.: A90720A, a Serine Protease Inhibitor Isolated from a Terrestrial
Blue-Green Algae Microchaete loktakensis. Tetrahedron 52: 395-404 (1996)
66. Matsuda, H., Okino, T., Murakami. M.. Yamaguchi, K.: Radiosumin, a Trypsin Inhibitor
from the Blue-Green Algae Plcctonema radiosum. J Org. Chem. 61: 8648-8650(1996)
67. Kodani, S., Ishida. K., Murakami. YE: Dehydroradiosumin, a Trypsin Inhibitor from the
Cyanobacterium Anabaena cvlindrica. J. Nat Prod. 61: 854-856 (1998)
68. Shin, El. E, Matsuda, IE. Murakami, M., Yamaguchi, K.: Circinamide, a Novel Papain
Inhibitor from the Cyanobacterium Anabaena aremalis. Tetrahedron 53: 5747-5754
(1997)
69. Kashman. Y., Hirsch. S., McConnell, O. J., Ohtani, E, Kusumi, T., Kakisawa, IE:
Ptilomycalin A: A Novel Polyeychc Guanidine Alkaloid of Marine Origin. J. Am. Chem.
Soc. 111:8925-8926(1989)
75 70. Jares-Erijman, E. A., Ingram, A. L., Carney, J. R., Rinehart, K. L., Sakai, R.: Polycyclic
Guanidine-containing Compounds from the Mediterranean Sponge Crambe crambe: Ehe
Structure of 13, 14, 15-Isocrambescidin 800 and the Absolute Stereochemistry of the
Pentacyclic Guanidine Moieties of the Crambescidins. /. Org. Chem. 58: 4805-4808
(1993)
71. Hanada, K., Tamai, M., Adachi, T., Oguma, K., Kashiwagi. K., Ohmura, S., Kominami,
E., Eowatari, T., Katsunuma, N., Medicinal and Biological Aspects, in Proteinase
Inhibitors. Tokyo, Japan Sei. Soc. (1983)
72. Moore, R. E., Bornemann, V., Niemczura, W. P., Gregson, J. M., Chen, J-L., Norton, T.
R., Patterson, G. M. L., Elclms, G. L.: Puxvainaphycm C. a Cardioactive Cyclic Peptide
from the Blue-Green Algae Anabaena BQ-16-1. Use of Two-Dincnsional 13C-13C and
13C-15N Correlation Spectroscopy in Sequencing the Amino Acid Units. /. Am. Chem.
Soc. 111:6128-6132(1989)
73. Helms, G. E., Moore, R. li, Niemczura, W. P., Patterson, G. M. L.: Scytonemin A, a
Novel Calcium Antagonist from a Blue-Green Algae. /. Org. Chem. 53: 1298-1307
(1988)
74. Okino, T., Matsuda, IE, Murakami. M.. Yamaguchi. K.: Microginin. an Angiotensin-
Converting Enzyme Inhibitor from the Blue-Green Algae Microcystis aeruginosa.
Tetrahedron Letters 34: 501-504 (1993)
75. Prinsep, M. R., West, M. I,., Wylie, B. E., Thomson, R. A.: Tolypodiol, an
Antiinflammatory Diterpenoid from the Cyanobacterium Tolypothrix nodosa. J. Nat.
Prod. 59:786-788(1996)
76 5 The Genera Nostoc and Tolypothrix
5.1 The Genus Nostoc
Division: Cyanophyta
Class: Hormogoniophyceae
Order: Nostocales
Family: Nostocaceae
Genus: Nostoc
The genus Nostoc Vaucher is more common as a terrestrial and subacrial algae than as an aquatic one. It is widely distributed on and in alkaline soils and on moist rocks and cliffs.
The filaments are embedded in round and formless mucilaginous sheath. The bead-like cells undergo generalized cell division that increases the length of the trichomes, the sheath of which may be yellow or brownish. The single cells are round, ox old, spherical, cylindrical, or barrel- shaped. The trichomes are unbranched and form heterocysts and akinetes as maturity. The heterocysts are intercalary arranged and regularly distributed throughout the trichomes.
There are two types of development in laboratory culture, the Nostoc piscinale and the Nostoc commune type. In the latter, development of the motile trichomc to form a mature aggregate of filaments takes place entirely within a matrix with a firm surface pellicle. 'Thus, the aggregate has a fixed shape that is absent in the Nostoc piscinale type. The mature trichomes of most species of
Nostoc akinetes produce that often occur in chains. Most of the representatives of the genus
Nostoc are able to grow as photoheterotrophs (1,2).
Figure 5.1 shows a microscopically picture of Nostoc commune (EAWAG 122b).
77 *^-%\*» -***«^y.
Figure 5.1 Nostoc commune 122b 1 100
Figure 5.2 Tolypothm Inssoidea 195 1 100
78 5.2 The Genus Tolypothrix
Division: Cyanophyta
Class: Hormogoni ophyceae
Order: Nostocales
Family: Scytonemataceae
Genus: 'Tolypothrix
sheath The genus Tolypothrix Kiitzing is most often aquatic. "The trichomes are enclosed in firm
in without that may be colored. The trichomes are characterized by false branching which, initiating cell division in a nexv plane, the trichomes or their hormogonia rapture or groxv through the sheath. The trichomes are usually heterocystous, but akinetes are rarely produced. The trichomes of Tolypothrix are of uniform diameter and enclosed in narrow sheaths (1, 2).
Figure 5.2 shows a microscopically picture of Tolypothrix bvssoidea (EAWAG 195).
79 53 Refrerences
1. Bold, EL, Wynne, M. J., Divisions Cyanophyta and Prochlorophyta, in Introduction to the
Algae. Engelwood Cliffs, New Jersey, Prentice Hall (1978)
2. Wartenberg, A.: Systematic der niederen Pflanzen. Thieme, Stuttgart: 61-75 (1979)
80 6 Collection and Cultivation
6.1 Collection
Thirty-six cyanobacterial strains, selected for cultivation and biological and chemical
investigation, were obtained from the Culture Collection of Algae of Ihc Swiss Federal Institute
for Water Resources and Water Pollution Control (EAWAG). Diibendorf, Switzerland (described
in detail in paper 1). Three additional samples, one Nostoc commune strain (screening number
38) and two mixed samples (screening number 33 and 34) were field collected in Zürich,
Switzerland, in May 1996. Seven different strains were isolated and cultivated as stock cultures
from these samples. Additionally, the field collected samples were freeze-dried after removal
from accompanying soil, extracted and used directly for the screening program.
The field-collected samples were directly placed in sterile screw-cap tubes (50 ml capacity).
Tubes with terrestrial samples were filled xvith some drops of sterile water to keep the interior
moist. The samples collected from an aquatic environment were filled in tubes with water from
the habitat to ensure that the samples were well submerged by the aqueous phase typical for their
environment.
Before - isolation procedure the samples were kept in dim light at room (20 25 °C) or lower
temperature (about + 4 °C) for cyanobacteria from cold habitats (e. g. river, waterfall).
6.2 Isolation
Isolation is defined as the separation of indixidual clones of cyanobacterial species from other photosynthetic organisms present in the crude material. There are different possibilities to isolate cyanobacterial clones such as the liquid enrichment technique, the direct manual isolation and the self-isolation of phototactically motile trichomes and cells (1).
81 6.2.1 Isolation by Liquid Enrichment
The Liquid Enrichment technique is useful for the rapid isolation of cyanobacteria if the aim is to
select those members of the population possessing particular physiological properties. Liquid
enrichments with special cultivation properties (e. g. light, temperature, special kind of medium)
chosen for selection contain only one or very few dominant cyanobacterial strains. Their isolation
and purification is relatively easy. The samples are treated from this stage in an identical manner
to those subjected to the direct isolation method (2).
6.2.2 Direct Manual Isolation
With the direct manual isolation method large filamentous forms can be cloned by direct manipulation of the filaments on agar plates under a dissecting microscope. Once a sample has been microscopically examined for his content, aliquots of single strains can be transferred
manually to solid media of appropriate choice. For planctonic species diluted lake water is used
an inoculum on agar plates. If the sample is rich in eukaryotic contaminants it is advisable to use
plates to which 0.05 - 0.1 g/L cyclohcximide (Actidione) has been added. Plates are incubated
under the light and temperature regime of choice. If they are growing as unicyanobacterial
cultures they can be transferred in liquid media and if they show good culturing properties they
can serve as stock cultures (2).
During the present work the liquid enrichment technique and the direct manual isolation
technique were applied to isolate unicellular and filamentous species from field collected mixed
samples.
82 63 Culturing
6.3.1 Stock Cultures
The maintenance of stock cultures does not require sparking with air or other gas mixtures. The
intention of stock cultures is a low growth rate. Thus, the light intensity is kept at a low level (3).
At the EAWAG the stock cultures are cultix ated under xveak illumination, with intervals between
reinoculations ranging from several days to several months depending on the cyanobacterial
strain.
Aliquots of 5 ml from stationary phase stock-culture s were used to inoculate 250 ml of media and
were incubated under continuous illumination xvith fluorescent lamps (Philips TEM/33 RS 40 W)
at 29 uE/sAm2. The alga cells were harvested every 4-6 weeks and separated from the medium
by decanting or filtration and were employed directly for biological screening.
6.3.2 Large-Scale Cultures
During this work two cyanobacterial strains, Nostoc commune 122b and Tolypothrix byssoidea
195 xverc further scalcd-up to 10-E bottles containing inorganic media. The cultivation bottles arc
equipped with three inlets for addition of air, CO, and culture medium, as xvell as an outlet for
the culture. At first a collecting sterile glass bottle xvas inoculated with about 0.5 E of an actively
growing culture and 0.5 L of sterile medium is added by pumping. Ehe volume of the culture is
approximately doubled xveekly by pumping in sterile medium. Ehe cultures were illuminated
' continuously with fluorescent lamps (Philips ELM 33 Rs 40 W) at 29 uE/s 7m aerated with a mixture of 2% air and C02in incubated at a temperature of 24 ± 1 °C. A nearly even distribution
of light inside the bottles is achieved by permanent intensive mixing with 'Teflon stirring bars.
The cultures were harvested after 25-30 days. The medium was separated from the cells by
filtration and freeze-dried. Yields of freeze dried cells were ranging from 0.5 to 1.5 g/L. The cell-
free culture medium was each time passed through a glass column filled with Ambcrlite XAD-2
and was washed successively xvith methanol ( IL) and dichloromethane (IL).
The cell material xvas freeze-dried and stored bv -20°C.
83 6.3.3 Composition o f Culture Media
Five different inorganic culture media designated Z, Z2, Z4, Z45, and Z454 were employed.
Individual tailoring and modifications are needed depending on the purpose intended. The basic culture medium is termed Z. Media Z2 is poor in nitrogen. Media Z4 and Z454 contain nitrogen only in the trace clement solution. Medium Z454 as well as Medium Z45 lacks sodium. The composition of media is given in Table 6.1 and 6.2. The culture media of the different cyanobacterial strains are listed in Table 63.
84 Table 6.1 Compositon of Culture Media Sait Medium Medium Medium Medium Medium
Za Z2a Z4a Z45° Z454a
CaCEEEO 0.00 0.00 37.00 0.00 37.00
NaNO, 467.00 8.50 0.00 467.00 0.00
Ca(NO,)y4IEO 59.00 59.00 0.00 59.00 0.00
KH2P04 0.00 0.00 0.00 41.00 41.00
K?HP04 31.00 31.00 31.00 17.00 17.00
MgS04'7H20 25.00 25.00 25.00 25.00 25.00
Na2CO, 21.00 21.00 21.00 0.00 0.00
FeEDTA 1,00 1.00 1.00 1.00 1.00
Gaffron 0.08 0.08 0.08 0.08 0.06
''Concentrations of salts are given in mg/E, FeEDTA solution and Gaffron solution are given in nil/L. The FeEDTA solution consists of 10 ml of 0.1 M FeCl, 6EEO solution in 0.1 N HCl and 10 ml of 0.1 M Na2EDTA solution in 1 L.
Table 6.2 Composition of Gaffron Solution
Salt Concentration (mg/L) H^BO^ 310.0 MnS04-4H,0 223.0 Na9W04-2H20 3.3 (NH4)6Mo70244HA9 8.8 KBr 11.9 KT 8.3 ZnS04'7H,0 28.7 Cd(NO,)2'4H20 15.4 Co(N01)2'6H20 14.6 CuS04-5H20 12.5 NiS04(NH4)9S04-6EEO 19.8 Cr(NO,)v7H,0 3.7 VOS04-2H,0 2.0 AE(SOj)3K,S0424HA) 47.4
85 Table 6.3 Culture Media of the différent Cyanobacterial Strains
Strain Medium Strain Medium
222 a Z 237 b Z 217 z 261 z 108 b Z 132 Z45 262 Z 178 Z 91-150 z 211 z 140 z 166 z
163 Z2 226 a z
164 z 102 a z 91/154 z 102 b z 122 b z 130 z
123 b z 154 a Z2
124 b Z4 179 a Z4 259 Z 200 b Z2
105 a z 236 Z4 144 z 180 Z454
137 a z 157 a Z4
139 a z 176 Z 165 z 195 Z45
256 jLJ 153 a Z45 257 z 88 Z2
258 z 96 c Z 260 z
86 6.4 References
1. Court, G. E, Kycia, J. H., Siegelmann, H. W., Collection, Purification and Culture of
Cyanobacteria, in The Water Etwironment. Algal Toxins and Health. New York, Plenum
Press(1981)
2. Rippka, R., Isolation and Purification of Cyanobacteria. in Methods in Enzymology. San
Diego, Academic Press (1988)
3. Castenholz, R. W., Culturing Methods for Cyanobacteria, in Methods in Enzymology. San
Diego. Academic Press (1988)
87 7 Methodology of Isolation Procedure
7.1 General Isolation Strategy
7.1.1 Isolation of Intracellular Compounds
The isolation of intracellular natural products, derived from microorganisms or higher plants, can be divided into three main stages, namely extraction, fractionation and purification.
The first stage, the extraction, is the release of compounds from the cell mass and the removal of bulk of the biomass. Most of the bulk exists as fairly inert, insoluble, and often polymeric
material, such as the microbial cell wall or the cellulose of plants. The first step of the extraction
is to release and solubilize the smaller secondary metabolites by a thorough extraction with
organic solvent or water. This can be done by a series of stepwise extractions, using solvents of varying polarity, which act as the first fractionation step, or by using a single general solvent such
as methanol, which should dissolve most natural products. Filtration or centrifugation can then remove the insoluble material. The resulting initial extract is usually still a wide complex mixture. Much of this material will be grossly different in polarity from the target compound.
The aim of the second step, the fractionation, is to remove a large portion of the unwanted material in a fairly low-resolution separation step. Such a fractionation method may involve an
open silica column, vacuum liquid chromatography or a series of liquid-liquid extractions. The
aim is to obtain a mixture containing all the natural products of interest.
The third general stage, the purification, is often a high-resolution separation to separate the
interesting compounds. Whereas the second stage might involve a general fractionation with
subsequent analysis and work up of the fractions, this third stage tends to involve preliminary
work modifying and altering conditions to achieve the desired separation before preparative work
is carried out. The final stage is often achieved by HPLC (1).
7.1.2 Isolation of Extracellular Compounds
Microorganisms typically produce an extracellular product at low concentrations (^ 3 %). The primary goals of the initial capture stage are to concentrate the product, separate it from the
88 biomass, and purify it from impurities. The first step of isolation is the separation of the broth
from the bacterial cell mass by means of filtration or centrifugation. The second step can be a
solvent- or a solid phase extraction. In the present work, the solid phase extraction was applied
for the isolation of extracellular compounds. The third step, a final purification, that separates
closely related impurities is typically achieved via preparative chromatography (2).
7.1.2.1 Solid Phase Extraction
Solid phase extraction utilizes adsorbents for sample cleanup, trace enrichment, and fractionation
of extracellular compounds in crude fermentation broth, which is a complex mixture of
component that often contains only trace amounts of a desired product. This method exploits the
same product/sorbent interactions used in chromatography. As with any chromatographic method, there is a direct relationship between floxv-rate and separation efficiency, but an inverse
relationship between flow rate and time required for separation. Lower flow- rates improve
separations. When flow rates are too high, the product may not have sufficient contact xvith the
adsorbent.
There are three general adsorbent classes used most often in solid phase extraction applications: polar, ion exchange, and non-polar. Each class exhibits unique properties of retention selectively based on interactive properties of the products and adsorbent surface. In the present work,
Amberlite XAD-2, a non-polar adsorbent xvas applied. Resin particles of the adsorbents are typically 40 pm in diameter with either 60- or 300 A pores. The adsorbent's retention capacity is
approximately 5 % of the adsorbent weight. Solid phase extraction columns in general are
conventional gravity driven columns containing the adsorbent. Prior to sample loading, the
column material should be prewashed xvith a strong eluent. After prexvash the material must be
equilibrated with an eluent of similar polarity to the sample solution. The sample should be
loaded onto the column material at less than 5 ml/min to achieve a narrow band of adsorbed products at the top of the column-bed. After loading the column should be washed with an eluent
of the polarity as the sample feed. For elution. a suitable solvent must be applied (2) (see Figure
7.2).
89 7.2 Chromatographie Methods
7.2.1 Normal Phase Chromatography
Nomial phase chromatography employs a polar stationary phase, usually silica, and less polar
(usually non-aqueous) chromatographic eluents. Solutes separate during passage through the column by adsorbing the surface of the stationary phase particles, from which they may be displaced by solvent molecules. The strength of interaction depends on the nature of the functional groups present in the solute molecules. Thus, as a general rule, non-polar analytes elute more quickly than polar analytes since they interact less strongly with the highly polar surface of the adsorbent particles. Elution is controlled by the polarity of the chromatographic solvent; retention time decrease with increasing sob ent polarity (3).
7.2.2 Reverse Phase Chromatography
Reverse phase (RP) is the reverse of normal phase operation; the stationary phase is non-polar and the eluent is polar. 'The eluent normally comprises mixtures of water and organic solvents.
Other additives, such as buffers, acids, bases, and ion-pair reagents can be added. The stationary phases, most commonly used in reverse phase HPEC arc known as "bonded-phase" materials and comprise fused colloidal silica particles chemically derivatized with an alkylsilyl reagent, which renders the surface hydrophobic (3).
7.2.3 Thin Layer Chromatography (TI C)
Of the widely used chromatographic methods TLC is the simplest of all to perform. A suitable closed vessel containing solvent and a coated plate are all that is required to carry out separations as well as qualitative and semiquantitative analysis. With optimization of techniques and materials, highly efficient separations and accurate and precise quantification can be achieved
(4). TEC separations can also be used to select and optimize column chromatography conditions.
TLC conditions that give useful R, values, xvith separation of some compounds from the majority of the other components without staying at the origin or at the solx ent front, can be approximately transferred to column chromatography. Identification of the target compound on the TLC plate
90 can be carried out by comparison with a standard, by chemical staining, or by an overlay assay carried out on the developed plate in the case of an unknown biologically active component (1).
The detection of biological active principles by TLC is a fast and cheap technique very often employed in analyzing natural material (5).
In the present study, TEC was used in routine laboratory work (identifying and monitoring the behavior of compounds during purification and at the final purification step) as well as for primary screening of extracts or fractions for their antibiotic potential.
7.2.4 Vacuum-Liquid Chromatography (VLC)
VLC is a simple open-column method that can be practiced as a first separation step for crude extracts. The column is prepared in a sintered glass funnel, using TEC grade packing (aluminum oxide, silica gel, or revcrsed-phase supports). Uniform packing is achieved by initially tapping the funnel and then by application of a x'acuum from below the funnel. The sample is applied uniformly at the top of the support and the floxv is maintained by vacuum (6). Since the head of the column is at atmospheric pressure, manipulations on the column such as solvent changes can be done easily and therefore step gradient elution can be used. Intermittent breaking of the vacuum allows the collection of the fractions. The advantages of VLC include the simplicity of the equipment, low cost of operation, low solvent consumption, and the speed of separation.
Nevertheless, this method is only suitable for a crude fractionation, since channeling, caused by necessary interruption of the vacuum; uneven sample application and limited resolution due to the shortness of the column impair the separation (1, 7). (see Figure 7.1).
7.2.5 Open Column Chromatography
The conventional gravity-driven, open column chromatography method is still xvidcly used in both rapid filtration and true separation methods. It requires relatively large amounts of solvents
(6). Nevertheless, it provides a mild, rapid and efficient technique for preliminary fractionation of crude extracts or fractions and in certain cases may yield pure products. Classical column chromatography xvith a step gradient elution sequence was employed in this study for fractionation of crude cyanobacterial extracts (1), (see Figure 7.2).
91 Column
Adapter
Vacuum
Figure 7.1 Vacuum Liquid Cartridge setup, according to Cannell (1 )
Sample Reservoir
Top Fritted Disc
Adsorbent Bed
Bottom Fritted Disc
Leur Tip
Figure 7.2 Solid Phase Extraction Cartridge, according to Cannell (1)
92 7.2.6 Gel Permeation Chromatography
Gel permeation chromatography (also called size exclusion chromatography or gel filtration)
separates molecules according to their size. Although xvhen applied to the separation of low molecular weight molecules, it also obeys the basic principles of chromatography (1). Examples
of commonly used solvents are chloroform, methanol, THF, toluene and water. Ehe column
packing is a porous material that allows the molecules of different sizes to penetrate into the gel to different extends. As solutes migrate through a bed of stationary phase, their speed of passage is governed by the extend of diffusion into and out of the porous gel matrix. Targe molecules have limited access to the pores and arc eluted near to the xroid volume of the column. The extend
of retardation is a function of molecular size, large molecules elutes more quickly than small
ones. For any gi\en gel. there is an exclusion limit, a molecular weight above which no penetration into the gel will occur (6). In general, molecules xvill be eluted in the reverse order of their molecular weight. If smaller molecules (MW ^ 1000) are chromatographed. other effects
such as polarity and adsorption also have influence on the elution order (8). The most popular gel in the isolation of natural products is Sephadex LH-20, a hydrophilic hydroxypropylated dcxtran
gel with an exclusion limit of about MW 5000. It can be used with water as xvell as with polar
organic solvents. In the present work gel-filtration with Sephadex1' LH-20 xvas used for
fractionation of a column filtration extract.
7.2.7 High Performance Liquid Chromatography (I1PLC)
Preparative High Performance (or High Pressure) liquid chromatography (HPLC) is a versatile, robust, and widely used technique for the isolation of natural products. The main difference between HPLC and other modes of column chromatography is that the diameter of stationary phase particles is comparatixely low (3 - 10 pm), and these particles are tightly packed to give a very uniform column bed structure. Due to the loxv particle diameter a high pressure is needed to drive the chromatographic solvent (or eluent) through the bed. However, because of the very high total surface area available for interactions xvith solutes (approximately 100 - 300 nrVg for a typical 5-pm-diameter stationary phase) and the uniformity of the column bed structure, the
resolving power of HPLC is very high.
93 Sampling Pump Device I]
ODdnh h don n n p ggo y Column
t ~ J w~i t« i L ~,J i J
<<
Recorder Integrator Pressure >40 bar
Detector
-k.
Solvent w Reservoir A
Figure 7.3 Scheme of a semiprepatatn e HPLC equipment
94 The modes of separation most commonly applied to natural product isolations are normal phase, reverse phase, and to a lesser extend, gel permeation and ion-exchange chromatography. The choice of packing material is governed by the physicochemical properties of the target metabolite and of the other metabolites present in the sample, as well as by factors such as costs, general applicability, scale of operation, and compatibility with the stationary phase used.
Chromatographic cluents used in HPLC typically comprise mixtures of organic solvents, or organic solvents and xvater, and often contain additives such as buffers, acids, bases, and ion-pair reagents (3), (see Figure 7.3).
73 References
1. Cannell, R. J. P., How to Appoach the Isolation of a Natural Product, in Natural Products
Isolation. Totoxva. Nexv Yersey. Humana Press (1998)
2. Gailliot, F. P.. Initial Extraction and Product Capture. /;; Natural Products Isolation.
Totowa. Nexv Yersey, Humana Press (1998)
3. Stead, P., Isolation by Preparative HPLC. in Natural Products Isolation. Totoxva, New
Yersey, Humana Press (1998)
4. Sherma, J., Basic Techniques, Materials and Apparatus, in Handbook of'Thin-Layer
Chromatography. New York, Marcel Dekker. Inc. (1996)
5. Rios, J. L., Simeon, S., Jimenez, F. E. Zafra-Polo, Vf. C. Vi liar, A.: Reagents for
Screening Medicinal Plants by Thin-Layer Chromatography. Fiioterapia 57: 153-159 (1986)
6. Ghisalberti, E. L., Detection and Isolation of Bioactive Natural Products, in Bioactive
Natural Products - Detection. Isolatation and Structural Determination. Boca Raton,
Florida, CRC (1993)
7. Eargctt, N.M.: Vacuum Liquid Chromatography: An Alternative to Common
Chromatographic Methods. J. Org. Chem. 44: 4962-4964 (1979)
8. Henke, IE: Präparative Gelchromatographie an Scphadex LH-20. AKZO Research
Laboratories, Oberaburg: 33 (1994) 8 Structure Elucidation
Various spectroscopic and spectrometric methods, such as ultraviolet (UV), infrared (IR), and nuclear magnetic resonance (NMR) spectroscopy as xvell as mass (MS) spectral experiments arc employed in this study for structure determination of pure compounds. Also physical methods like optical rotation measurements and N-ray crystallography, which provides the exact three- dimensional molecular structure of crystalline substances, arc used to complete the structural
informations.
8.1 Spectroscopic and Spectrometric Methods
8.1.1 Ultraviolet (LJV) Spectroscopy
Ehe principle of UV spectroscopy is the measurement of the interactions between molecules and electromagnetic radiation. Molecular absorption in the ultraviolet spectrum (200 - 380 nm) is dependent on the electronic structure of a molecule and for the most part limited to conjugated systems. Thus, UV spectra of substances generally do not have a high degree of specifity and are rather used for quantitative assays than for structure elucidation of unknown compounds.
Nevertheless, informations on the extend, shape, and substituents of 7t-configurations are provided (1).
8.1.2 Infrared (IR) Spectroscopy
' Infrared radiation in the range of 107)00 - 100 cm is absorbed and converted by an organic molecule into energy of molecular vibration. Since particular bonds of functional groups show
specific IR absorptions, interpretation of an IR spectrum is useful to confirm the presence of functional groups xvithin a molecule in conjugation xvith other spectral data (2).
8.1.3 Mass Spectrometiy (MS)
Mass spectrometry (MS) bases on the generation of gas-phase positive or negative ions from
organic molecules, separation of these ions according to their mass-to-charge ratio, collection of
96 and the ions, recording relative abundance of each resolved ion species. With this mass
spectrometry technique it is possible to obtain not only the molecular weight of a compound but
also structural information regarding functional groups or subunits within the structure. Very
high sensitivity is an additional feature, xvhich makes mass spectrometry an attractive analytical
Mass EI technique. (electron impact), FAB (fast atom bombardment) and ESI (electron spray
ionization) are the most common operation methods, but also CI (chemical ionization) and
MALD/I (matrix laser desorption/iomsation) and MS-MS techniques are also employed
depending on the lability and volatility of the compound (3).
8.1.3.1 Electron Impact (EI)
A compound is bombarded xvith electrons from a glowing filament at low source pressure to
produce a positively charged molecular radical ion of high internal energy, which usually
fragments to form positively charged fragment ions, neutral fragments, and radicals. Their
relative abundance, together with their isotope ions forms a characteristic EI1 spectrum
fragmentation pattern for a given organic molecule under defined conditions. Due to the
instability of M it sometimes may be undetectable. The fragment masses may have structural
significance for given compounds (4).
8.1.3.2 Fast-Atom Bombardment (FAB)
This method involves placing a fexv microliters of the sample, dispersed in a liquid matrix
(usually glycerol, thioglycerol, or 3-nitrobenzyl alcohol), on an inert surface (commonly stainless
xvith steel). Bombardment fast atoms (argon or xenon) displaces often positive [M I EI]' or negative [M - Hf ions from the surface, fhe sample is not x'olatilized as such. In the positive-ion mode + and -+ ions [M Na]' [M K] may also be observed. The general applicability to very polar
compounds (polysaccharides, polyphenols), labile polar compounds and high-molecular weight biological compounds (polypeptides, polynucleotides) has made FAB the method of choice for
biological organic chemistry (4).
97 8.1.3.3 Matrix Laser Desorption/Ionisation (MALDI)
Desorption techniques, like laser desorption, succeed to transfer essential nonvolatile, fragile molecules into the gas phase with no or only limited fragmentation.
Lasers throughout the xvavelength range from the far ultraxiolet to the far infrared. With pulse durations from continuous waxe down to the femtosecond range they are used to irradiate solid or liquid material in vacuum or under ambient pressure. The intention is to couple energy from the laser beam into the sample via linear or non-linear absorption and to transfer some of the sample material into the gas phase. Direct laser desorption of intact organic molecules without a matrix seems to be limited to molecular masses of about 1000 Da. This mass range limitation gave rise to the development of the matrix-assisted laser desorption ionisation. This opens the possibility for a selective deposition of the energy into the matrix in xvhich the sample is dissolved. This leaves also high-molecular weight compounds to be analyzed xvithout alteration (5).
8.1.3.4 Electron Spray Ionization (EST)
An electron spray is generally produced by application of a high electric field gradient to a small flow of liquid (generally 1 to 10 liEinin) coming from a capillary tube. A potential difference of 3 to 6 kV is typically applied between the capillary and a counter electrode 0.3 to 2 cm away. The electric field disrupts the liquid surface and produces highly charged droplets that may be positively or negatively charged, depending on capillary polarity. Solvent evaporation is aided by pumping and using a nebulizing "curtain" of gas. Ions and clusters are generated that are then sampled by the mass spectrometer (4).
8.1.3.5 Chemical Ionization (CI)
Chemical ionization (generally of much lower flux than for EI ) is used to generate positive ions in a reagent gas at relatively high pressure (0.1 to 1 torr) inside the MS source. Ions are formed by charges exchanged between the reagent gas ions and the target molecule by proton transfer, charge exchange, electrophilic addition, or anion abstraction (4).
98 8.1.3.6 Mass Spectrometiy/Mass Spectrometiy (Tandem Mass Spectrometry, MS-MS)
To ionize large polar molecules, such as peptides, methods are used that impart little excess energy on the molecule (e. g. FAB-MS, MALDI-MS). Furthermore, most of these ionization techniques produce the charged particle by addition or removal of a proton to form the very stable, cven-electron-ions (M + EI) or (M - H)~. which hax c little tendency to fragment. This lack of fragmentation is very useful for the unambiguous determination of the molecular weight of high-molecular weight compounds. The resulting absence of stmctural infomiation could be overcome by transferring additional energy sufficient to force fragmentation of the stable molecular ion formed in the initial ionization process. Collision with a neutral gas is presently most widely used leading to collision induced dissociation (CID). The resulting fragment ions
(product ions) are then mass analyzed in a second mass spectrometer. The necessary energy can be aquired by the recursor ion, either by multiple collisions at low energy (eV) or single collisions at high energy (keV). see Figure 8.1 (6, 7).
99 MSI MS II Mass dispersion Mass dispersion of of parent ions daughter ions
Reaction" Ion Detec¬ Region tor
Mass spec¬ MS/MS spec¬ trum trum
A diagram of an MS'MS experiment depicting the two different stages of mass dispersion, according to Bush (7)
100 8.1.4 Nuclear Magnetic Resonance (NMR) Spectroscopy
Nuclear magnetic resonance (NMR) spectroscopy is by far the most powerful spectroscopic technique to obtain detailed structural information about organic compounds. Its particular strength lies in its ability to differentiate between most structural, conformational, and optical isomers. Given a molecular mass, NMR spectroscopy can usually provide all the additional information to unambiguously identify a completely unknown compound.
The proton NMR detection technique is also quantitatixc with individual areas in spectra being proportional to the number of contributing nuclei. One drawback of this technique is the relative low sensitivity in comparison to other spectroscopic techniques such as mass spectrometry (8).
The necessary information is obtained by measuring, analyzing and interpreting high resolution
NMR spectra recorded on liquids of low viscosity or in some cases solids by using special techniques and instruments that have been dex-eloped in the last few years.
NMR is a technique for studying nuclear magnetic moments. Nuclei that have a nonzero spin quantum number (e. g. lVV nC / lvN/ riV) are placed into a strong magnetic field, where they can occupy either of two possible energy states. Depending on their surrounding electron density, the observed nuclei are able to absorb energy if they are irradiated with the correct radio frequency.
This results in an emission signal from the excited nuclei. The signal is known as the free induction decay (FID). Fourrier transformation of this decay yields the NMR spectrum (9).
The NMR technique defines not only the number and types of nuclei present in a molecule but also supplies informations about their indixidual chemical enxironments and their connections by showing neighboring relationships (scalar and dipolar coupling).
8.1.4.1 One-Dimensional NMR Spectroscopy
One-dimensional NMR spectroscopy includes 'H-, nC and DEPT (Distortionless Enhancement by Polarization Transfer) experiments.
The aim of a standard 'H experiment is to record a routine proton NMR spectrum in order to get
structure-related informations for the protons of the sample, like chemical shifts, spin-spin couplings, and intensities.
101 The aim of a standard nC NMR experiment is to record a nC NMR spectrum with proton broad¬
band decoupling and data accumulation in order to indicate stmctural fragments and to confirm
the presence of nonprotonated carbon atoms whose presence in a molecule can only be detected
in the nC spectrum.
With DEPT experiments the number of hydrogens bonded to each carbon atom can be
determined. A DEPE 135 spectrum shows all protonated carbon signals with Cff and CH
resonances being positive and all CH, resonances being negative. A Dept 90 spectrum shows
only positive CH and negative CH, resonances. Fully substituted carbons do not give signals in a
DEPT spectrum (10, 11).
8.1.4.2 Two-Dimensional NMR Spectroscopy
One-dimensional NMR experiments are limited to the figure of response intensity as a function
of the observation frequency. In contrast, 2D-NMR spectra have available a second frequency
domain, which opens vast new horizons in regard to the infomiation content of the spectrum.
8.1.4.2.1 Homonuclear Experiments
The COSY (Correlation Spectroscopy) pulse sequence generates a 2D NMR spectrum in which
the signals of normal 'EI NMR spectra are correlated with each other. Cross-peaks appear if spin
coupling is present; thus the COSY sequence detects coupled pairs of protons. Homonuclear
correlation as described for the COSY' technique correlates protons via a geminal or vicinal spin coupling.
The TOCSY (TOtal Correlation SpectroscopY) or UOHAEEA (HOmonuclear HArtmaim IIAhn)
experiment gives a total correlation of all protons of a spin system with each other. The technique is therefore mostly used for peptides or oligosaccharides, since there it serves for the identification of single residues.
The Nuclear Overhauser Effect (NOE) alows the determination of local neighbourhood of the nuclei within a molecule and to infer infomiation about the distances between atoms. Ehe use of one-dimensional NOEÎ difference experiments, or. more frequently, the two-dimensional NOESY or ROESY techniques give answers to many stereochemical problems. Ehe NOESY (Nuclear
Overhauser Enhancement SpectroscopY) yields correlation signals, xvhich are caused by dipolar
102 cross-relaxation between nuclei in a close special relationship. In NMR studies of peptides and
proteins NOESY is the essential method for determining peptide conformations or tertiary
structures of proteins. The NOESY technique has the disadvantage that for molecules with a
molecular mass in the order of 1000 to 3000 the cross-signals may disappear, since the NOE
changes its sign depending on the molecular correlation time. Hoxvcver, the nuclear Overhauser
effect in the rotating frame under spin-lock conditions is alwavs positive. This effect is used in
the ROESY (Rotating Frame Overhauser Enhancement SpectroscopY) experiment. This
experiment gives identical results as the NOESY technique, but in a shorter time, due to the
shorter mixing period during which the spin-lock is used (12).
8.1.4.2.2 Heteronuclear Experiments
Multidimensional NMR in the form of EIMQC (Heteronuclear Multiple Quantum Coherence) or
EISQC (Heteronuclear Single Quantum Coherence) and HMBC (Heteronuclear Multiple Bond
Correlation) offer new ways to identify 'II - p,C pairs in a molecule. 'Ehe EIMQC experiment is
the simplest form of an inverse H. X correlation technique. The suppression of unwanted signals
is performed by the phase cycle. No nC broad band decoupling is applied during the aquisition
and the 2D spectrum is recorded without quadrature detection in FT, which requires the
magnitude mode of data processing. Using only four different radio frequency pulses it
demonstrates the sensitivity advantage of the inverse detection mode. The HMQC sequence was
designed to correlate proton and carbon nuclei xia './ (CH). To obtain long-range EI, C
correlations via M (C, H) and \I (C. IT) one can use the special EIMBC pulse sequence. Its
purpose is to suppress correlations via v/(C, H) (12).
8.1.4.3 High Perfomiance Liquid Chromatograph} Proton Nuclear Magnetic Resonance On-
Line Coupling (LC-NMR)
Recent developments have enabled the use of NVIR spectroscopy directly coupled with high
performance liquid chromatography (HPLC). Ehe experimental arrangement used in online
EIPLC-NMR coupling is shown in Figure 8.2.
One of the advantages of using NVIR in combination xvith HPEC is that both are conducted in
solution, and no evaporation and heating procedures are required. Therefore, the investigation of
103 air-and UV-sensitive compounds is possible. One of the serious disadvantages of the NMR
detection principle is the low sensitivity of NMR, leading to larger detection volumes. Because
the introduction of extra column dispersion effects leads to a decrease of HPLC separation, the
NMR cell volume should be as small as possible. Therefore, a continuous-flow NMR detection
probe is a compromise between the needs of H PTC and NMR.
In flowing liquids nuclei within a distinct volume exhibit a defined residence time x. At a
constant flow rate complete exchange of all nuclei in the detection volume occurs after the period
%. The residence time t is defined by the ratio between the detector volume Vd and the flow rate
u:
t-Vj/u (8.1)
A shorter residence time x within the NMR measuring coil results in a reduction of the effective
residence time of the particular spin states. Thus, the effective relaxation rates 1/Tn are increased
by 1/x:
l/Tnc(fccme==l/T+l/x (8.2)
The influence of the floxv rate can be described as a relaxation phenomenon. Due to the limited
residence time % of a nucleus in the NMR detection xolumc, the spin-spin relaxation times (T,),
are reduced in the flowing mode.
l/T! now=l/T, static -f 1/x (8.3)
According to Eq. (8.3) an increased flow rate (I t) results in a reduction of T,. An increase in
relaxation rates (l/T]) result in a higher population of the ground state and thus lead to an
increase in N MR signal intensity.
In structure elucidation of unknown compounds, the application of two-dimensional assignment
techniques is necessary. Because the diffusion constant of water is very small, ETPLC separations
can be stopped resulting in small dispersion effects. Therefore, it is possible in an on-line ITPLC-
NMR experiment, after determination of the maximum peak concentration in the NMR flow cell,
to stop the flow and to record a 2D-correIated spectrum.
A further application mode of on-line EIPEC-NMR coupling is a combination of continuous- and
stopped-flow techniques. First, a continuous-flow run gives information on the 'H NMR signals
of the chromatographic peaks. Storing the retention data, a series of interrupted HPLC
separations can be performed, bringing each interesting peak in the NMR detection volume.
Corresponding 2D spectra of all interesting peaks can be recorded. Thus, extremely valuable information about unknown compounds can be obtained overnight.
104 The majority of HPLC separations are perfonned with reverse-phase columns using binary or ternary solvent mixtures. The use of deuterated eluent is too expensive for routine analysis.
Therefore proton-containing solvents such as acetonitrile, methanol and water are used in on-line reverse phase HPLC experiments. To get rid of dynamic range problems of the receiver of the
NMR instrument, the proton NMR signals of the solvent have to be suppressed (8).
In the present xvork both, on-floxv and stop-flow, methods of on-lme HPEC-NMR are applied to detect different compounds in a fractionated cyanobacterial extract.
105 Continous flow Probe stopped flow
injection valve
on¬ flow
foi a chromatographic Figure 8.2 Fxpenmental ai-rangement coupling to Albert (8) separation system with an NMR spectrometer, according
106 8.2 Physical Meth ods
8.2.1 X-ray Crystallography
The technique of single crystal X-ray diffraction proxades the three-dimensional molecule structure of a pure compound. X-rays are electromagnetic radiations of wavelength between 0.01
- 100 A°. Electrons of indixidual atoms act as scattering centers when a collimated beam of X- rays strikes a crystalline object. Ehe result is a characteristic diffraction pattern, because the atoms in crystalline solids are arranged in a regular array. X-ray diffraction patterns can be observed as spots reflections of radiation, cither by photographic film or a detector in a diffractometer system. The limiting step for a X-ray analysis is the preparation of a suitable single crystal of the compound. The X-ray diffraction experiment is suitable for the structure elucidation of natural compounds, especially for the determination of their stereochemistry. The knowledge of detailed three-dimensional structure information is also of interest for the investigation of relating biological activity to the structure (13).
8.2.2 Optical Rotation
Chiral chemical substances are able to rotate an incident plane of polarized light so that transmitted light emerges at a measurable angle to the plane of the incident light. The specific rotation [a)D is a characteristic physical constant. Optical rotation measured under defined conditions using the sodium D-line (589.5 nm) is also of interest in the structure elucidation process (14).
83 Chemical Methods
In order to support the spectral findings and to arrive a final conclusion for a structure, conventional chemical methods are also often of choice. In the present study derivatization reactions (Marfey's Method) were utilized (15). The work-up and the other details are presented in chapter 14.
107 8.4 References
1. Henke, H.: Preparative Gelchromatographie an Sephadcx LH-20. AKZO Research
Laboratories, Obernburg: 33 (1994)
2. Hesse, M., Meier. IE, Zeh, B.: Spektroskopische Methoden in der organischen Chemie.
Ehieme, Stuttgart: 29-70 (1995)
3. Hesse, M.. Meier, IE, Zeh, B.: Spektroskopische Methoden in der organischen Chemie.
Thieme, Stuttgart: 219-307 (1995)
4. Bloor, S. E. Porter. L. E. Mass Spectrometry, in Bioactive Natural Products. Boca Raton,
Florida, CRC (1993)
5. Hillenkamp, F., Karas, M., Mass Spectrometiy of Peptides and Proteins by Matrix-Assisted
Ultraviolet Laser Desorption Ionization, in Methods in Enzymology. San Diego, Academic
Press (1990)
6. Biemiann, K., Sequencing of Peptides by Tandem Vlass Spectrometry and High-Energy
Collision-Induced Dissociation, in Methods in Enzymology. San Diego, Academic Press
(1990)
7. Bush, K. L., Glish, G. E., McLuckey, S. A.: Techniques and Applications of Tandem Mass
Spectrometry. VCH, New York: 120 (1995)
8. Albert, K.: On-Linc Use of NMR Detection in Separation Chemistry. J. Chromatography A
703: 123-147(1995)
9. Friebolin, H.: Basic One- and Ewo-Dimensional NMR Spectroscopy. VCH, Wcinhcim: 1-
39(1993)
10. Friebolin, H.: Basic One- and Two-Dimcnsional NVIR Spectroscopy. VCH, Wcinhcim:
181-221 (1993)
11. Braun, S., Kalinowski, H.-O.. Berger, S.: 100 and More Basic NMR Experiments. VCH,
Weinheim: 33-65 (1996)
12. Braun, S., Kalinowski, H.-O., Berger. S.: 100 and More Basic NMR Experiments. VCEI,
Weinhein: 273-341 (1996)
13. Wong, R. Y.. Gaffield. W., Determination of Three-Dimensional Structure and
configuration of Bioactive Natural Compounds by X-Ray Crystallography, in Bioactive
Natural Products. Boca Raton. Florida, CRC ( 1993)
108 14. Schmidt, W.: Spezielle Methoden der Optischen Spektroskopie. VCH, Weinheim: 326-361
(1994)
15. Marfey, P.: Detemiination of D-Amino Acids. Use of Bifunctional Reagent, 1,5-Difluoro-
2,4-Dinitrobenzene. Carlsberg Res. Commun. 49: 591-596 (1984)
109 9 Biological Investigation
9.1 Screeningfor Biological Active Metabolites and Determination ofPure Compounds
9.1.1 Introduction
The detection of biologically active metabolites is the starting point for a strategic approach in the search for compounds, which are potentially useful for the treatment of diseases and human health care. In the search for bioactive natural products, a common tendency is to use primary screening techniques that monitor biological activity toward a problem of current interest. The appropriate choice of any in vitro or in vivo detecting system is a determining factor for a successful outcome. There are several criteria to be met for a useful front-line screening bioassay.
It must be rapid, convenient, reliable, inexpensive, and require little amount of material.
These preliminary screening programs enable one not only to find biologically interesting organisms for further investigation but also to trace and follow a specific activity found in an extract. This bioactivity-guided isolation strategy starts xvith preparation of appropriate extracts of selected organisms and application to a range of relevant bioassays. Following the determination that an extract is active in one of these preliminary screening tests, it can be subjected to bioactivity-guided fractionation. Each fraction obtained in a chromatographic separation is submitted to the same screening system, to isolate the active principles. After isolation of the single active componcnt(s). verification of its purity and the detailed structure determination is essential. After that, it can be systematically evaluated for toxicity and biological activity using in vitro and in vivo bioassays.
Many factors can complicate matters when using bioassay or bioactivity-guided fractionation.
The most obvious is that the solubility of the extracts or fractions is usually limited and the selection of solvents is critical if a meaningful assay is to be carried out. Other factors can be due to one event or a combination of events, including synergistic effects, chemical changes during extraction and separation of extracts, and the canceling of activity by certain concentrations of substances (1,2).
The following test systems were used to monitor biological activities as in detail discussed in publication 1 (Chapter 18).
110 9.1.2 Brine Shrimp Lethality Test
A general bioassay that appears capable of detecting a broad spectrum of bioactivity present in crude extracts is the brine shrimp lethality bioassay. The technique is easily mastered, inexpensive, rapid, and utilizes small amounts of test material. The aim of the method is to provide a front-line screen that can be backed up by more specific and more expensive bioassays once the active compounds have been isolated (1).
Bioactive compounds are almost always toxic in high doses. Thus, in vivo lethality in a simple zoological organism might be used as a rapid and simple monitor during the fractionation of bioactive extracts. Ihc eggs of brine shrimp, Artcmia sa Una (Leach), arc commercially available at low cost and can remain viable for years in the dry state. Upon being placed in a brine solution, the eggs hatch within 48 h, providing large numbers of larvae (nauplii) that are attracted toward artificial light sources (3, 4, 5).
Brine shrimps have been previously utilized in xarious bioassay systems. Among these applications have been the analysis of pesticide residues (6), mycotoxms (7), cytotoxic compounds (8) and active plant constituents (4).
Today, brine shrimps are suggested as a convenient monitor for the detection and isolation of bioactive natural compounds, which may be manifested as toxicity towards the newly hatched nauplii (3).
9.1.3 Bioassays for Antibiotic Activity
Antimicrobial actixity of extracts from natural sources can be detected by obserxing the growth response of various microorganisms to those extracts.
The choice of test organisms xxill obviously depend on the purpose of the mx estigation. If the investigation is of a general character, the test organisms selected should be as diverse as possible and preferable representative of all important groups of pathogenic bacteria and yeasts according to their chemical and physical composition and resistance pattern.
Standard microorganisms should be preferably used as test bacteria or yeasts, so that the screening could be repeated everywhere. Consequently, all results can then be compared to another. In contrast, if one is interested in finding nexv products, which are selectively active against problem microorganisms causing certain diseases, e.g. Pseudomonas aeruginosa, it is
111 clearly appropriate to employ the corresponding isolated pathogenic microorganisms. Primary purified cultures of the isolate should be lyophyhsed and stored, so that a control experiment always can be carried out at a later stage.
Test selection of test organisms used in the present work are the gram-positive cocci
Staphylococcus epidermidis. and Micrococcus Intens, the gram-positive spore-forming rod
Bacillus cereus. the gram-positix'e asporogenous rod Mycobacterium fortuitum, the gram- negative rods Escherichia coli and Pseudomonas aeruginosa, as well as the yeast Candida albicans (1, 9).
9.1.3.1 Diffusion Method
In the diffusion technique a reservoir containing the plant extract to be tested is brought into contact with an inoculated medium (agar). After incubation the diameter of the clear zone around the reservoir (inhibition diameter) is measured.
In the present work the agar-well method xvas employed for antimicrobial screening. Advantages of the diffusion methods are the small size of the sample used in the screening and the possibility of testing five or six compounds per plate against a single microorganism.
It is useful to compare the zones of inhibition xxith those obtained for antibiotics to establish the sensitivity of the test organisms. A comparison of the antimicrobial potency of the samples and antibiotics cannot be drawn from this since a large inhibition zone may be caused by a highly active substance present in quite small amount or by a substance of comparatively low activity but present in high concentration in the plant extract (9).
9.1.3.2 Agar Overlay Method
The agar overlay method is a bioautographic method to localize antibacterial activity on a chromatogram. A developed thin-layer chromatogram plate is covered xxith agar that has been inoculated with the test organism and incubated for 24 h. The agar overlay method was employed for the antimicrobial testing of fractions during the bioactixity-guided isolation procedures (1).
Zones of inhibition are xisualized with an aqueous solution of INT reagent for both test methods.
112 9.1.3.3 Method for MIC Determination of Pure Compounds with Antimicrobial Activity
For pure isolated compounds it is necessary to detect the Minimal Inhibition Concentration
(MIC) by means of a dilution method.
The samples being tested are mixed with a suitable medium, which has previously been inoculated with the test organism. After incubation, growth of the microorganism may be determined by direct visual (e.g. aqueous solution of INT reagent) or turbidimetric comparison of the test culture with a control culture which did not receive an addition of the sample being tested, or by plating out both test and control cultures. A series of dilutions of the original sample in the culture medium is made and then inoculated xvith the test organism. After incubation the endpoint of the test is taken at the highest dilution x\hich xull just prevent perceptible growth of the test organism (9).
9.1.3.4 Assay for MoUuscicidal Activity
Schistosomiasis, commonly known as bilharzia, is a parasitic disease, xvhich is endemic in about
76 countries. Propagation is via contaminated water and occurs principally during childhood in the areas concerned. Three species of schistosomes are important parasites in humans:
Schistosoma mansonii (intestinal bilharzia). S. faponicum (intestinal bilharzia), and S. haematobium (urogenital bilharzia).
The schistosomes lay eggs in the human body and these are excreted xvith the faeces or urine. In areas with poor sanitary conditions the eggs reach water sources and hatch into miracidia which require the presence of an intermediate host for further development. This host is a freshwater snail of the genera Biomphalariu. Once in the snail, the miracidia produce thousands of cercaria, which arc shed into the water and eventually penetrate the skin of humans m contact with the water. The cycle is then ready to repeat itself.
One attempt of controlling schistosomiasis is to destroy the intermediate host (the mollusc) with moUuscicidal agents. This interrupts the parasite's life cycle and prexents infections of humans in contact with water in high-risk areas.
Tests for moUuscicidal activity basically involve introducing the substance or extract into water containing bilharzta-transmitting snails and obserxing their mortality (10).
113 9.1.3.5 Assays for Cytotoxic Activity
Some confusion exists in the use of the terms cytotoxicity, antitumor and antineoplastic activity.
The National Cancer Institute (NCI) has defined these terms precisely. Cytotoxicity refers to in vitro toxicity to tumor ceiE. xx hile antineoplastic and antitumor should refer to in vivo activity in experimental systems.
In vitro preliminary screenings for cytotoxicity haxe been employed to facilitate fractionation, but do not distinguish cytotoxicity from antitumor actixity. Although many cytotoxic compounds have been isolated through these screening procedures, few of these were found to be clinically effective against slow-growing solid tumors. This is a consequence of using rapidly dividing tumors as the primary screens. On the other hand, screening with in vivo assays is impractical due to the time required and the high cost. The trend is to deemphasizc the use of typically insensitive in vivo animal models and concentrate on very sensitix'e disease-oriented human tumor cell lines as primary screens (1).
In this study, the KB and Caco-2 cell lines arc applied in the pre-screcning for the evaluation of cytotoxicity. The epithelial-like KB cell line, derixed from a human nasopharyngal carcinoma, has been useful for many years as a preliminary screen, being more sensitive to most antitumor agents than in vivo assays (11). The epithelial-bke. Caco-2-cell line, obtained from a human colon adenocarcinoma (12), xvas chosen in order to get a broader infomiation on cytotoxicity. For pure isolated compounds with cytotoxic actixity it is necessary to determine the ED50 value.
9.1.4 Antiplasmodial Activity
Useful in vitro tests for antiplasmodial actixity have become available only recently. Since 1947,
600 plants from 126 families were extracted, and the extracts were tested for in vivo activity
animal against malarias. Species from over 30 genera were found to haxre some activity, but the extrapolation to human malarias is questionable. The continuous m vitro culture of the human parasite Plasmodium falciparum represented a significant advance xvhich led to a technique for quantitative assessment of the actixity. A modification of this technique relies on measuring the ability to inhibit the incorporation of'H-hypoxanthine into plasmodia (1).
114 9.2 References
1. Ghisalberti, E. L., Detection and Isolation of Bioactive Natural Products, /// Bioactive
Natural Products - Detection, Isolation and Stmctural Determination. Boca Raton, Florida,
CRC (1993)
2. Waterman, P. G., Natural Products from Plants: Bioassay-guided Separation and
Stmctural Characterization. /// Drugs from Natural Products- Pharmaceuticals and
Agrochemicals. London. Ellis Horwood Limited (1993)
3. McLaughlin, J. L., Croxvn Gall Tumors on Potato Discs and Brine Shrimp Lethality: Two
Simple Bioassays for higher Plant Screening and Fractionation, in Methods in Plant
Biochemistry. London, Academic Press Limited (1991)
4. Meyer, B. N., et ab: Brine Shrimp: A Convenient General Bioassay for Active Plant
Constituents. Planta Med. 45: 31 -34 (1982)
5. Sam, T. W.. Toxicity Testing using the Brine Shrimp: Artemia salina, in Bioactive
Natural Products - Detection, Isolation and Structural Determination. Boca Raton, Florida,
CRC (1993)
6. Grosch, D. S.: Poisoning with DDT: Effect on Reproductive Performance of Artemia.
Science 155: 592-593 (1967)
7. Harwig, E. Scott, P. VI.: Brine Shrimp Larxae (Artemia salina L.) as a Screening System
for Fungal Toxins. Appl. Microbiol! 21: 1011-1016 (1971)
8. Soils, P. N., Wright, C. W., Anderson, M. M., Gupta, P. VE, Phillipson, J. D.: A
Microwell Cytotoxicity Assay using Artemia salina (Brine Shrimp). Planta Med. 59: 250-
252(1993)
9. Vanden Berghe. 1). A.. Vlietinck, A. E, Screening Methods for Antibacterial and Antiviral
Agents from Eligher Plants, in Methods in Plant Biochemistry. London. Academic Press
Limited (1991)
10. Marston, A., Hostettmann, K., Assays for MoUuscicidal, Cercaricidal, Schistosomicidal,
and Piscicidal Activities, in Methods in Plant Biochemistry. London, Academic Press
limited (1991)
11. Perdue, R. E.: KB Cell Culture. Role in Discox-ery of Antitumor Agents from Higher
Plants. J. Nat. Prod. 45: 418-426 (1982)
115 12. Eaboisse, C. L., Differentiation of Colon Cells in Culture, in Cell and Molecular Biology
of Colon Cancer. Boca Raton, Florida, CRC (1989) 10 Diterpenoids
10.1 Introduction
Diterpenoids are, by definition, Cy, compounds based on four isoprene (C5H8) units. Ehe term diterpenoid, has come into considerable usage after 1955 and is now considered to be synonymous with ditcrpene and. is the preferred generic name for this class of natural products
(1).
Diterpenoids to be first investigated were the resin acids, occurring in resin (colophony), the steam-nonvolatile residue from the traditional manufacture of terpentine from the oleorcsinous exudate of trees belonging to the family Pinaceae. These acids occur as rather complex mixtures of compounds of similar structure and hence their separation proved quite difficult. Thus, a somewhat impure specimen of abictic acid, the most important constituent of resin, had been obtained in 1824. It xvas not before 1910 that pure abietic acid (Figure 10.3) was isolated. At the end of the 19th century txvo more resin acids had been obtained pure: dextropimaric acid (1887) and lcvopimaric acid (1887).
Though structural investigations on these acids started xvith the availability of abietic acid and the pimaric acids, no real headway could be made until the introduction of the method of dehydrogenation. The first application of the dehydrogenation method was made in the diterpenoid field, when abietic acid was subjected to sulfur dehydrogenation (1903) and retenc was obtained. Several years later it was characterized l-methyl-7-isopropylphenanthrene (Figure
10.1). The application of this method to the study of diterpenoids proved most basic for further development of diterpenc chemistry. Thus, these investigations revealed that the tricyclic diterpenoids possess a perhydroxyphenanthrenc skeleton and further belong to two distinct classes: those (abictic acid, levopimaric acid) giving retene, and those (dextropimaric acid) generating primanthrene (Figure 10.1) on dehydrogenation. Bicyclic ditcrpenes, such as sclareol and agathenedicarboxylic acid, furnished agathalene under similar conditions (Figure 10.1), often accompanied by primanthrene or 1.7.8-trimethylphenanthrcne (Figure 10.1). These results coupled with the isoprene rule, according to which tcrpene structures are formally divisible into
"isoprene units", paved the way for elucidation at the parent skeleton of these diterpenoids. 'Thus, formation of retene on dehydrogneation abictic acid revealed the position of eighteen out of 20
117 carbon atoms and the carbon skeleton could be extended (Figure 10.2) on the basis of the isoprene rule. This vindication of isoprene rule led to its general acceptance as a good working hypothesis for exploring terpene structures. The position of two ethylenic linkages of abietic acid, as well as the site of the carboxyl function, which had been known to be tertiary could be established 1942, to reveal the gross structure of abietic acid (Figure 10.3).
The evolution of this methodology was a remarkable achievement to clarify the gross structures of many of known cyclic diterpene acids and alcohols which had been obtained pure: for example manool (Figure 10.3, 1936), levopimanc acid (Figure 10.3. 1940), sclareol (Figure 10.3, 1942), agathenedicarboxylic acid (Figure 10.3, 1943), (1. 2).
With the developement of nexver and more effective separation techniques and powerful spectroscopic methods of structural analysis, coupled xvith newer advances in organic chemistry theory and practice the variety of diterpenoid structural types that arc known increased enormously. Of the over 170 carbon frame-works known for diterpenoids at present, seven structures (Figure 10.4) account for some 50°o of the known diterpens, labdanc (Figure 10.4) accounting for the largest number. Diterpenoids of marine origin have yielded a rich harvest of new carbon frame works, especially higher isoprenologues of several well-known sesquiterpene types.
Special mention may be made of xenicin (Figure 10.5), from Australian soft coral, Xenia elongata, dilophol (Figure 10.5) from the Sicilian brown alga Dictvota ligulatus, dictyolene
(Figure 10.5) from the Hawaiian brown alga Dictvota acutuloba, pachydictiol-A (Structure 10.5) from the Pacific seaweed Pachvdictwn coriaceunu dilopholone (Structure 10.5) from the brown alga Dictvota prolificans, and xeniaphyllenol (Structure 10.5), a metabolite of the soft coral
Xenia maerospiculata.
Several halogenated diterpenes, isolated from marine flora and fauna, are known at present.
Diterpenoids are widely distributed in the plant and animal kingdoms, both terrestrial and marine, and have even been isolated from fossils. They occur m several families of higher plants. Since gibberellins, a group of closely related diterpene acids, arc natural plant growth hormones, they occur in most plants. Conifer resins (Pinaceae, Araucariaccae. Taxodiaceae, Cupressaccae, and
Podsocarpaceae) are especially rich sources of diterpenoids. Equally rich are some angiosperm
such resins, as those from the families Cistaceac, Leguminosae. and Burseraceae. They are also
in the wide-spread families Labiatae and Euphorbiaceae. Diterpenc alkaloids are widely
118 distributed in the two genera Aconitum and Delphinium of the family Ranunculaceae.
Diterpenoids are also elaborated by a number of fungi, bryophyta and algae. Nevertheless, diterpenoid compounds are very uncommon in cyanobacteria. Tolypodiol is the first reported diterpenoid compound, isolated from a strain of the terrestrial cyanobacterium Tolvpothrix nodosa.
In recent years xvide distribution of diterpenoids in marine animals of the order Alcyonaceae (soft corals, sea fans), and sponges have been discovered (1).
119 retene primanthrene agathalene
1,7,8 trimethy 1 ph en anthren e
Figure 10.1
Figure 10.2
120 HOOC HOOC
abietic acid manool levopiniaric acid
COOH
HOOC
sclareol agathenedicarboxyhc acid
Figure 10.3
121 labdane
Figure 10.4
122 ,\x*-\>'OAc AcO
O niOAc
OAc
xenicin dilophol
OH
dictvelene pachydictol A
HO
dilopholone xeniaphyllcnol
Figure 10.5
123 10.2 Biosynthesis
It was generally assumed that diterpenes, like all other naturally derived terpenoids, are biosynthesized via the classical acetate'mevalonate pathxvay. However, a totally different route,
which is called triosephosphat pyruxate pathway xvas found in early steps of isoprenoid biosynthesis in bacteria, green-algae and in higher plants.
10.2.1 Acetate/Mevalonate Pathway
Ewo molecules of acetyl-coenzyme A, derived by carbohydrate, fat, or protein catabolism,
condense to yield acctoacetyl-coenzyme A. Further condensation of acetoaeetyl-coenzyme A with another molecule of acetyl-coenzyme A, noxx in an aldol type reaction, results m ß-hydroxy-
ß-methylglutaryl-cocnzyme A (EIMG-CoA), xxiiich is irreversible reduced through intervention of
NADPH to R-mevalonic acid, the building block of almost all isoprenoids. Phosphorylation of mevalonic acid (MVA) by ATP, in two steps (two enzymes) leads to mevalonic acid-5- pyrophosphat. The latter reacts on the enzyme xxith ATT generating isopentenyl pyrophosphate
(1PP), the biological isoprene unit. This elimination reaction proceeds by concerted trans-
elimination. Next, IPP is converted by an enzyme-catalyzed prototropy into an equilibrium mixture with dimethylallyl pyrophosphate (DMAPP). m xvhich the latter predominates. 'The two intermediates, IPP and DMAPP react with the aid of a prenyl transferase generating gcranyl pyrophosphate in a stercospecific condensation. Further condensation of gcranyl pyrophosphate
and IPP will lead to farnesyl pyrophosphate and so on. Geranyl pyrophosphat represents an acyclic precursor for further elaboration into cyclic diterpenoids (I), see Figure 10.6.
10.2.2 Cyclization
Attack of an eicctrophilic species, such as IT at the C-14, C-15 olefinic linkage in geranylgeraniol-pyrophosphate (GGPP) can trigger cyclization leading to a mono-, bi-, or tricyclic system depending on the conformation of the substrate ordered by the cyclase.
Diterpenes of all these three types occur in nature. It may be further noted that the absolute
124 stereochemistry of the resulting cyclic diterpenes will be dictated by the nature of the cyclase and unlike and tiiterpeiies steroids, for many diterpene classes both antipodal types occur in nature
(1), sec Figure 10.7.
125 X 11 to Sto\ ni torn m \ç0\j CoASii
CI! tO SCo\
to\SH
WDP N4DPII 11
C°\ HOOC Z nXKiCoA R \te\ demie îcid
? \TP
\DP
OPP ,t H OPP ,CH Ov l\IP) .—\Z
-IIb r>-A (/ j co, iro P) AÜP ] moi i îi
H) n kopenttmi p\i jphosphile (IPP)
w
r Hd
Guimlsiuiml p\tO) h isph lit
Figure 10-6 Acetate Adexalonate Pathway
126 H,OH
HpOH
t J
^ Isoagathclactonc
Figure 10.7 Cyclization 10.2.3 Triosephosphate/Pyruvate Pathway
In the triosephosphat/pyruvate pathway the C, isoprenic units IPP and DMAPP would not be formed via HMG-CoA and MVA, but by direct condensation of thiamine-activated acetaldehyde arising from pyruvate decarboxylation on a C\ unit derived from a triose phosphate, followed by a transposition step (3).
It was proposed that the two different pathways might be separated by compartimentalization (4).
At present it is being hypothesized that this alternatix'e pathway is located in the plastids and involved in the formation of mono-, di-, and tctraterpenoids. whereas sesquiterpenes and the biogcnctically related sterols are formed in cytoplasm via mevalonatc pathway. Elcnce, many secondary metabolites of diterpene origin, such as taxan derivatives from Taxus ehinensLs (4), marubiin, a furanoid labdane (Marubium vulgare) (5), as well as Carotinoids, phytol, and the prenyl chain of plastoquinone were shoxvn to be biosynthesized via the novel IPP pathway, see Figure 10.8.
128 H—-CX— CX ^P/C
HO.
HO C- ±1)
- C02 H-.C CH,
TT * Oihydioxyacctonc hq- OP phiwphate
H-,C OPP CH-, il'P
HO, H O C-
OPP D>- DMAPP OH
,OH OP OPP
H H
Figure 10.8 Triosephosphate/PyruvatePathway References
Dev, S., Misra, R.: Handbook of Terpenoids, Diterpenoids. CRC Press, Florida. 3: 7-25
(1986)
Hanson, J. R.: Methods in Plant Biochemistry, Terpenoids. Academic Press, London. 7:
263-287(1991)
Rohmcr, VI., Knani, M.. Simonin, P.. Sutter. B., Sahm, IE: Isoprenoid Biosynthesis in
Bacteria: A Novel Pathway for the Early Steps Leading to Isopentenyl Diphosphate.
Biochem. J 295: 517-524 (1993)
Eisenreich, W., Menhard, B., Hylands, P. E, Zenk, M. FE, Bacher, A.: Studies on the
Biosynthesis of Eaxol: The Eaxane Carbon Skeleton is not of Mevaloiioid Origin. Proc.
Natl. Acad. Sei. USA 93: 6431-6436 (1996)
Knöss, W., Reuter. B.. Zapp, E: Biosynthesis of the Labdane Diterpenes from Marrubium vulgare viaNon-Mevalonate Pathway. Biochem. J. 326: 449-454 (1997)
130 11 Anthraquinones
11.1 Introduction
Anthranoids are common secondary metabolites. They occur widely m the subclass Asteridae, comprising among others the plant families Rubiaccae, Gesneriaceae and Scrophulariaceae. In these plants they are considered to be biosynthetically derived from shikimic acid and mevalonate. Anthranoids not from the subclass Asteridae, e. g. those which occur in the
Rhamnaceae, Polygonaccae, Leguminosae and in fungi and lichen, are polykctides. Several hundred different anthranoids are knoxvn. differing in the nature and positions of the substituents
(1).
Substances of the anthraquinone type xvere the first to be recognized, both in free state and as glycosides. Further work showed that natural products also contain reduced derivatives of the anthraquinones (oxanthrones, anthranols, and anthrones) and compounds formed by the union of two anthrone molecules (i. e. the dianthrones).
Derivatives of anthraquinones present m purgative drags are dihydroxy phenols such as chrysophanol (Figure 10.1), tri hydroxyphenols such as emodin (Figure 10.1), or tetrahydroxy phenols such as carminic acid. Other groups are often present - for example, methyl in chrysophanol, hydroxymethyl in aloe-emodm and carboxxl m rhcin and carminic acid. Occurring as glycosides, the sugar is attached in various positions.
Since glycosides are often easily hvdrolyzable, the earlier xvorkcrs tended to isolate products of hydrolysis rather then the primary glycosides. The following aglycons have long been established: chrysophanol or chrysophanic acid from rhubarb and cascara; aloe-emodin (Figure
11.1) from rhubarb and senna; emodin or frangula-emodin from rhubarb and cascara; rhein
(Figure 11.1) from rhubarb and senna. Improx-ed extraction methods led to the isolation of the main senna glycosides, sennosides A and B (2).
131 OH O OH
CH3
Figure 11.1 Chrysophanol:R - CH, Emodin: R = OH
Aloeemodin: R - CTEOH
Rhein: R-- COOH
11.2 Biosynthesis
11.2.1 Shikimi Acid-Mcvalonate-Pathxvay
Shikimi acid leads to an intermediate compound that reacts with a-ketoglutaric acid to ortho- succinyl-benzoic acid. This product reacts xvith itself to a naphtohydrochinon derivative and takes a prenyl rest coming from mevalonic acid. Cyclisation to tricyclic compound leads, under oxidative degradation of a methyl group at the prenyl rest and additional oxidation steps, to the anthraquinone derivative alizarine (3), see Figure 11.2.
11.2.2 Polyketidc Pathway
Anthraquinones, dcrixred by the polvketide pathxxav, are formed by the sequential condensation of acetyl CoA xvith sex en molecules malonyl CoA to a C,,,-polyketoacid. After decarboxylation the primary cyclisation product leads to emodinanthrone. whose anthraquinone is emodin.
Emodin and the other compounds of this class arc characterized through a 1.8-dihydroxylation and the in C,-substitution position 3 of the anthracene ring (3). see Figure 11.3.
132 coo ^coo ^coo
coo
a ketoelulai ic acid
fOOII
'"""OH
711,0 H°H>
mevalonic acid
Figure 11.2 Shikimi Acid-Mex alonate Pathway
0 0 0 OH 0 OH
_C0 SCoA COOH
CH, HO CH-, O O O HSCo-X 2H 0 activated Cib-Pohketoautl mtennediate pi oduct
_»• CO,
OH O OH
X
HO ^CH3
Emodmanthioiic Figure 11.3 Polyketide Pathway
133 References
Schripsema, J., Dagnino, D.: Elucidation of the Substitution Pattern of 9,10-
Anthraquinoncs through the Chemical Shifts of Peri-Hydro xyl Protons. Phytochemisiiy
42: 177 - 184(1996)
Evans, W. C: Trease and Evans' Pharmacognosy. Baillièrc Tindall, London, Philadelphia,
Toronto, Sydney. Tokyo: 395 - 418 (1989)
Schneider, G, Hiller, K.: Phannazeutische Biologic. Spektrum, Heidelberg, Berlin 2: 158 -
171 (1999)
134 12 Cyclic Peptides
12.1 Introduction
Cyclic peptides and depsipeptides are rapidly emerging in importance as sources of potentially important new drugs. A great number of cyclic peptides, having interesting biological activities and novel structures have been isolated from various origins. Some of the most exciting natural products discovered in recent years, cyclosporin A (1) and FK-506 (2) from microorganisms, cycloheonamides (3), didemnins (4), and dolastatins (5) from marine invertebrates, are amino acid derived metabolites. Because of their attractive biological profile, didemnins have been the focus of considerable attention. Didemnim B was the first marine natural product to enter clinical trials (6).
Recently, cyanobacteria have been demonstrated to be a rich source of unique and interesting bioactive cyclic peptides. The best-knoxvn cyclic peptides, the microcystins and congeners, have been isolated from various cyanobacteria (Microcystis. Oscillatoria, Anabaena. and Nodularia), and over 40 microcystin-typc peptides have been reported to date. Other prominent examples are laxaphycinc A and B (Figures 4.11 and 4.12), two related antifungal cyclic peptides, isolated from Anabaena laxa CI) or the positive inotrope chlorine containing cyclic decapeptide puwainaphycin (Figure 4.39) from Anabaena sp. (8).
12.2 Biosynthesis
12.2.1 Nonribosomal Biosynthesis ofUnusual Peptides (Thiotemplate Mechanism)
Some natural peptides have very atypical structures, including D-amino acids, unusual amino acids, covalent linkages other than peptide bonds, and acyclic polypeptide chains. Most of these peptides are antibiotics, which inhibit competing or predatory species. In this case, peptides
somewhat different from ordinary proteins - and thus resistant to proteases and other defense mechanisms - have probably been very useful in the biological warfare that takes place between
species.
135 Microorganisms are able to use a simple non-ribosomal method of peptide bond synthesis. While perfectly effective for the peptide formation, the system lacks the highly ordered apparatus provided by the ribosome and tRNA structures. As such, only small peptides are synthesized by this means. The antibiotic gramicidin S (Figure 12.1), a cyclic decapeptide from Bacillus brevis, providing an important example.
Most peptide antibiotics are synthesized on large, multifunctional enzymes that cany out all the steps. ATP generally activates the carboxyl groups of the amino acids by forming amino acyl adenylates as used m ribosomal synthesis. Subsequent binding of the amino acids to an SEEgroup of an enzyme via a thiol ester is more likely than to a tRNA. Either D- or I,-amino acids can be used because the enzymes can catalyze their isomerization. The activated ammo acids are then added sequentially to the groxving polypeptide chain, w hich is attached as a thiol ester to the -SH group of the cofactor 4'-phosphopantcthein. The co factor is thought to function as a swinging ami to deliver the growing peptide to different sites on the enzyme where the various amino acids are added sequentially. The enzyme determines the sequence of the peptide generated, presumably by the amino acid specifities of the sites that add the residues.
The enzymes required to produce peptides of 6-15 residues in this way are very large, consisting of multiple polypeptide chains with molecular w eights from 100 000 to 450 000. This type of mechanism of peptide synthesis would clearly be impractical for proteins in general; each protein would require a larger protein to make it. Nex ertheless such a mechanism is apparently practical for the biosynthesis of a few atypical peptides.
There is a limit practical to the size of the peptide that could be made in this way. Unusual
than peptides larger about 20 residues are made on ribosomes in the usual way, using the normal
20 L-amino acids, and are then modified posttranslationally (9).
The - biosynthesis of gramicidin involves two enzymes, a light enzyme (MW 10 000) and a heavy (MW 280 000).
Quaternary interactions between the two enzymes allow the two amino acids to get close enough for peptide bond formation. This process is repeated five times to form the txvo halves of the molecule. The protein complex connects the two identical chains to give finally the cyclic peptide.
136 D-Phc L-Pio -*- L-Val L Om -*- L~I eu
I -I cu L-Oni L-Vdl -«- 1 -Pio ~* D-Phc
,NH3
- Om = ornithine Hi 0 G 0 CH H; H2 H, V
COO
Figure 12.1 Gramicidin S
Synthesis begins on the light enzyme, which also functions as a 'racemase', converting L- phenylalanine to the D-enantiomer (Figure 12.2). A thiol nucleophil on the light enzyme attacks an activated phenylalanine (ATP and the amino acid reacted to an anhydride) to give a high- energy thiol ester.
,NH^ / NH3 ,C CH •SH - \ S G-— CH CH- CH2
- \MP
Ad V^ K OH OH
Figure 12.2
- In the next step, proline attached to the heavy enzyme by a thiol ester linkage - attacks the light enzyme, thus transferring the dipeptide to the heaxy enzyme. The function of the light enzyme is completed (Figure 12.3).
137 Figure 12.3
Similarly, on the heavy enzyme, the amino function of valine (attached as thiol ester) attacks the dipeptide, fomiing a tripcptide product. The energy for peptide bond formation is being derived from the aminolysis of the thiol ester (Figure 12.4).
Ehe next step involves an 'arm' on the heavy enzyme. This arm attacks the tripcptide via its thiol function. It is then attacked by Ornithin to form a tetrapeptide. picks up the tetrapeptide (via the same thiol) and is attacked by leucine to form a pentapeptide. The arm retrieves the pentapeptide, but this time the pentapeptide is attacked by a complementary pentapeptide (attached to another arm) to give rise to a cyclic decapeptide (9).
Prer-^Phe ,SH
,NH~
,s—C—Cl -Vah—pro—phe
-CH,
CH,
Figure 11.4
138 The thiol containing 'swinging arm' in the heavy enzyme consists of ß-mercaptoethylamine and panthothenic acid, esterified to a serine phosphate of the enzyme (Structure 12.5), (9).
O CH, OH O CH2
Figure 12.5
123 References
1. Wera, S., Belayexv, A., Martial, J. A.: Rapamycin. FK506 and Cyclosporin A Inhibit
Human Prolactin Gene Expression. FEES Lett. 358: 158-160 (1995)
2. Tanaka, TE. et al.: Structure of FK506: A noxel Immunosuppressant Isolated from
Streptomyces. J. Am. Chem. Soc. 109: 5031-5033 (1987)
3. Fusetani. N., Matsunaga, S.: Cyclotheonamidcs. Potent Thrombin Inhibitors, from a
Marine Sponge Theonc/ts sp. J Am. Chem Soc. 112: 7053-7054 (1990)
4. K. I J. J. S. Rinehart, ., Gloer, B., Cook, C. Mizsak. A.. ScahiU, T. A.: Structures of the
Didemnins. Antiviral and Cytotoxic Depsipeptides from a Caribbean Tunicate. J. Am.
Chem. Soc. 103: 1857-1859(1981)
5. Pettit, G. R., et al.; The Isolation and Structure of a Remarkable Marine Anomal
Antincoplstic Constituent: Dolastatin 101a../, Am Chem. Soc. 109; 6883-6885 (1987)
6. Kodani, S., Ishida, K., Murakami, M.: Dehydroradiosumin. a Trypsin Inhibitor from the
Cyanobacterium Anabaena cvlindrica. J Xat. Prod. 61: 854-856 (1998)
7. Frankemöhlc, W. P., et al.: Antifungal Cyclic Peptides from the Terrestrial Blue-green
Algae Anabaena iaxa. Isolation and Biological Properties. J. Antibiot. 45: 1451-1457
(1992)
8. Moore, R. E.. et al.: Puxvainaphycin C, a Cardiosciective Cyclic Peptide from the Bluc-
Grcci\ klg-àc Anabaena BQ-lbA. J im. Chem. Soc 111 : 6128-6132 (1989)
139 9. Creighton, T. E.: Proteins - Structures and Molecular Properties. Freeman, New York:
100-102(1997)
140 13 Introduction
The experimental part describes the results of the biological screening of cyanobacteria and the detailed chemical and biological investigations of the culture medium and the cell material of
Nostoc commune (EAWAG 122b) and of the culture medium of Tolypothrix byssoidea (EAWAG
195).
141 14 Extraction
14.1 Material for the Biological Screening
'The lyophilized cyanobacteria cultivated for the biological and chemical screening (3 - 4 g dry material) were mazerated with DCM/MeOH 2:1 (extract A), followed by McOHTEO 7:3 (extract
B). The solvents were removed at ambient temperature (30 °C) in vacuo. Crude extracts were stored in the freezer at -80 °C.
The lyophylizcd cells of the fixe field collected samples were treated the same.
14.2 Materialfor Phytochemicai Investigation
The large-scaled cultured and freeze dried cyanobacterial cells of Nostoc commune (EAWAG
122b) and Tolypothrix byssoidea (EAWAG 195) were homogenized in DOM/McOH 2:1 using an
Ultra-Turax® (Janke and Kunkel). The resulting suspensions were filtered and the wet material was mixed with about 1000 g of quarz sand. This mass xvas then filled into a MPLC column
(Beech, 80 x 5 cm) containing 1 cm of quartz sand at the bottom. After filling, the top of the column was covered by a second layer of quarz sand and the more polar solvent (DCM/MeOEI
2:1, extract A) was pumped through the column by means of an MPLC pump (Eabomatic 2-4 bar). Each time the color of the solvent became brighter, the pump was stopped for a few hours to allow maceration and afterwards restarted. After three days, the extraction solvent was changed to McOEl/IfO 7:3 (extract B) and processed the same manner.
Removal of the solvents at ambient temperature (30 °C) in vacuo yields two extracts for each cyanobacterial strain. Crude extracts were stored in the freezer at - 20 CC.
142 15 Biological Screening
Extract A and B of the 43 cultured cyanobacterial strains as well as the five field collected samples were screened in in-house bioassays for their antimicrobial and moUuscicidal activity, brine shrimp lethality, and cytotoxicity. The results of the biological screening of the 43 cultivated strains are discussed in publication 1 (Chapter 18). Assignments and results of the field-collected samples and their isolates are outlined in Table 15.1, 15.2. and 15.3.
Extract A and B as well as the MeOH- and DCM-cxtracts resulting the Amberiite elution of the large scale cultured strain Nostoc commune (EAWAG 122b) were submitted to assays against
Trypanosoma rhodesiense. T. cruzi. Leishmania donovani, and Plasmodium falciparum, performed at the Sxviss Tropical Institute in Basel, (Sxxitzcrland). The results are listed in Tables
15.4.
Table 15.1 Assignment of the field collected samples and the resulting isolated strains Sample Isolated Strain (s)
1 256 Oscillatoria amocna 257 Oscillatoria limosa
2 258 Oscillatoria tenuis
3 259 Nostoc commune 4 260 Oscillatoria formosa 261 Phormidium favosum 5 262 Lvngbya putcalis
Ehree isolated strains (258, 259, 262) lost their bioactivity completely. The bioactivities of the
strains 256, 257. 260, and 261 are comparable xxith the results from the field-collected samples
they were isolated from.
143 Table 15.2 Biologicalactivitiesof the field-collectedsamples Sample Extract ilAntib acte rial/an tifungalactivity bMolluscicidal cBrine shrimp activity leth ali ty/dcytotoxicity B.c. S.e. P.a. E.c. C.a. Biomph.glabr. B.s. KB Caco-2 0.6 mg 0.6 mg 0.6 mg 0.6 mg 0.6mg 100 ppm 500 ppm 50 pmni 50 ppm 1 A
B f ++ 2 A + + B
**> z> A T 4 -\ B 4 A + B + J_ 5 A ++ 66% 4- B 4 4 control 5 5 fig JLlg 5 Mg 5 fig 5 Mg Cc 4-4-4- +++ Tc + +++ Mc J L Pt 4-4- 100% 4- + A = dichloromethane/metlianol 2:1 extract; B = methanol/water 7:3 - = - extract: Cc Chloramphenicol; Tc Tetracycline: Mc = Micona/ole: Pt Podophyllotoxin: '*+ = zone of inhibition 2-6 ++ = of inhibition 6 b++ - total mm; zone > mm; lethalityof all snails: c(%) = lethality; % = inhibition;B.c. - Bacillus cereus: S.e. = P.a. = Pseudomonas E.c. = Escherichia Staphylococcusepidermidis: aeruginosa; coli; C.a.= Candida albicans; Biomph. glabr.= Biompludaria glabrata;B.s. - Brine shrimp Table 15.3 Biologicalactivitiesof the isolated strains Strain Extract "Antibacterial/antifun gal activity bMollusckidal cBrine shrimp activity Iethality/dcytotoxicity B.c. S.e. P.a. E.c. C.a. Biomph. glabr. B.s. KB Caco-2 0.6 mg 0.6 mg 0.6 mg 0.6 mg 0.6mg 100 ppm 500 ppm 50 pmrn 50 ppm 256 A .p B 4-4- 257 A B 4- A 258 B 259 A B 260 A
B -t 4 14 £ 261 A „ B 262 A 4-4- 66%, B 4-
control 5 fig 5 pg 5 ug 5 fig 5 fig Cc 4-4-4- Tc 4- 4-4-4- Mc 4-4- Pt 4-4- 100%, 4- ~ A - dichloromethane/melhanol 2:1 extract: B tnethanoPwater 7.3 extract; Cc = Chloramphenicol: Tc = Tetracychne: Mc = Miconazole: Pt = Podophyllotoxm: total a4- = zone of inhibition 2-6 mm: ++ = zone of inhibition > 6 mm: l+~ = lethality of all snails: c(%) = lethality:c • = inhibition; B.c. - Bacillus cereus; S.e. = Escherichia = = Staphylococcusepidermidis;P.a. = Pseudomonas aeruginosa; E.c. = coli: C.a.= Candida albicans: Biomph. glabr. Bwmphaiaria glahraia:B.s. Brine shrimp Table 15.4 Antiplasmodial activities Strain Extract T. rhodesiense T. cruzi L. donovani P. falciparum TCS0
122b A n. a. n. a. n. a. 6.0 (Kl); 2.6 (NF 54)
122b B n. a. n. a. n. a. n. a.
122b C n. a. n. a. n. a. n. a.
122b D n. a. n. a. n. a. n. a. Control mclarsoprol MIC-0.011; lC5iJ - 0.0004 benzmdazol MIC -3.7 pentostam ICS0 30
= T. rhodesiense = Trypanosoma rhodesiense; T. cruzi = Trvpano^ona cruzi; L. donovani Leishmania donovani; n.a.
= = = = extract of the Amberiite no activity; A DCM/MeOH 2:1 extract; B MeOH H;0 7:3 extract: C MeOH elution;
D = DCM extract of the Amberiite elution
146 16 Phytochemicai Investigation of Nosctoc commune (EAWAG 122b)
16.1 Fraction ait on and Isolation
Fractionation and isolation were carried out with the MeOH extract resulting from the Amberiite
elution of the culture medium (extract C) and xxith extract B of the cell material. Phytochemicai investigations of these extracts furnished nine compounds. Eight of them were novel to the
literature and one xvas new as a natural product.
VLC on normal phase material (Si gel) for extract B or Sephadcx LH-20 open column
chromatography for extract C, respectively, xvas utilized for initial fractionations. For purification of the fractions prior to HPLC separations normal phase VLC and open column chromatography with Si gel were employed. Final purifications x\ere carried out using HPLC monitored with UV detection (A, 254 nm). TLC was successively used for the selection of the eluent for isocratic
HPLC separations. The optimized mobile phase was then transferred to scniipreparativc HPLC, via an analytical HPLC prerun. Fractionation procedures were conducted by the help of bioactivity-guided fractionation (antimicrobial assays), TLC and 'H NMR.
16.1.1 Fractionation of Extract B
Publication 4 (Chapter 21) describes the work-up of the MeuH/H,0 (7:3) extract in detail. A flow chart including the isolation procedure of all isolated metabolites is given m Figure 16.1.
16.1.2 Fractionation of Extract C
Fractionation and isolation procedure of the MeOH-soluble material of the Amberiite elution is described in publication 2 and 3 (Chapter 19 and 20). A complete isolation protocol for the metabolites of this extract is illustrated on a separate floxv chart (Figure 16.2).
147 Cell Material Nosctoc commune (EAWAG 122b) 43 g
DCM/MeOH2:1 MeOH/H20 7:3 Extract Extract Extract A Extract B 6g 3g
VLC step gradient Silica Gel hexane/EtOAc
hexane/EtöAc hexane/EtOAc 60:40 30:70
Pf, | -4 Fr. 5 Fr. 6 Fr. 7-11 90 mg 80 mg
Open Column CHCI3/MeOH Silica Gel (45:55) Open Column CHCI3/MeOH Silica Gel 30:70"
Fr. 5.1-5.4 Fr. 5.5 Fr. 5.7-5.8 30 mg CHCI3/MeOH 30:70
RP HPLC MeOH/H20 Fr. 6.1-3 Fr. 6.4 Fr. 6. 65:35
RP HPLC MeCN/H20 60:40
NC-2 NC-8 NC-9 4 mg 3 mg 3 mg
Figure 16.1 Isolation protocol for the cell material of Nostoc commune (EAWAG 122b)
148 Culture Medium Nostoc commune (EAWAG 122b) 85 L Amberiite XAD-2 LMeOH 2. DCM
MeOH Extract DCM Extract Extract C Extract D i-2g 0.6 g
step gradient MeOH/H20/acetone
MeOH/H20 MeOH/H20 MeOH/H2C 40:60 90:10 75:25
Fr. 1-6 Fr. 7 Fr'9 Fr. 10-13 Fr! 14 Fr. 15-16 Fr * 8 79 40 50 mg m9 mg
RPHPLC MeOH/H20 RP HPLC MeOH/MeCN/H20 60:40 63:25:12
RP HPLC MeOH/MeCN/H20 63:25:12
NC-t> NC-7 3 4 mg mg NC-5 3.5 mg
Fr. 9.1 Fr. 9.2 Fr. 9.3
NC"1 RP HPLC MeCN/H20 8m9 80:20
N C-3 NC -4 8 mg 9 mg
Figure 16.2 Isolation protocol for the culture medium of Nostoc commune (EAWAG 122b)
14b 16.2 Structures of the Isolates
As mentioned before, the phytochemicai inx'cstigation of the large-scale cultured cyanobacterial strain Nostoc commune (EAWAG 122b) yielded nine compounds. Seven of them, NC-i - 7, comprise two unprecedented diterpenoid skeletons. NC-8 is a new unusually substituted anthraquinone and NC-9 an indan-denvate that is not x et reported as a natural compound.
In order to facilitate the structural comparison of the isolates the diterpenoids are presented altogether in Figure 16.3 - 16.6, irrespective of the number of the publication or the extract from which they were isolated.
The non-diterpenoid compounds are shown in Figure 16.7 and 16.8. A general overview of the isolates is given below (Table 16.1).
Table 16.1 Key for the isolates of Nostoc commune (EAWAG 122b)
Compound Trivial name Novcltv Isolated from Publication no. extract NC-1 Noscomin A noxel C 2
NC-2 Noscomin B novel c 4
NC-3 Coninostiii A noxel c 3
NC-4 ComnostinB noxel c
NC-5 Comnostin C novel c *>
NC-6 Comnostin D nox el c z>
NC-7 Comnostin E novel c 3
NC-8 _ nexx B 4
NC-9 - nexv as natural prod. B 4
150 The diterpenoid part of the skeleton of NC-1 and NC-2 represents a phenanthrene derivative, which is connected with a/wra-substituted aromatic ring. Compound NC-1 is connected via a
CH2-group at position 14, while compound NC-2 has two connections to the aromatic ring, a
CIC-bridge at position 14 as xvell as an ether connection between C-7 and C-21, and therefore comprises an additional 7-ring.
Figure 16.3 NC-1 Figure 16.4 NC-2
The diterpenoid skeleton of NC-3 - 7 represents a naphtalene derivative, xvhich is connected to the saine^ara-substituted aromatic ring, xvhich can be found in compounds NC-1 and NC-2. It is connected via a CFE bridge at position 13. NC-3 -7 are very closely related compounds. NC-3 shoxvs an alcohol moiety, NC-4 an aldehyde instead of the carboxy group in position 23 of NC-5.
NC-6 reveals an acetal group at position 23. It is possible that the acetal is generated by methylation of the aldehyde group of NC-4 during the extraction procedure xvith MeOH.
Therefore NC-6 may be an artifact of isolation and not a genuine natural product.
Compared with NC-3. NC-7 lacks the methyl group at C-3. An acetyl group replaces the secondary alcohol moiety. 27 ÇOOH
5 ~
H3COC H 21
NC-3: R = CH,OH, NC-4 R-CHO
NC-5: R-COOH; NC-6 R - CH(OCH,);
Figure 16.5 NC-3 - 6 Figure 16.6 NC-7
The 1,4-substituted NC-8 and the 4.7-substituted anthraquinone indan-derivate NC-9 comprise a similar unusual substitution Most of the natural pattern. derixed anthraquinones shoxv 1.2- or 1,3- instead of 1,4-substitution of ring A.
16.7 Figure NC-8 Figure 16.8 NC-9
152 16.3 Structure Determination
Structure determination was carried out xvith spectroscopic and spectrometric (UV, IR, EIMS,
ESIMS, ID and 2D NMR) as well as xvith physical methods (optical rotation and X-ray ciystallography). Complete experimental data of the presented compounds NC-1 - 9 are reported in publications 2. 3, and 4 (Chapter 19. 20, and 21). A more detailed description including NMR and MS spectra of the different structural types is gixren in this section.
16.3.1 Structure Determination of NC-1
The 'H NMR spectrum contained signals of fix-e methyl groups, four tertiary (H-,-22, H-,-23, FFr
24, If,-25), one secondary (H,-26) as well as signals indicating a 1,2,4-substitutcd aromatic ring
(11-17, 11-19, FI-20). Furthermore one proton attached to an oxygenated carbon (Fl-3) and an olefinic CH resonance (CII-l 1) could be detected in addition to a number of aliphatic signals. 'H
NMR spectrum measured in CD-,OD and the signal assignment of NC-1 are shoxvn in Figure
16.9.
In the nC NMR spectrum a total of 27 carbon atoms could be observed. The spectrum shows the presence of one carboxy group (C-27), one tertiary aliphatic oxygen-substituted C atom (CFf-3), and one C-:C double bond (C-9, CF1-11). flic observation of three low-field quaternary carbon signals (C-16, C-18, and C-21) and three mcthine carbon signals (CH-17, CFt-19, and CFf-20) confirmed the presence of a tri substituted aromatic ring (see Figure 16.10).
The TOCSY spectrum revealed spin system A (IF-26, Fl-13. FI,-12, and H-l 1), spin system B
(FT-1, FC-2, and 1T-3). and spin system C (II-8, 11,-7. FF-6. H-5). TOCSY correlations of NC-1 arc presented in Figure 16.11.
The three spin systems were assembled by an HMBC experiment. The correlations are shown in
Figure 16.12 and 16.13. The relative stereochemistry was deduced from a TROESY experiment.
153 The structure was confirmed with ESI- and El-MS. NC-1 gave the [M-H]" ion peak at m/z 425.2 by ESI-MS. An El-MS spectrum revealed the molecule fragments.
154 h3-24
23 22 H3-23
H3-26
H?-12 1 H?2 H, ?, H2-12 \ \ Hr15 H?-1 \ H?J5i
If II i-a A' v '\J V -A 'J A/ r-l » V ^,
3. 0 2.5 2. 0 1.5 1.0 ppm
H-19 H-20K tj yj-LM Ä~V' v^jU^ÄÄ
Figure 16.9 1H NMR spectrum oi NC-1, CD3OD, 300 MHz, 298 K CH2-7
, CH3 22 CH2 12 CH21 CH325
ÇH3 24 CH^ 2 I ,C14 I 15 I CH, ÇH13 I CHse CHj^1! ' \ 1/
l,^',' Jy fo-Viv^y.;*h\ l"AlVji
?3 22
CH 19
CH 17J ^
ß%fV^#w N#W^Vi*^'^''1^
169 140 130 3 0 71 170 150 120 110 100 90 70 60 50 40 iG ?0 prjro
Figure 16.10 isC NMR spectrum of NC-1, CD3OD, 75 MHz, 298 K i !
iuOl aJ ^\flj
spin syslern A H326 co spin system C spin system E jß#' 5 H27 1 r-,»c^ r î> H2 1 W'I rp*çn «m « H13 ! H212 f)H22
': H2 1 A' A-, Ö 9 HO«,,,; #
J) o ^ rf
.4.5
60- .5.5
5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm
Figure 16.11 Part of TOCSY spectrum of NC-1, CD3OD, 300 MHz, 298 K H2-15 Hî-15
;r15/CH3-25 _ 20
H2-15/CH 13
———- H2 15/CH !
Jjj 15/C 16 23 ?2 Ü2 15/CH 17
H2-15/C-21 H2-I5/C-21
3.0 2.9 2.7 2.6 2.5 2.4 ppm
Figure 16.12 Part of HMBC spectrum of NC-1, CD3OD, 300/75 MHz, 298 K ppm
H 5/CH3 23 15 H325/CH, 26 j
^20
H 5/CH3 24H2 I/CH3 24 25
H3 23/CH3 22
30 H3 26/CH2 12
H3 26/CH 1j 25 ru zr^z> H, X~jt 35
H3 25/CH? 15 r
8 H2 7/CH 8 H3 26/CH 8 H,2R/CH H3 22/C 4 23/C 4 -" *-, ' H3 H3 24/CH2 1 ~
H5/CH324 H_5/ÇH322 H5/CH3 23
-50
r i
1.4 1.3 1-2 1.1 1-0 0.9 0.8 0.7 0.6 ppn>
Figure 16.13 Part of HMBC spectrum of NC-1, CD3OD, 300/75 MHz, 298 K 16.3.2 Structure Determination of NC-2
The 'II NMR spectrum contained signals of five methyl groups, four tertiary (FI,-22, Hr23, H-,-
24, 11,-25)), one secondary (11,-26) as well as signals indicative of a 1,2,4-substituted aromatic ring (FI-17, Ff-19, H-20). Furthermore three protons attached to an oxygenated carbon (H-3, H-7 and FI-11) could be detected in addition to a number of aliphatic signals. The 'FI NMR spectrum measured in CD,OD and the peak assignment of NC-2 are shown m Figure 16.14.
In the nC NMR spectrum a total of 27 carbon atoms could be observed. The spectrum shows the presence of one carboxy group (C-27), three tertiary aliphatic oxygen-substituted C atoms (CH-3,
CH-7, CII-11), and one C-C double bond (C-8. C-9). The observation of three low-field quaternary carbon signals (C-16, C-18, and C-21) and three methmc carbon signals (CTI-17, CH-
19, and CFI-20) confirmed the presence of a tnsubstituted aromatic ring (see Figure 16.15).
The TOCSY spectrum revealed spin system A (Fl,-26. H-13, H?-12, and FI-11), spin system B
(H2-l, FF-2, and 11-3). and spin system C (H-7. 11,-6. H-5). TOCSY correlations of NC-2 are presented in Figure 16.16.
The three spin systems were assembled by an HMBC experiment. The correlations are shown in
Figure 16.17 and 16.18. The relative stereochemistry was deduced from a TROESY experiment.
The structure was confirmed with FSI- and El-MS. NC-2 gave the [M-Hf ion peak at m/z 439.1 by ESI-MS. An El-MS spectrum re\ealed the molecule fragments.
160 CO A>
C7
-o
x —=_^_
c^ _
en _
>fc» i
1 1 U) 1 Ï \
Ki _:
i~i
S ^ S
161 CH322 /C4 CH212 ch .CH2 ct324 r" Ch2K | F.CH325 g L-CH 3 N fi [^CH25^ ,,CH„2
* *-' i ' > "* i ["'fA1'"-" * i •• li'' H0/„, 'JUCOOH CHI 7 CH19 CH3 C '8 C27 C21 C^j ^ II 160 350 j41 l->0 12J Jlj j.Jj 90 80 / 60 50 O 30 J ,[m Figure 16.15 «C NMR spectrum of NC-2, CD3OD, 75 MHz, 298 K HO/,,, __j \ Pï>m spi^ system A B Js H 26 spir s/sterr H? 1d ^=~ i C sprn System çj W, i2 - H 5 J =p ^5 H26b 2.0 Ö H26a H2 la m —~~-fp^'— — =/»- p 3.5 •I <*- , ~_ T 5.0 4.8 4.6 4.4 3.6 3 .2 ppm Figure 16.16 Part of TOCSY spectrum of NC-2 CD30D, 300 MHz, 298 K H2-15 A l\ H215 / V ppm HiJI£tt25 ulU'S!"1»25 H215/CH3J5 h215/ch325 [ 30 H215/CH13 H2 15/CH 13 H, 15/C 14 15/C Hz M H?J5£14 H2 15/C 14 _ 40 . 50 f I 60 t r- 70 I I 80 SO 100 1 no L120 H215/C16 H2 15/C 16 H2 15/C 16 H 15/C 16 H2 15/CH 17 Ha 15/CH 17 H2 15/CH 17 H, 15/CH 17 [130 H2I5/C8 H^JSICS H2 15/C 8 H2 15/C 8 1 140 -150 1 f 160 15/C 21 H2 H2 15/C 21 H2 15/C 21 H? 15/C 21 1 2.80 2.75 2.70 2.65 2.60 ppm 16.17 Part of HMBC spectrum of NC-2, CD3OD, 300/75 MHz, 298 K H3 26 ri322 \ \ H 25 Hj24 JWC ppm 15 i-h 22/CH3 23 20 -25 ri 23/CHj/2 HO,, 30 10 Hj2f/CH 3 H^5/CH13 3 o 35 Hj 24/CH2 1 H3£5/CH 15 H» 26/CH2J2 |"J>H3 2J/C 4 40 H3 26/C 14 M3H325/C^5/ 4 H3 24/C 5 H3^3/C 5 Hj 22/C 5 45 O 0> O 50 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 O.t 0.7 ppm 16.18 Part of HMBC spectrum of NC-2, CD3OD, 300/75 MHz, 298 K 16.3.3 Structure Determination of NC-3 - NC-7 The ]H NMR spectrum of NC-3 contained signals of five methyl groups, four tertiary (11,-21, II,- 22, H,~25, H,-24), one secondary (H,-26) as well as signals indicative of a 1,2,4-substituted aromatic ring (H-16, Fl-18, Fl-19). Furthermore, the methylene protons of a secondary alcohol group (H2~23) could be detected In addition to a number of aliphatic signals. The 'H NMR spectrum measured in CO(OD,): and the peak assignment of NC-3 arc given in Figure 16.19. In the nC NMR spectrum a total of 27 carbon atoms could be observed. The spectrum shows the presence of one carboxy group (C-27) and a secondary alcohol moiety (CFF-23). The observation of three low-field quaternary carbon signals (C-15. C-17, and C-20) and three low field methine carbon signals (CII-16, CH-18, and CIi-19) confirmed the presence of a tri substituted aromatic ring (see Figure 16.20). The TOCSY spectrum revealed spin system A (FE-26, H-12. FF-11, and Hr10), spin system B (H,-9, H2-l, and FF-2), and spin system C (H-7, IF-6, FF-5). TOCSY correlations of NC-3 are given in Figure 16.21. The three spin systems were assembled by an HMBC experiment. The correlations are shown in figure 16.22 and 16.23. The relatrve stereochemistry was deduced from a TROHSY experiment. The structure was confirmed with ESI- and FI-MS. NC-3 gave the [M-IIp ion peak at m/z 427.2 by ESI-MS. An El-MS spectrum revealed the molecule fragments. NC-4 -NC-7 are closely related to NC-3. 'FT and nC NVIR spectra and Ihc peak assignments are shown in Figure 16.24 - 16.31. 166 _3 3 ce o c 3 167 y i v 2 I *- \ o "o c CD Q_ o Ol ID 3 168 H26 M2 b t\ \.A u / N J'^' \j L^- ppm ~i [ J. 4 _0.6 spin system C H 7 H 7 „ a _0.8 i _1.0 _1.2 i län% '-' i -x 1.4 I 1.6 i _1.8 2.0 ! I ' J !) . 2.2 --9 . 2.4 \ ^ rmcJK L2.6 1 1 _2.8 _3.0 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 ppm Figure 16.21 Part of TOCSY spectrum of NC-3, CO(CD3)2, 300 MHz, 298 K H2-23 H2-14 H2-14 ./ .J feiâffih!3-25 ÜSÜEÜ3-25 i- 20 H2-23/CH3-22 j L 30 Hj^23/ÇH2-2 H2-14/CH-12 H2-14/CH-12 r Ä£4 L 40 [- 50 "- 6 0 70 l o-J 80 .- 90 ^100 I 110 \. I t_120 H2-14/C- H?-14/C-17 |_130 H2-14/CH-16 H214/CH-16 Ll40 ^150 f H2-14/C-20 H2-14/C-20 L160 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 Figure 16.22 Part of HMBC spectrum of NC-3, CO{CD3)2, 300/75 MHz, 298 K e a, a, v o\J "Of I ~5 f? x) it îloifejj „-, tA 6X Ü M O r- X O o g© - xf\T C) 5(0° y X et X 5|\ CO CM ^ w n Y x fi :~Uj fi (9x _ 171 CO CO CM N I o o m Q Ü O ü 6 z o E o CD Q. CO 3 0) 172 — -*• »— CM „ 173 il /H324 Ha23'[ HOOC? \7i H321 23 Ha2f 2 H 9 H,2 H25 , H25 H26 pA.y\ 7 I f {.'J H/ 14 H2 6 'if I )J d \ - " i t —i r r —r r1 t r — 3 0 ? 9 2 3 21 2 S 2 5 2 1 2 3 2 2 2 1 ppn • CH 19 - CH-16 s CH 18 Figure 16.261H NMR spectrum of NC-5, CD3OD, 300 MHz, 298 K /CH2 14 CH322 ^CH325 CH,24\ /CH3 26 /CH 12 CH CH24l N 1 2 i, %%^!kii 1, f\if VA yfipf'ft'o^fJtiJ^ß^ HOOC r2 21 23 L^-CH 9 A'AAj ' .' A"* ''A>w.fV/^Ä 5 < S i) Û3 J b EtC OH 18 C-?3 C 27 ^ I VMii»^/|N^M^Wft'Wl*4fy II/| H4 Kf'^#tW' iV ?#,*" 70 .60 150 140 30 120 110 100 70 60 40 30 20 Don Fig.16.27 13C NMR spectrum of NC-5, CD3OD, 75 MHz, 298 K H326; -H322 -H327 Z3CH 1 H.,28 H3Cg'AcH3 2 H25 XI 1 'to H22 H25 > l\J A/ lJ Ns 6 i h i H? ,mAcA KU l/l \ > I rf f / / 0 i « a- H3 25 /, 14 _aA_A/_ 42 40 38 3o 34 32 3 0 2 8 2 5 P'îm r "I I,III ! |i -,_JW ppm Figure 16.281H NMR spectrum of NC-6, CD30D, 300 MHz, 298 K OH2-14 /CH3-26 CH3-27^/CH3-2I ,XH2-10 /CH3-28 ^CH: CH, 22 CA| CH2-6 0H2-E 'CH2-11 /CH22 C-13 C-8 ] \ i li I 23CH^22 iätäwhWV^*0$M H3Cg OCH3 20 sût ^f^fm0pf-ßßf$/ m 51 St Si S3 56 55 5i 5! pjr C-15 C 17 ^/^fil^f^ yètfmt^wti 170 160 150 140 130 120 110 100 90 BO 7C 50 50 40 30 20 ppir Fig.16.29 «C NMR spectrum of NC-6, CD3OD, 75 MHz, 298 K S1 '_TZZD -J> -fi ,-rs I o CO D) 178 H,COC H ?1 22 CH326 " CH325 CHJ l,| CH/10/.CH?2 rCH*>AAHA , kCCH25 24 NI', ri, QHf ^CH323 ICHAI Äff ilirVt,V¥w^/Ji^ VH#,Jt>'w«l'',*!ïWJ Uw V'VJ/^W* V1-'/i'V./- 19 Cj-!18 /CH M^i ,C 15 ty^flflfty^^ MliMpPi 220 200 ISO j.60 140 20 100 BO 60 ' 0 Fig.16.31 13C NMR spectrum of NC-7, CD3OD, 75 MHZ, 298 K 16.3.4 Structure Determination of NC-8 The 'H NMR spectrum contained signals for one methyl group attached to an aromatic ring (Hr 11) as well as two aromatic doublets (H-2 and H-3) and three aromatic double doublets (H-5, H- 6, and H-7). The 'H NMR spectrum measured in CO(CD-3>> and the signal assignment of NC-8 are given in Figure 16.32. In the nC NMR spectrum a total of 15 carbon atoms could be observed. The spectrum shows the presence of one methyl group (CH,-11). two carbonyl groups (C-9 and C-10). and Iwo aromatic oxygen substituted carbons (C-1 and C-8). Additionalh'. four low-field quaternary carbon signals (C-4a, C-8a, C-9a, and C-lOa) and five low-field methine carbon signals (CH-2. CH-i, CI 1-5, CH-6, CH-7) could be detected (see Figure 16.32). The structure of NC-8 was assembled by means of HMBC correlations (see Figure 16.33). NC-8 gave the [M+Tl]" ion peak at m/z 255.2 by FTMS as well as a fragmentation pattern that confirms the structure of the molecule. 180 °c i - O/ 7 X o < ' 1 * 3» 4 i i ".} 1 r JA^A x "A " CO A^< (J) CM 3: N Î I ö o CO r~ 3. Q ^A O O Ü cd 1* O °L z cm m o Co ro I I o t CD o- -Ü - A V Q. CO CC a> / \*t =/en °\= o= =0 Ü \ ______/ra CO ? \t~ I / \ o- if> DC h- © CM CO .81 Oi o CM ro -«ai m ^o t~~ co en _ q. OlgHO O T-t H O t-HO H O H O tH O H O »HO g g »Z~x f- O c/" ?p Ie y.\M^i*fW^^^ 182 16.3.4 Structure Determination of NC-9 The 'H NMR spectrum contained signals for one methyl group attached to an aromatic ring (Hr 10) as well as two aromatic (H-5 and H-6)) and two aliphatic (IL-2 and H2-3) doublets. 'H NMR spectioim measured in CDX)D and the peak assignment of NC-9 are given in Figure 16.34. ' In the 'C NMR data were assigned indirectly by anah/ing HMQC and HMBC spectra The structure of NC-9 was assembled by means of HMBC correlations (see Figure 16.35 and 16.36) NC-9 gave the [M+H]" ion peak at m z 163.1 by EIMS as well as a fragmentation pattern that confirms the structure of the molecule. 183 OH -H3-f 4^ If" Ik ^ W/Jf Vs Aw <-/ fj ^J '.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 ppm Figure 16.341H NMR spectrum of NC-9, CD3OD, 300 MHz, 298 K ppm 20 H2-2/CH2-3 40 _120 H3-8/C-7 -140 OH H3-8/C-5 j« H3-8/C-8 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 ppm Figure 16.35 Part of HMBC spectrum of NC-9, CD3OD, 300/75 MHz, 298 K !! JA- ppm -110 1115 -120 CH -125 H-5/C-7 -130 .135 -140 OH -145 1150 1155 -160 H-6/C-4 -165 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 ppjn Figure 16.36 Part of HMBC spectrum of NC-9, CD3OD, 300/75 MHz, 298 K 16.4 LC-NMR Experiments To exclude potential uncertainties from possibly incomplete axenic cultures, the cyanobacterial origin of the medium derived compounds was pro\en by conducting LC-NMR-expcriments on the DCM-McOH (2:1) extract of lyophylizcd cyanobacterial cells. The diterpenoid skeleton could be detected as a major compound in the cell material but in a minor concentration in comparison to the extracellular isolates. A detailed description of the experiments is given in publication 3 (Chapter 20). An LC-NMR chromatogram and the IH NMR spectra of NC-3 and NC-4 are shown in Figure 16.37. 187 peak 1 = NC 3 peak 2 = NC 4 I i CO CO <=> ''IM ff^y\ u (sec) 7 6 5 4 3 Z -1 -2 -3 -i 8 R RI « a s s « (PP») Chromatogram of a fraction containingNC-2 and NC-3 iH NMR of peak 1 and peak 2 16 37 LC-NMR experiments 16.5 Biological Testing and Results of the biological testing are outlined in paper 2, 3 and 4 (Chapter 19, 20, 21). 189 17 Phytochemicai Investigation of Tolypothrix byssoidea (EAWAG 195) 17.1 Fractionation and Isolation Fractionation and isolation were carried out from the MeOH extract resulting from the Amberiite elution (Extract C). Phytochemicai investigations of these extracts yielded two novel cyclic peptides. Initial fractionation was carried out with open column chromatography on normal phase material (Si gel). For further purification HPLC monitored with VV detection (X 220 nm) were employed. TLC was successively used for the selection of the eluent for isocratic HPLC separations. The optimized mobile phase was then transferred to semipreparativc HPLC, via an analytical HPLC prerun. Fractionation procedures were conducted by the help of bioactivity (antifungal activity against the yeast Candida albicans). TLC and 'H NMR. Fractionation and isolation procedure of the MeOH-soluble material of the amberiite elution is described in detail in publication 5 (Chapter 22). A complete isolation protocol for the metabolites of this extract is illustrated on a separate flow-chart (Figure 17.1). 190 Culture Medium Tolypothrix byssoidea (EAWAG 195) 90 L Amberiite XAD-2 LMeOH 2. DCM MeOH Extract DCM Extract Extract C Extract D 0.8 g 0.4 g Open Column step gradient Silica Gel CHCIß/MeOH Fr. 1-5-5 Fr .6 Fr. 7-10 80 mg RP HPLC MeCN/H20 1:1 Fr. 6.1 -6.2 Fr. <3.3 Fr. 30 mg RPHPLC MeCN/MeOH/H20 60:20:20 TB -2 TB -1 3 m g 8 ITig Figure 17.1 Isolation protocol for the culture medium of Tolvpothrix byssoidea (EAWAG 195) 191 17.2 Structures of the Isolates TB-1 is a cyclic tridecapeptidc containing one unusual amino acid designated as didehydro- homoalanine (Dhha), one csterified amino acid (acetyl-threonine, AcThr) and 11 units of naturally occurring amino acids. The absolute stereochemistry was determined with Marfey's method and revealed L-contiguration for nine amino acid residues (Phe, Val', Val3, Vaf, AcThr, Thr', Thr2, Arg, île1, Ile2) and D-configuration for two amino acid residues (Leu and Pro). L-Phe L-lle2 L-lle1 H3' Dhha pH2 H4C-—CH H ;H= «sX r L-Val1 ,CH- c ç N c——,C N 0—-C— NH -cm H H H H '\7 CH L-Thr2 h3c— c- ;h - CH3 OH NH HC—— L-AcThr h C—CH3 H D-LOU h3C~Ç C~—"CH H OH H2 CH3 H ^C^^CH3 L_Thr1 t—^c I H I H C 'CH, HC CH CH, CH3 D-Pro .-Vab L-Val2 HN=i L-Arg Figure 17.2 TB-l 192 TB-2 is also a tri cyclic decapeptide containing didehydrohomoanalinc and 12 naturally occurring amino acids. All ammo acid residues show L-configuration. L-Aig L-Tyr ./ OH H H Dhha ^ CH H _CH, L-Met r L-Val5 r -NH- LI -NH—CH —r—NI Hjf HO- C! H pH—-pH—( n3 L-Val' L-Phe H G CH CH3 H (H3 C=0 — cm—ch3 L-lhr1 L-Val4 H3C HO (H OH O o o o N: „NH —— \\p HN—-~'Ù C"~ 0-— HO—— HN- - H,C CH HoG CH Hü CH HjC CH CIÏ ( H CH) H2 L-Val L-Thr? L-VaP CH L-Ile Figure 17.3 TB-2 193 17.3 Structure Determination 17.3. ! General Structure Detemiination Strategy of Peptides by means of NMR 17.3.1.1 Choice of the NMR Solvent The choice of the NMR solvent depends on the polarity of the peptide. Hydrophilic peptides should be measured in 90% ILO/10% D-.0 at pH 4-5 in order to prevent the exchange of the labile NH protons. Globular folded peptides are changing much slower, because the amid protons are involved in H-bridges or find themselves in the core and are therefore protected from water. Normally the N-terminus has a free NH-group. which exchanges so fast, that the NH-protons of the first amino acid are not detectable. Of course this is not the case for cyclic peptides. Alternative DMSO and CD,OH are good solvents for hydrophilic peptides. A common problem is the aggregation of the peptide molecules due to the high concentrations necessary for NMR measurements. This problem can be reduced by the appropriate choice of buffer or salt addition, respectively. 17.3.1.2 Sequence Specific Sequential Resonance Assignment of Peptides The data obtained from DQF-COSY, TOCSY, and NOESY (ROESY) spectra build the basis of the assignment of peptide proton resonances. The first step is the identification of the spin systems. Here, the criteria are the length of the spin systems, the chemical shifts, and characteristic peak patterns. The most important part of the spectra is the NH region, because the spin systems are best separated there. The spin systems of short peptides (less than 15 amino acids) can be identified this way and a pre-sclection of the amino acids can be done. For analyzing short peptides the NH(F2)- H-aliphatic area in the TOCSY spectrum is necessary to define the spin systems of the amino acids. Combining TOCSY and COSY it is mostly possible to assign all resonances within one spin system. 194 Additionally the 'random coil shifts' ('H chemical shifts for the 20 common amino acids when followed by alanine), which give the chemical shifts of the protons in amino acids of non- structured, peptides, can be useful for the detection of the amino acid belonging to each spin system. The spectrum derived chemical shifts may differ from the coil due to local anisotropic effects in structured peptides. Some amino acids, (Ser, Thr, Ala) can directly be assigned by analyzing their chemical shifts. The localization of the NH resonances extremely depends on temperature and there is a good chance to get overlapped signals separated by measuring at a slightly changed temperature. Aromatic spin systems in Phe, Tyr, His, and Trp can be connected to the backbone spin system by determining the NOLs from the ß-protons to the ring protons. Good identification criteria for spin systems are the presence of methyl groups (Ala, Thr, Val, Leu, and lie). Gly is the only natural occurring amino acid whose amino resonance exhibits a triplet. There is no amid proton in Pro. but it shows a characteristic spin systems in the TOCSY and correlations in the COSY spectrum. Analyzing a ['H^Cl-correlation spectrum (HSQC. HMBC) a statement of the kind of the spin systems due to the nC shifts can be given. Additionally gemmai protons can be identified as well as the spin systems be connected. Connection of the spin systems is also possible with the NOB (or ROE) signals. Here observation of short sequential NH (1). Ha (i-1) distances is possible. Of course also non-sequential short distances can be observed m the NOBSY (ROESY) spectrum. Thus, only strong or middle-strong NOEs should be used for the sequence assignment. For interpretation of the spectra it is necessary to use identical expansions of the NH/Ha of the COSY and NOESY (ROFSY) spectra (1. 2. 3). 17.3.2 Residue Masses of Neutral Amino Acids Residue masses (g/M) of neutral amino acids are 99,1 for valin (Val). 113.2 for leucin (Leu), 113.2 for isolcucine (lie), 131.2 for methionine (Met), 97.1 for proline (Pro), 147.2 for phenylalanine (Phe), 101.1 for threonine (Thr), 163.2 for Pvrosmc (Tyr), 156.2 for arginine (Arg), 143.1 for acetyl-threonine (AcThr), and 83.0 for dehydrohomoalanine (Dhha) (3). 195 17.3.3 Structure Determination of TB-1 and TB-2 The peptidal nature of TB-1 and TB-2 was recognized by the characteristic peak patterns of the 'H NMR spectra. Measured in CD.OD and 90% ILO'10% D.O several NH doublets and one Nil singulet could be observed in the characteristic NH region between 10 and 7 ppm. These peaks disappeared m the spectrum measured in CD,OD. Additionally a complex set of multiplets was detected in the a-H region of peptides between 5 and 4 ppm. 'H NMR spectra measured in CD^OH and the peak assignment of TB-1 are shown in Figure 17.4 17.7 and of TB-2 in Figure 17.19 - 17.22, respectively. In the nC NMR spectrum of TB-1 a total of 71 carbon atoms could be observed. In the carbonyl region between 180 and 165 ppm 14 signals could be assigned. By using the DEPT135 and DEPT90 experiments 27 methine. 10 metlrvlene. and 17 methyl signals were identified additionally to three quaternary carbon signals Six aromatic signals, belonging to Phe, were detected in the aromatic region. nC NMR spectra of fB-1 measured in CD,OH and the peak assignment are presented in Figure 17.8 - 17.11. The carbon signals of TB-2 were detected indirectly by means of HMQC and HMBC correlations. Detemiination of the spin systems of the ammo acid residues belonging to TB-1 and TB-2 was possible analyzing the TOCSY spectra measured in CD,OFI or 90% H,O/10% IYO, All amino acids, except proline, which has no H \ could be assigned this way in the NH (F2)/a-FI-aliphatic region. TOCSY' spectra and peak assignment of TB-1 measured in CD,OD and 90% II2O/10% D20 arc shown in Figure 17.12-17.13 and of TB-2 in Figure 17.23. The proline unit of TB-1 was determined using the 'H-'H COSY spectrum (see figure 16.14). The determination of the amino acid residues was confirmed by comparison of the assigned signals with the 'random coil shifts' and by analyzing the HMQC and HMBC spectra to give a statement of the kind of the spin systems due to the nC shifts. The sequence assignment was done analyzing the HMBC correlations (Figure 17.15) between a- FI (i)/C-0 (i) and a-H (i)/C"-0 (i-l) and confirmed by NOT signals which could be observed for short sequential NH (i)/ll-a(i-l) distances presented in Figure 17.17 for TB-1 and in Figure 17.24 for TB-2. 196 MALDI-MS and MS-MS confirmed the gross stracture of the peptides. The MALDI MS gave the pseudo molecular peaks at 1488.69 [M+Na]+ analyzing for C71H]16N16017 for TB-1 and a molecular peak at 1491.20 [Mf analyzing for C7,HmN16016S for TB-2 (sec Figure 17.17 for TB- 1 and Figure 17.25 for TB-2). Detailed analysis of the MS-MS spectra of TB-1 and TB-2 are given in Figure 17.17 and 17.25, respectively. 197 Aromatic Region FF Region H ! Reg on ,( «,»/ ni ovr ,i A'jv'',,;. Jv J v_y O LvJt _Lj^_mjJ w ja J9 S 7 6 5 3 2 1 ppm Figure 17.4 'H NMR spectrum of TB 1,CD3OD, 500 MHz, 298 K Arg -VaP ' Thr2 Thri ,i \ r VaP ij AcThr ' rJ [J W ^ rrr - n |-rr rp Phe 7 18 8.1 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 Dhha 3 ppia ' lle^NH J\ 75 7 4 7 3 72 7 1 7 0 6 9 6 8 6 1 ppm Figure 17.5 'H NMR spectraof TB-1, CD3OD, 500 MHz, 298 K . 1 . . . O . 1 3 2 1 1 1 O 9 O . 8 O . V PPm Figure 17.6 'H NMR spectraof TB-1, CD3OD, 500 MHz, 298 K U-" Thr? 2 , _ i | , „ Ac Thr 2 V*-? He« 2 I I | N | \ I Ac Thr 2 \ \ i I V\!l 'I Ile22 ! I i __,Arg2 , , Iff! |V I VI I ^Val3 2 y; ''V O (J s -4.7- -?.ö -4.Ä 4.4 4.3 4.2 4. 1 4.0 3.9 3.8 3.7 pp>m Figure 17.7 'H NMR spectrum of TB-1, CD3OD, 500 MHz, 298 K ' vis!*>***«tH-*i'>~w*J*{**tt-*-'~ v#* v-v,* j iU^-l *î/f'^JHWV"'~W^ •*<~^^<~J*>f\**1»»jA^^'^^^y^vA^'^V^^f-^i'iV^'i-^*rl'lV^Ww^ Ws^". Wf-1" ^•*«H*^v^^*N*r^N^^Vv* *[*""''/' tfkW/tJjWHSyw ^jvV^f*^-« v4*^^^*WWv*sv^A^W^ ^J,'**/^^^ W^v^y*]^ Aromatic Rf gion Ca^box/l Region Ha Region iM^-v ^wu^ny^v^M rt*tiWfi*üWv ^ ^i^'iWVJf 170 160 150 140 130 120 110 100 90 SO 70 60 50 3 0 2 0 ppm Figure 17.8 '^C NMR, DEPT95, and DEPT135 spectraof TB-1, CD30H, 500 MHz, 298 K Arg1 o a A i \Jr I X ,, ' M s A t1 f / / rs 0 j.7" 5 174 0 l"j 5 173 0 172 5 P2 f, -71 5 V± C 170 5 cpm Dhha 3 I ] Dhha 2 i " ' l"lJ 60 bc bO 143 140 135 130 eft Figure 17.9 "C NMR spectraof TB-1, CD3OH, 500 MHz, 298 K Thr2 2 Thr2 3 oto ' " > ', nwwwvi am"<> 73 72 71 70 69 68 67 66 65 64 63 62 61 60 59 58 57 56 55 ppm Figure 17.10 "C NMR spectrum of TB-1, CD30H, 500 MHz, 298 K Arg 5 Val,33 Val1 3 Ile2,3 Val2 3 Arg 3 V\t\nu VlUJ',',',/ ' .''/ ji\(fjl V^'I'II'V^J/'IV,!,.^' ' i '' I I i '' I to n o I I « 42 « « 39 3^ 17 36 3^ 34 33 32 31 30 • /aP 4 Ag4 ,/al 4 I" 4-^ Lou o [ i/ Dhh?4 Leu 4 \ Ile' 5_ ' -AcThr 6 Pro 4 i ' v ' 1L i, j Y fCi»/w M' ' 28 27 26 25 24 23 22 21 20 19 i7 i6 i; 13 12 ppm | Figure 17.11 "C NMR spectraof TB-1, CD.OH, 500 MHz, 298 K \û( T -r Pi I 1 X ' m M- co , ! i- f t+ -1 206 M Phi y Ile2 ft Val2 a AcThr jJ y La. pp H35TTH'6 oto -o Figure 17.13 'H-'H TOCSY spectraofTB-1, H20 90%/D2O 10%, 500MHz, 298 K WWr> fQr oto oo Figure17.14 >H->H DQF-COSY spectraof TB-1, CD3OH, 500MHz, 298 K LU O ^" cc > .7 Q T X X 0 O O O- PQ o • ci) O < c/) .2 \ Vx -A cd 13 CD o o U .2f 209 g ft ft «CSb» "X) ()HN (i )«h 1H NH fc 210 25000 148889 i 20000 15000 c o i Lü 4 1024 66 000 5000 i 1068 52 •504 6 1375 85 1124 95 tma. 4tiOw*iA4*to*<à*W^ 1000 1200 140« 1600 1800 2000 Mass (m/z) figure 17 1" MALDI MS spectiurro^TB 1 m rTTTTTTTTT *TT * pTTTTTT" ~TT^r T T~rTTTT7~'7- q ni-] 11 rrrn o m q «o o m O m o in o m o o o> 35 oo 00 CD ro cm cm 212 ! Aroma'ic Region H« Region HN Rpgiori ' ; v / ^ y,Ï^J> W'LJ! j'xj^j^v^^fjxAyjv^ 9 7 5 3 2 ppm Figure 17.19 'H NMR spectrum of TB-2, CD3OH, 500 MHz, 298 K IleHN Thr' HN \ , Thr? H" / \ l\\i VaPHN < ,Tyr HN Val ff' / Xal2HI? Phe H^< ' i« ilP\ i f!/ \ ! 1 ' « !! Ji to 3 6~ i.4 ? ? 9a ? ? S fS 4.4 P ? 8.0 c Dpn 4^ - Phe 5/9 -Phe 6/8 Tyrî -Ty9 ^Tyr6 -Ty-8 ArrjNH \i \ L_^/v U!i L_J 'JU ' ' l I ' T I I I 7.6 7.4 7 2 7.0 6 8 6 6 6 6 2 ppm Figure17.20 'H NMR spectraof TB-2, CD30H, 500 MHz, 298 K ~M»OH Thri 4 T< Thr? 4 . WJ \'Jn Ja 14 \ , /a? 4 \l I — ya 5 4 I //al 4 Met i i yi 7 \ I Va 3 /an 4 Metp i i \ 7 /* »i ! 7 Ij / / "^J / i> ) ~!le3 J "Va < 3 I \ Jafi 3 ya! •/aP 3 2 tf ?o 24 2 2 2 0 18 16 là T 2 TO 0 a ppm Figure 17..21 iH NMR spectraof TB-2, CD3OH, 500 MHz, 298 K to r r- 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 ppm Figure 17.22 'H NMR spectrum of TB-2, CD3OH, 500 MHz, 298 K Met Arg "1 Arg II Ty Phe _^W IPV^'A/P"-y~-' y /7r 7 J d ^j H3 6 Li A h 4_&4^)r-iïaaJA |=^h34 1.0 H23 1.5 H,3 : 2.0 _2.5 4 0 4.5 _5.0 9.0 8.2 8.0 7.0 6.8 6.6 ppm Figure 17 23 'H 'H TOCSY spectrum of TB 2 CD OH 500MHz 298 K 11, Avr\7' _A L ppm 3 ^3.9 4.0 _4.X 4.2 4.4 Dhha II ^4.6 _4.7 yx L4.8 9.6 9.4 9.2 9.0 8.8 8.S 8.4 8.2 8.0 7.8 7.6 7.4 Figure 17.24 NOESY spectrum of TB-2, H20 90%/D2O 10%, 500MHz, 298 K s m en en *- O) '"m CO ©or: o>Sr>55 com o ? i O >J CO \ OT h- o o S' o o o a CM Oi * O o ! '8 o o o«- o to eouepunqv aw}6|3y 219 m H ii *- ^ I to O 6 I T T O O u .1. J. O u u souepunqv aAijeiay fe 220 17.3.4 Determination of the Absolute Stereochemistry of the Amino Acid Residues according to Marfey 17.3.4.1 Introduction The peptide must be splitted into free amino acids by acid hydrolysis. l-Fluoro-2.4- dinitrophenyl—5-L-alanine (FDAA) can be used for the reaction with L- and D~ amino acids and RP HPLC can separate the resulting diastereomers. which arc obtained in quantitative yield. The L-diastereomers were eluted from the reverse-phase column before D-diastereomers. The reason for this behavior is probably due to a stronger intramolecular H-bonding in D- than in l,- diastereomers. One can expect that the carboxyl group can hydrogen bond cither to an ortho- situated nitro group producing a 9-membered ring or, more likely, to the carbonyloxygen of the mcta-situated L-Ala-NH-, forming a 12-membered ring. Stronger H-bonding in a D-diastercomer would produce a more hydrophobic molecule, which would be expected to interact more strongly with the reverse-phase column and thus have a longer retention time than an L-diastereomer (4, 5, 6). An outline of the derivatization of L- and D-isomers of amino acids is given in Figure 17.27. 221 :—- NH? O.N Separation of two isomers Figure 17.27 17.3.4.2 Acid Hydrolysis of TB-1 and TB-2 For amino acid analysis. 200 ug of the peptide was dissolved in 1 ml 6 N HCl and heated at 110 °C for 16 h. The reaction mixture was dried under a nitrogen stream. 17.3.4.3 IIP! C Analysis of the Marfey Dem ati\ es To the acid hydrolysate 100 uT of I -FDAA in McCO (10 mg'mL) and 200 \xL of 1 N NaHCO, were added, and the mixture was kept at 80 "C for 3 min. To the reaction mixture, 100 u,L of 2 N HCl and 400 uL of 50% MeCN were added and analyzed by RP HPLC: Lichrosorb RP-18, (250 x 4 ram); gradient elution from MeC\ FLO'TFA 95:5:0.1 to MeCN H,0/TFA 40:60:0.1 in 60 min; UV detection at A 340 nm, flow: 1.5 mL min. The chromatograms of the Marfey dérivâtes n) of TB-1 and TB-2 are shown in Figure 17.27 and 17.28. Retention times of the Marfey derivatives are given in Table 17.1 and Table 17.2. 17.3.4.4 Preparation of the Acetyl Derivatives of Threonine (AcThr) Threonine (5.6 mg), anhydrous pyridine (0.5 ml.) and acetic anliydridc (0.5 mL) were kept in the dark at room temperature for 1 8 h. The reaction mixture was diluted with 2 mL of water and stored at 4 °C for 1 ft. Then it was applied on a Scp-Pak' tC18 cartridge, which had been washed with 10 mL of water. Pyridine, acetic anhydride and not acetylated Thr were eluted with water, followed by the elution of acetylated compound with chloroform. Evaporation of chloroform with reduced pressure below 30 °C left 2.5 mg of the acetyl derivative, which was completely dried in vacuum drying oven at 25 - 28 °C. The identity of AcThr was proved by 'H NMR spectroscopy. In comparison to the Tl NMR spectrum of non- acetylated Thr the derivatized compound shows an additional CH, signal at 1.80 ppm measured in CD,OD. Table 17.1 Retention Times of the Marfey Derivatives of Standard Amino Acids L-Amino Acids Retention time (mm) j D-Amino Acids Retention Time (min) L-Leu 41.42 D-Leu 45.31 L-Phe 41.76 D-Phe 44.51 L-lle 40.70 D-Ilc 44-94 L-Val 37.07 D-Val 40.56 L-Pro 31.44 D-Pro 32.78 L-Arg 27.82 D-Arg 28.68 L-Thr 26.84 D-rhr 28.42 L-AcThr 25.27 D-AcThr 26.05 L-Mct 37.07 D-Met 38.25 L-Tyr 48.56 D-T\r 50.96 223 Tabic 17.2 Amino Acids of TB-1 Amino Acid Retention Time (min) D-Lcu 45.31 L-Phe 41.76 L-llc 40.70 L-Val 37.07 D-Pro 32.78 L-Arg 27.82 L-Thr 26.84 L-AcThr 25.27 Table 17.3 Ammo Acids of TB-2 Amino Acid Retention Time (mm) L-Phe 41.98 L-Ue 40.96 L-Val 36.86 L-Arg 27.98 L-Thr 26.84 L-Met 37.07 L-Tyr 48.56 to to IM Figure 17.28 HPLC chromatoeram of the Marfev derivatives of TB-1 226 17.4 Biological Testing Both peptides show antifungal activity against the yeast Candida albicans. The MIC values were detected to be 32 ppm for TB-1, 64 ppm for TB-2. and 8 ppm for the reference compound miconazole. 17.5 Referen ces 1. Cavanagh, J., Fairbrother, W. .1., Palmer, A. G., Skclton, N. J.: Protein NMR Spectroscopy. Principles and Practice. Academic Press, London (1996) 2. Wüthrich, K.: NMR of Proteins and Nucleic Acids. Willcy, London (1986) 3. Zcrbe. O.: NMR spektroskopische Identifizierung von Peptiden und Proteinen. Lecture Script, Swiss Federal Institute of Technology (Efll), Zürich (1997) 4. Aberhart. D, J., Cotting, J.-A., Lin. H.-J.: Separation by FTigh-Performance Liquid Chromatography of (3R)- and (3S)-ß-Leucine as Diasteremeric Derivatives. Analyt. Biochem. 151:88-91 (1985) 5. Brückner, IL, Kellcr-Hoehl, C: HPLC Separation of DL-Amnio Acids Derivatized with N2-(5-Fluoro-2,4-Dinitrophenyl)-L-Amino Acid Amides. Chromatographic! 30: 621-629 (1990) 6. Marfey, P.: Determination of D-Amino Acids. Use of Bifunctional Reagent. 1,5-Difluoro- 2,4-Dinitrobenzene. Carlsberg Res. Commun. 49: 591-596 (1984) 227 1 8 Publication 1 Biological Screening of Cyanobacteria for Antimicrobial and MoUuscicidal Activity, Brine Shrimp Lethality, and Cytotoxicity B. Jaki l, J. Orjala A FAR, Biirgi 2 and O. Stichcr Department ofPharmacy, Swiss Federal Institute of fccliiiologv (ETH) Zurich, CH-8057 Zürich, Switzerland " Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG), CH- 8600 Dübendorf, Switzerland Current affiliation: AgraQuest Inc.. 1105 Kennedy Place. Davis, CA 95616 ^Author to whom correspondence should be addressed: Phone: h 41 1 635 6050, FAN: h-41 1 635 6882. E-mail: [email protected] 228 Abstract A total of 86 lipophilic and hydrophilic extracts obtained from 43 samples of cultured and field collected freshwater and terrestrial cyanobacteria have been screened for their biological activities. Antimicrobial evaluation demonstrated 16.3% to be active against Gram-positive and 5.8% against Gram-negative bacteria, while 10.5% possessed antifungal activity. A lethal effect (>60%) against brine shrimp (Artemia salina Leach) was exhibited by 8.1% of all extracts at 500 ppm. Cytotoxic activity against KB cells was shown by 1.2%, and 8.1% were active against Caco-2 cells at 50 ppm. A moUuscicidal effect against Biomphalaria glabrata was found for 5.8% of the extracts (LCjoo < 100 ppm). Introduction A large number of interesting metabolites of wide chemical diversity has been isolated from cyanobacteria (Carmichael, 1992). Several cyanobacterial metabolites have shown biological activity in various bioassays. Some prominent examples described in recent literature are the antimicrobial active polychlorinated phenols, ambigol A and B, isolated from Fischerella amhigua (Falch et al, 1995) and compounds with moUuscicidal activity, like barbamidc, obtained from Lvngbya majuscula (Orjala and Gerwick. 1996). Lyngbya majuscula is also a source of curacine A, a thiazoline-containing lipid with antimitotic, brine shrimp toxic and antiproliferative activity (Gerwick et al, 1994). Strong cytotoxic activity was observed for the depsipeptide cryptophycine A, from Nostoc sp., with an IC50 of 3 pg/ml against KB cells (Trimurtulu et al., 1994). We arc currently investigating the biological potential of various species and strains of terrestrial and freshwater cyanobacteria. For this purpose several assays were used to detect antimicrobial activity, brine shrimp lethality, cytotoxicity against cultured KB and Caco-2 cells and moUuscicidal activity against the snail Biomphalaria glabrata, a host oi Schistosoma mansoni. Materials and Methods Collection. Thirty-six cyanobacterial strains were obtained from the Culture Collection of Algae at the Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG), Dübendorf, Switzerland. Seven cyanobacterial strains were isolated from samples obtained by field collections in Zurich. Switzerland. In general, the enrichment technique and the selective media method (Moore et a!.. 1988) were used to isolate the cyanobacterial strains. Specifications of species and their original habitats are listed in fable 1. 229 Cultivation. At the EAWAG, stock cultures of cyanobacteria were cultivated under weak illumination, with intervals of several months between reinoculations. The cultures were grown in different media (Hughes et al, 1958). Aliquots of 5 ml from stationary phase stock cultures were used to inoculate 250 ml of media. The field collected samples (strains 256, 257, 258, 259, 260, 261, 262) were inoculated in 250 ml of media directly after isolation. Preparation of Extracts. Algal cells were harvested every 4-6 weeks and separated from the medium by filtration and freeze dried. The lyophilized material (2-5 g) was extracted by maceration with dichloromethanc/mcthanol 2:1 followed by methanol/water 7:3. The extracts were dried in vacuo. Bioassays Antimicrobial Assay. Antimicrobial activity was determined by a well-assay (Rios et al, 1988). The test organisms are outlined in Table 2. An aliquot (30 ul) of bacterial or fungal stock suspension was transfered into 10 ml broth (BBL Nutrient Broth, Bcckton Dickinson, Microbiology Systems, Cockeysville. MD 21030. I1.S.A. for bacteria; Sabouraud Liquid Medium. OXOID, UK for Candida albicans, respectively) and incubated at 37°C. Nutrient agar plates (Miillcr-Hinton Agar, OXOID, UK for bacteria; Malt Extract Agar, OXOID, UK for Candida albicans, respectively) were inoculated with 25-100 ul of the microbial culture in the exponential growth phase (100-lOOOfold diluted). The crude extracts (600 tig in 50-75 ul DMSO) were applied into a well (0 6 mm) on an agar plate and diluted with sterile water up to 100 fil. The plates w;ere incubated at 37°C for 24 h (36 h for Mycobacterium fortuitum). As antibacterial standards, chloramphenicol (Siegfried. Switzerland) and tetracycline hydrochloride (Fluka. Switzerland), and as an antifungal standard, miconazole nitrate (Sigma, USA) were used. Zones of inhibition >2 mm were considered active. MoUuscicidal activity. MoUuscicidal assays were performed with freshwater snails of the species Biomphalaria glabrata (Hostettmann ct ah, 1982). The snails had an equal size with a shell diameter of 8-10 mm. Samples were dissolved in 120 ul ethanol/PEG 300 (2:1) and then diluted to 20 ml with distilled water. The extracts were tested at a final concentration of 100 Ug/ml. After 24 h, the snails were monitored by observing their heart beat under a microscope. Brine shrimp (Artemia salina) lethality assay, 'fhe brine shrimp (Artemia salina Leach) lethality-assay was performed at a concentration of 500 ppm as previously outlined (Meyer et 230 al, 1982; McLaughlin, 1991; Orjala et al, 1994). Extracts causing a mortality higher than 60% were considered active. and et Cytotoxic assay. The cytotoxicity of the extracts (Swanson Pezzuto, 1990; Orjala a/., 1994) was assessed using the KB cell line (ATCC CCL 17: human nasopharyngal carcinoma) The volumes as well as the Caco-2 cell line (ATCC FITB-37, human colon adenocarcinoma). Medium were modified for cultivation in 24-well plates with Eagles Minimum Essential (21090- 022; Gibco. LifeTechnologies. Switzerland) for KB cells and Dulbecco's Modified Eagles Medium (31966-021, Gibco, LifeTechnologies. Switzerland) for Caco-2 cells. Results and Discussion Cultivation and Extraction. The selected freshwater and terrestrial strains of blue-green to five different algae provide a representative choice of species of cyanobacteria, belonging families: Nostocaceae (strains 122b.l23b, 124b. 259. 105a, 144, 137a, 139a, 165), Oseillatoriaccae (strains 262. 91/150, 140, 163. 164. 256. 257. 258. 260, 261, 132, 130, 178, 211, 176), Scytonemataceae (strains 108b. 222a, 217. 154a, 179a, 200b, 236, 180, 157a, 195, 153a, 88. 96c. 237b, 166). Rivulariaceae (strains 226a, 102a, 102b) and Phenocapsaceac (strain 91/154). The investigated cyanobacteria originated from different habitats and geographical locations, mainly from Switzerland and Nepal (Table 1). All samples were cultivated in inorganic media under constant illumination at 1500 lux. The growth period lasted 4-6 weeks to ensure the vast majority of cultures were in stationary phase and therefore most likely producing secondary metabolites (Lincoln et al. 1996). Harvested cells were freeze-dried and then successively extracted with solvents of increasing polarity. The resulting cyanobacterial extaicts were subsequently tested for their biological activities (Table 3). Antimicrobial activity. The chosen combination of bacteria and yields contains 6 groups of pathogens. Such a microbial battery is representative of human pathogenic microorganisms. Weil-plate assays allowed the detection of antibacterial and antifungal extracts on agar plates. The extracts were considered active with a zone of inhibition >2 mm. Of 86 extracts, 18 (20.9%) showed antibacterial activity. Fourteen thereof (16.3%; 9 DCM/MeOII 2:1 and 5 MeOHALiO 7:3 extracts) showed activity against Gram-positive bacteria. Only one of them, a MeOH/LbO 7:3 extract from strain 195, was active against Mycobacterium forluitum. Five extracts (5.8%: 3 DCM/MeOH 2:1 and 2 MeOH H20 7:3 extracts) exhibited activity against Gram-negative bacteria. Nine extracts (10.5%; 6 DCM MeOH 2:1 and 3 MeOH/H20 7:3 extracts) showed antifungal activity. 231 MoUuscicidal Activity. Biomphalaria glabrata is an intermediate host of the tropical parasitic disease schistosomiasis. The snails are essential for the life cycle of schistosomes. Local treatment of freshwater sources with moUuscicidal extracts is a simple and cheap method of removing the snail vectors (Marston et al, 1996). 'fhe WHO criteria for an efficient plant molluscicidc stipulate that an extract must be active at 100 ppm or less in water, after 24 h (Duncan and Sturrock, 1987). Five extracts (5.8%; 5 MeOHTAQ 7:3 extracts ) fulfilled this condition. Brine shrimp lethality. The brine shrimp {Artemia salina) lethality assay is considered to be a useful tool for preliminary assessment of cytotoxicity (Solis et al, 1993). Brine shrimp assays have also been used for the detection of fungal toxins and their active metabolites (Harwig and Scott. 1971), active plant constituents (Meyer et al, 1982), for the analysis of pesticide residues (Grosch. 1967), and to monitor the toxicity of organic waste to marine organisms (Hood et al, 1960). Seven extracts (8.1%; 5 DCM/MeOFI 2:1 and 2 MeOH H2Q 7:3 extracts) showed significant activity (lcthality>60%) against Artemia salina nauplii at 500 ppm. Cytotoxic assay. Cytotoxicity indicates inhibition of the proliferation of tumor cells in vitro (Suffness and Pezzuto, 1991). The KB and Caco-2 cell assays were used to screen for cytotoxic activity. Only one lipophilic extract (strain 260) showed activity against KB cells, while seven lipophilic extracts (8.1 %) exhibited cytotoxic effects against Caco-2 cells. Conclusion. Of a total of 15 strains belonging to the family Oscillatoriaceae, 11 demonstrated bioactivity. The 9 Nostocaceae strains revealed 4 active strains. From a total of 15 Scytonemataceae strains, 6 were active in at least one of the test systems. No bioactivity was detected with the strains of the Rivulariaceae and the Phenocapsaceac selection. The bioactivity rate in the antimicrobial assay was 46.5% for lipophilic and 27.9% for hydrophilic extracts. Eight extracts were active against cocci and 9 against rods, while only one extract was active against cocci and rods. These results express that the activity of most of the extracts is specific for one group of bacteria. MoUuscicidal activity was found only in hydrophilic and cytotoxic activity was only observed for lipophilic extracts. Brine shrimp lethality was detected for 5 hydrophilic and 2 lipophilic extracts. There was no correlation between brine shrimp lethality and cytotoxicity against KB cells and only two extracts were active against brine shrimp and Caco-2 cells. Regarding these results it may not be possible to monitor cytotoxicity using the brine shrimp bioassay rather the cytotoxic assays. The results of this study showed that 21 of 43 investigated cyanobacteria strains (48.8%) arc able to induce a response in at least one of the screening systems applied and therefore support 232 the use of biological screening to assist in the selection of samples for further chemical analysis. Based on our results, large-scale cultivation of the most active samples is in progress and further the isolation of the biological active compounds by a bioassay-guided fractionation is planned. Acknowledgements. We thank Dr. M. Bosh and Mr. F. Sunder (EAWAG) for providing cyanobacterial material. We are also grateful to Mr. M. Wasescha for technical assistance. 233 References Carmichael, W. W. (1992). Cyanobacteria secondary metabolites. J. Appl. Bacteriol. 72: 445- 459. Duncan, J. and Sturrock, R. F. (1987). Laboratory Evaluation of Potential Plant Molluscicidcs. In: Plant Molluscicidcs. (Mott. K. E.. ed.). pp. 251-265, John Wiley, Chichester. Falch, B. S., König. G. M.. Wright. A. D.. Sticher. O., Angerhofer, C. K., Pezzuto, J. M. and Bachmann, H. (1995). Biological activities of cyanobacteria: Evaluation of extracts and pure compounds. Planta Med. 61: 321-328. Gerwick, W. H., Protean. P. J., Nagle. D. G.. Flamel, E., Blokhin, A. and Slate, D. L. (1994). Structure of curacinc A, a novel antimitotic, antiproliferative and brine shrimp toxic natural product from the marine cyanobacterium Lvngbva majuscula. J. Org. Chem. 59: 1243-1245. Grosch, D. S. (1967). Poisoning with DDT: effect on reproductive performance of Artemia. Science 155: 592-593. Harwig, J. and Scott, P. M. (1971). Brine shrimp (Artemia salina L.) larvae as a screening system for fungal toxins. Appl Microbiol. 21: 1011-1016. Hood, D. W.. Duke, T. W. and Stevenson B. (I960). Stream pollution. Measurement of toxicity of organic wastes to marine organisms. J. Water Pollution Control Fed. 32: 982- 993. Hostettmann, K., Kizu, H. and Tomimori, T. (1982). MoUuscicidal properties of various saponins. Planta Med. 44: 34-35. Hughes, E. O., Gorham, P. R. and Zehnder, A. (1958). Toxicity of an unialgal culture of Microcystis aeruginosa. Can. J. Microbiol. 4: 225-236. Lincoln, R. A., Struoinski, K. and Walker, J. M. (1996). The use of Artemia nauplii (Brine shrimp larvae) to detect toxic compounds from microalgal cultures. Int. J. Pharmacog. 34: 384-389. Marston, A., Dudan. G., Gupta. M. P., Solis, P. N., Correa, M. D. and Hostettmann, K. (1996). Screening of Panamanian plants for moUuscicidal activity. Int. J. Pharmacog. 34: 15-18. McLaughlin. J. L. (1991). Crown Gall Tumours on Potato Discs and Brine shrimp Lethality: Two Simple Bioassays for Higher Plant Screening and Fractionation. In: Methods in Plant Biochemistry. Vol. 6. (Hostettmann. K„ ed.). pp. 1-32, Academic Press, London. Meyer, B. N., Ferrigni. N. R., Putnam, J. IA Jacobson. L. B., Nichols, D. E. and McLaughlin, J. L. (1982). Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med. 45: 31-34. Moore, R. PL Patterson, G. VI. L. and Carmichael W. W. (1988). Biomedical Importance of Marine Organisms. In: Memoirs of the California Academy ofScience. Vol. 13, (Fautin, D. G., ed.). pp. 73-102, California Academy of Science. San Francisco. 234 Orjala, J. and Gerwick, W. H. (1996). Barbamide, a chlorinated metabolite with moUuscicidal activity from the Caribbean cyanobacterium Lvngbya majuscula. J. Nat. Prod. 59: 427-430. Orjala, J., Wright, A. D., Behrends, IL, Folkers, G., Sticher, O., Ruegger, FI. and Rali, T. (1994). Cytotoxic and antibacterial dihydrochalcones from Piper aduncum. J. Nat. Prod. 57: 18-26. Rios, J. L., Recio, M. C. and Villar, A. (1988). Screening methods for natural products with antimicrobial activity: A review of the literature.,/. Ethnopharmacol 23: 127-149. Solis, P. N., Wright. C. W.. Anderson, M. M.. Gupta. M. P. and Phillipson, J. D. (1993). A microweil cytotoxicity assay using Artemia salina (brine shrimp). Planta Med 59: 250-252. Stiffness. M. and Pezzuto, J. M. (1991). Assays Related to Cancer Drug Discovery, in: Methods in Plant Biochemistry. Vol. 6, (Hostettmann, K., ed.). pp. 71-133, Academic Press, London. Swanson, S. M. and Pezzuto. J. VI. (1990). Bioscreenmg Fechnique for Cytotoxic Potential and Ability to Inhibit Macromolecule Biosynthesis. In: Drug Bioxscreening: Drug Evaluation Techniques in Pharmacology. (Thompson. F. B., ed.). p. 273, VCH, New York, Weinhcim, Basel. Cambridge. Trimurtulu, G., Ohtani, I., Patterson, G. M. L., Moore, R. E., Corbett, T. H., Valeriote. F. A. and Demchik, L. (1994). Total structures of cryptophycins, potent antitumor depsipeptides from the blue-green alga Nostoc sp. strain GSV 224. J. Am. Chem. Soc. 116: 4729-4737. 235 Table 1. Investigatedspeciesand their geographicalorigin. Strain3 Species, family Origin 222a Calothrix parietinaLliuret,Scytonemataceae Traunstein (AU), on rocks,1977 217 Dichothrix orsiniana Born, et Flah.. Scytonemataceae Davos (CH), on rocks,1976 108b Fischerclla ainbigua(Nag.)Gomont, Scytonemataceae Mellingen(CH), shallow depression,1965 262 LvngbyaputealisMont., Oscillatoriaceae Zürich (CH), brook, 1996 91/150 Lvngbya sp., Oscillatoriaceae Neckcr (CH), on rocks,1991 140 Lyngbya sp., Oscillatoriaceae Kathmandu (Nepal),on rocks,1965 163 Lyngbya sp. Oscillatoriaceae Kathmandu (Nepal),graniteblock, 1967 164 Lyngbya sp., Oscillatoriaceae Kathmandu (Nepal),graniteblock, 1967 91/154 Myxosarcina sp.. Phenocapsaceae Necker (CH), on rocks, 1991 122b Nostoc commune Vaucher, Nostocaceae Mellingen(CH), plainhollow,1965 123b Nostoc commune Vaucher. Nostocaceae Mellingen (CH), plainhollow, 1965 124b Nostoc commune Vaucher, Nostocaceae Mellingen(CH), plainhollow,1965 259 Nostoc commune Vaucher, Nostocaceae Zürich (CH), roadside,after rain,1996 105a Nostoc inuscorum Ag., Nostocaceae Lutisbach (CH), riverbank,1964 144 Nostoc sphaericumVaucher,Nostocaceae Morteratsch near Pontresina (CH), on rocks,1965 Table 1 continues Strain3 Species, family Origin 137a Nostoc sphaericumVaucher, Nostocaceae Morteratsch near Pontresina (CH), on rocks,1965 brick 1967 139a Nostoc sp., Nostocaceae Kathmandu (Nepal), wall, brick 1967 165 Nostoc sp., Nostocaceae Katlimandu (Nepal), wall, 256 Oscillatoria amoena Com., Oscillatoriaceae Zürich (CH), small pond, 1996 257 Oscillatoria limosa Ag.,Oscillatoriaceae Zürich (CH), small pond. 1996 258 Oscillatoria tenuis Ag., Oscillatoriaceae Zürich (CH), small pond. 1996 260 Oscillatoria formosa (Bory),Oscillatoriaceae Zürich (CH), brook, 1996 237b Petalonema a/atnm Berk.. Scytonemataceae Nufenenpass (CH), on rocks, 1979 261 Phormidium favosum (Bory) Gom., Oscillatoriaceae Zürich (CII).brook, 1996 1965 132 Phormidium sp., Oscillatoriaceae Berninapass(CH). graniteblock, (Nepal), 1967 178 Phormidium sp., Oscillatoriaceae Kathmandu on claysoil, ruin 1975 211 Phormidium sp., Oscillatoriaceae Baden (CH), stone, brick 1967 166 LHectonema sp., Scytonemataceae Kathmandu (Nepal), wall, 226a Rivularia biasolettiana Meneghini,Rivulariaceae Aegerisee(CH), riverbank. 1978 rock, 1964 102a Rivularia sp., Rivulariaceae Verzascatal,(CH), gneiss Table 1 continues Strain3 Species Origin 102b Rivularia sp., Rivulariaceae Verzascatal (CH), gneissrock,1964 130 Schizothrix sp., Oscillatoriaceae Berninapass(CH), graniteblock,1965 154a Scytonema burmanicum Skuja,Scytonemataceae Kathmandu (Nepal),brick wall,1967 179a Scytonema myoclirons(Dillw.)Ag., Scytonemataceae Bürglcn (CFI),on rocks,1969 200b Scytonema myochrous (Dillw.)Ag., Scytonemataceae Nufencnpass (CH), gneissrock, 1979 236 Scytonema myochrous (Dillw.)Ag., Scytonemataceae Nufenenpass (CH), gneissrock, 1979 180 Scytonema stuposiun (Kütz.)Born., Scytonemataceae Pokhara (Nepal),graniteblock,1967 1967 to 157a Scytonema sp., Scytonemataceae Pokhara (Nepal),graniteblock, oo 176 Symploca museorum (Ag) Gomont, Oscillatoriaceae Kathmandu (Nepal),on claysoil,1967 195 Tolypothrixbyssoidea(Hass.)Kirchn.. Scytonemataceae Pokhara (Nepal),graniteblock,1967 153a Tolypothrixdistorta Kütz.,Scytonemataceae Linz (Au),small pond, 1968 88 Tolypothrixdistorta Kütz.,Scytonemataceae Pokhara (Nepal),graniteblock,1967 96c Tolypothrixdistorta var. peniciilataLemm., Scytonemataceae Aegerisee(CH),riverbank,1963 aDesienated t>tiam numbeis accoidine to the Culture Collection of Alaae of the EAWAG Table 3. Biologicalactive cyanobacterialextracts. c " e * Strain Extract Antibac terial/anti imgal activity MoUuscicidal Brine shiimp lethality/cytotoxicity activity B.c. S.e. M.l. P.a. E.c. M.f. Ca. Biomph. glabr, B.s. KB Caco-2 0.6 mg 0.6 mg 0.6 mg 0.6 mg 0.6 mg 0.6 mg 0.6 mg 100 ppm 500 ppm 50 ppm 50 ppm 108b A r V + + 94% ' B ++ 60% 91/150 A j.. B ++ 140 A 60% B j- _!_ 163 A B 164 A (++) B + 122b A ++ 80% (+) B 123b A 80%, B b+ 124b A + b + B 259 A ++ B 256 A + B ++ 257 A B ~r 260 A (+) B 4- -j- ++ 261 A + f B +4- 178 A (A B Table 3 continues e * c " Brine Strahl Extract Antibac terial/anti xing al activity MoUuscicidal shrimp lethality/ cytotoxicity activity Caco-2 B.c. S.e. M.l. P.a. E.c. M.f. C.a. Biomph. glabr. B.s. KB 50 50 0.6 ma 0.6 mg 0.6 mg 0 6 mg 0,6 mg 0,6 ma 0.6 mg 100 ppm 500 ppm ppm ppm 211 A A) B 200b A X 76% B 176 A A B j_ -L- T 195 A A A B _ 70 % + 153a A 1 B -La 4- 88 A B 96c A + ! f B I 5 1 control 5 gg 5 jig 5 M-g 5 ug 5 ug ug ug 1 i i , * ; Co¬ i , a. : le 4- i.(. Mc r-:- ~\—r~ + _j_ Pt 100% c+ Cc = Tc - Mc = Pt = = zone A -= dichloroniethaiie/methanol 2:1 extract; B = methanol/water 7:3 extract; Chloramphenicol; Tetracycline; Miconazole; Podopliyllotoxin; = <+ = all e(%) = A = total inhibition, of inhibition 2-6 mm, ++ -= zone of inhibition >6 mm. (+) partialinhibition; lethalityof snails; lethality; E.c. = M.f. S.e. = loccus M.l. = Micrococcus luteus;P.a. = Pseudomonas aeruginosa; Escherichia coli; (+) = partialinhibition;B.c. - Bacillus cereus; Staph} epidermidis; = B.s. - Brine = Mycobacterium forhutum; C.a. = Candida albicans; Biomph. glabr. Biomphalariaglabrata; shrimp Table 2. Test organisms for antimicrobial assays. Microorganism Origin Group of microoganism Abbreviation Gram-positivespore-fonning Bacillus ccreus ATCC 10702 rods B.c. S.e. Staphylococcusepidermidis ATCC 12228 Gram-positivecocci Micrococcus lutcus ATCC 9341 Gram-positivecocci M.l. Gram-positiveasporogenous acid fast M.f. Mycobacteriumfortuitwn apatientmaterial rods, Pseudomonas aeruginosa ATCC 27853 Gram-negative rods P.a. Escherichia coli ATCC 25922 Gram-negative rods B.c. Candida albicans H29 ATCC 26790 Yeast C.a. Obtained fiom the Institute of Vledical Miciobiology, Univeisity of Zürich 19 Publication 2 A Novel Extracellular Diterpenoid with Antibacterial Activity from the Cvanobacterium Nostoc commune Birgit Jaki, Jimmy Orjala1' and Otto Sticher ' Department of Pharmacy, Sw iss Federal Institute of Technology (ETLI) Zurich, CH-8057 Zürich, Sw nzerland f Current affiliation: AgraQuest Inc.. 1105 Kennedy Place. Da\is, CA 95616 * Author to whom correspondence should be addressed. Phone: +-»41 I 635 6050. FAX: M 41 1 635 6882. E-mail: sticher(apharma.etlv.ch 242 A novel extracellular metabolite with an unprecedented diterpenoid skeleton, 8-[(-5-carboxy-2- hydroxy)-benzyl]-2-hydroxy-1,1,4a,7,8-pentamethyl-1,2,3,4,4a,6,7,8,8a,9, 1 0,10a- dodecahydro-phcnanthrene. has been isolated from the culture medium of the terrestrial cyanobacterium Nostoc commune Vaucher (EAWAG 122b) by means of bioguided isolation. The molecule was designated as noscomin. The structure was determined by spectroscopic methods, mainly NMR and mass spectrometry. Noscomin exhibited antibacterial activity against Bacillus cereus, Staphylococcus epidermidis and Escherichia coli. Cyanobacteria are known to be a rich source of secondary metabolites with a wide variety of biological activities including toxins, antibiotics, fungicides and antineoplastic agents,1 The majority of these metabolites have been found in association with the cyanobacterial cells. The occurrence of terpenoids in cyanobacteria is rather uncommon. A few examples are the tritcrpenoid bactcriophanes-isolated from stneral species of cyanobacteria. The antifungal hapalindoles,3,4 hapalindolinones5 and ambiguines6 as well as the welwitindolioones7 are metabolites of mixed biosynthetic origin, containing isoprene units and have been found in several species of the family Stigonemataceae. The only diterpenoid compound reported from cyanobacteria is the antiinflammatory tolypodiol'5 from Tolvpothrix nodosa. This report describes the isolation of noscomin (I), a novel antibacterial metabolite with an unprecedented diterpenoid skeleton, from the culture medium of a cultured Nostoc commune Vaucher strain. This strain was selected on the basis of a biological screening of 43 different cyanobacterial cultures. Nostoc commune (EAWAG 122b) was cultured in an inorganic medium. The culture medium was separated from the cells and subjected to a solid-phase extraction on Amberiite XAD-2 resin. The resin was subsequently eluted with MeOH. fhe biologically active MeOH extract was fractionated on Sephadex LH-20. The final purification step was performed by reversed- phase HPLC as outlined in the experimental section. Noscomin gave the |M-H]~ ion peak at m/z 425.2 by ES1MS, The IR spectrum revealed bonds for hydroxyl (3416 cm1), carboxyl (1686 enr1) and aromatic (1603 and 1546 cm-1) moieties. The Al-NMR spectrum contained signals for the meth\l groups, four tertiary (50.67, 0.88, - 0.89, 1,11. each s). one secondary (5 1.05 d,,/ 6.7 tlz) as well as signals indicative of a 1,2,4-substitutcd aromatic ring (5 6.74 d. J - 8.3 Hz. 5 7.70 d, J-- 6.6 II/, 5 7.80 s). Furthermore one proton attached to an oxygenated carbon (5 3.19 dd./- 4.76. 11.07 Hz) and an olefinic CH resonance (5 5.38 m) could be detected m addition to a number of aliphatic The ''C-NMR signals. spectral data of 1 showed the presence of one carboxy group (5C 172.0 s), one tertiary aliphatic oxygen-substituted C atom (5C 79.8 d). and one C-C double bond (5C 116.4 152.1 The d, s). observation of three quaternary carbon signals (5C 126.9 s, 161.1 s, 243 127.8 s) and three methine carbon signals (5c 130.1 d, 115.3 d, 135.3 d) confirmed the presence of a trisubstituted aromatic ring. Analysis of the DQF-COSY and the TOCSY spectra revealed spin system A (H3A6, H-13, II2- 12, H-l 1), spin system B (H3-I, H2-2, H2-I) and spin system C (H-8, H2A, H2-6, HA). The structure was assembled by an HMBC experiment. In particular, the spin systems B and C were connected by two fragments. One was determined by the correlations observed between H-8 (5 2.44, m, ß) and C-9 (5C 152.1 s) and between C-1 (40.8 t), C-5 (5C 45.7 d), C-9 (5C 152.1 C-10 39.2 and The second is on the basis of s) , (5C s) H^-24 (5 1.11, s). generated interactions from the quaternary carbon 0-4 (5C 40.2 s) to 11^-22 (5 0.89 s) and H3-23 (5 0.88, s), which both further correlated to (A3 (5 79.8 d) and C-5 (545.7 d). Couplings from H2-15 (52.46. d, J 13.2 Hz; d 2.89, d../- 13.2 Hz) to C-8 (5C 39.8 d). C-13 (5C 34.5 d), C-14 (5C 39.3 s), C-16 (5C 127.8 s), C-21 (5C 161.1 s) and C-25 (5C 15.8 q) established that the aromatic ring was attached by a CH2-group (CH2-i5) to C-14. which is also the connecting carbon between the spin systems A and C. Further correlations from C-27 (5C 172.0 s) to H-l7 (5 7.80 s) and IT-19 (5 7.70, d, J - 6.6) enabled us to position the carboxy group at C-18 (5C 126.9 s). Interactions between lb-15 and C-21 located the aromatic hydroxy-substitutcd carbon (5c 161.1 s). The relative stereoconfigurational structure of 1 was detemiined by a 2D-LROFSY experiment. The t- ROESY spectrum exhibited the presence of ROEs indicating that H-13. CILr23, CH3A4 and CH3-25 were oriented on the same face of the diterpene plane (a), while H-3.11-5, H-8, CH2- 15, CH3-22 and CH3A2 were m ß-position. Noscomin showed antibacterial actnity against Bacillus cerens (MIC 32 ppm), Staphylococcus epidermidis (MIC 8 ppm) and Escherichia coli (MIC 128 ppm). These MIC values for noscomin arc comparable with those obtained for the standards chloramphenicol (B. cere us, MIC 8 ppm; 5. epidermidis, MIC 4 ppm) and tetracycline {E. coli, MIC 64 ppm). Experimental section General Experimental Procedures. Optical rotation was recorded with a Perkin Elmer 242 Polarimeter using MeOH as solvent. The IR spectra were measured on a Perkin Elmer system 2000 FT-IR infrared spectrometer as a pressed KBr disc, fhe UV spectmm was recorded in MeOH using an UVÏKGN 930 spectrophotometer. ES1MS spectra were measured on a Finnigan fSQ 7000 mass spectrometer, EIMS spectra on a Hitachi-Pcrkin Elmer- RMTJGM mass spectrometer at 70 eV. 'II- and 13C-NMR spectra were recorded with a Broker AMX-300 spectrometer operating at a basic frequency of 300 MHz. using solvent (OD3OD, [H d 3.31, J3C d 49.0) as a reference. HPLC separations were performed with a Waters model 244 590 pump connected to a Rheodyne HPLC injector, a Knauer variable wavelength monitor and a Knauer HPLC column (Hypersil ODS, 3 um, 250 x 16 mm). Sephadcx LH-20 (Pharmacia) was used for open column chromatography (column 4 x 100 cm). For TLC controls, RIM 8 F254 precoated sheets (0.25 mm, Merck) were used. All solvents were HPLC grade. Organisms and Culture Conditions. Nostoc commune Vaucher, designated strain EAWA.G 122b. was isolated from a sample collected a1 Mellingen, Switzerland, 1965. The culture is deposited in the Culture Collection of Algae at the Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG), Dübendorf, Switzerland. The cvanophyte was cultivated in 10-L glass bottles containing a modified inorganic culture medium (Z).9 The cultures were illuminated continuously with fluorescent lamps (Philips TLM/33 Rs 40 W) at 29 umol/s/m2, aerated with a mixture of 2% C02 in air and incubated at a temperature of 24 + 1 o°C. The cyanobacterial cultures were harvested after 25-30 days. The supernatant was separated from the cells by decanting and adsorbed on a column filled with 250 g Amberiite XAD-2 resin (non-polar, surface area 330 m-'g). Subsequently, the column was washed with McOH. Isolation of noscomin (1). The McOH extract (1.2 g) obtained from 85 L microscopically cell-free culture medium was applied to a gel-filtration column (4 x 100 cm, Scphadex LH-20, Pharmacia). Elution was carried out with 1.5 L MeOH ILO 1:1, 1 L MeOH/HoO 75:25, IL MeOH, 500 mL MeOH/acctone 1:1 and 200 mL acetone 100% to obtain 16 fractions (20-40 mg). Bioactive fraction 14 (40 mg, eluted with MeOH ILO 75:25) was subjected to reversed- phase LIPLC using MeOH/MeCN/H20 63:25:12 as an eluent to yield 1 (7.6 mg) as an amorphous white solid with purification by TLC analysis (RP-18, ACN/H20 7:3, Rf - 0.33). Antibacterial Assay. The MIC determination for noscomin were performed as previously described.10 Test organisms were Bacillus cereus (ATCC 10702. Gram-positive), Staphylococcus epidermidis (ATCC 12228. Gram-positive) and Escherichia coli (ATCC 25922, Gram-negative). Noscomin: white amorphous solid (7.6 mg): [«]2D5 -l 16e (c 0.1, MeOH); IR (ICBr),Vmax 3416, 1686, 1603. 1546 cmA UV ^°H 2$2 nm. EIMS (McOH) m/z (rcl. int.) [MA absent, 409 [M-OII1+ AD. 380 [M-C00H-H]4- (<1), 365 [M-C02-OH)|f (A), 275 lM~C8FI703i+ (75), 257 [M-C8H703-H20r (97), 107 [M-Cl9H3iO]+ (100); FSTMS [M-Hl" (MeOH) m/z (rel. int.) 425.2 (100); Al-NMR (300 MHz, CD3OD) and 1-A-NV1R (75.5 MHZ, CD3OD), sec Table 1. 245 Acknowledgments. The authors thank Dr. Hans-Rudolf Bürgi, Dr. Marianne Bosli and Frank Sunder (EAWAG, Dübendorf/Switzerland) for providing and cultivating the cyanobacterial material, Dr. Oliver Zerbe (ETH Pharmacy Department) for assistance in NMR measurements, Dr. Engelbert Zass (ETH Chemistry Department) for performing literature searches, Mr. Oswald Greter and Dr. Walter Amrein (ETF1 Chemistry Department. Mass Spectral Service) for recording mass spectra. References (1) Namikoshi, M.; Rinehart, K. L. J. Appl Phycol 1994, 6, 151-157 (2) Simonin, P.; Jürgens, IT. L; Rohmer. M. Tetrahedron Lett. 1992, 33, 3629-3632. (3) Moore, R. E.; Cheuk. C; Patterson, G. L. M. J. Am. Chem. Soc. 1984. 106. 6456- 6457. (4) Moore, R. E.; Cheuk, C; Yang. X. G.; Patterson G. M. L. J. Org. Chem. 1987,52, 1036-1043. (5) Schwartz, R. E.; Hirsch, C. F., Springer J. P.; Pcttibone. D. J.; Zink D. J. Org. Chem. 1987.52, 3704-3706. (6) Smitka, T. A.; Bonjouklian, R.; Doolm, L.; Jones, N, D.; Dectcr, J. B.; Yoshida. W. Y.; Prinsep, M. R.: Moore. R. F.; Patterson. G. M. L. J. Org. Chem. 1992, 57, 857-861. (7) Stratmann, KA Moore. R. L.; Bonjouklian. R.; Deeter. J. B.; Patterson, G. M. L.; Shaffer. S.; Smith, C. D.: Smitka. T. A. ./. Am Chem Soc. 1994, //6, 9935-9942. (8) Prinsep. M. R.; fhomson, R. A.; West. M. L.; Wylie, B. L. J. Nat. Prod. 1996, 59, 786-788. (9) Hughes, E. ().; Gorham. P. R.; Zehnder. A. Can. ./. Microbiol 1958, 4, 225- 236. (10) Rios. J. I..; Rccio, M. C; Villar, A. J. Ethnopharmacol 1988. 23, 127-149. 246 Table 1. Al- and 13C-NMR Chemical Shift Assignments (ppm) of 1 iH-NMR chemical shift d 13C-NMR position (mult,,.7-Hz) chemical shift 51 1 1.37 (HI. m, b), 40.8 t 1.93 (1H, m. a) 2 1.66 (1H. m. b), 20.8 t 1.84 (1H, m, a) 3 3.19 (1FI. dd, ./--4.7, ll.l) 79.8 d 4 40.2 s 5 J.44(lH,m, b) 45.7 d 6 1.61-1.69 (2H)b 28.9 t 7 1.25 (1H, m, a). 20.4 1 1.29(1 H, m, b) 8 2.44 (1H, m, b) 39.8 d 9 152.1 s 10 39.2 s 11 5.38 (lFLm) 116.4 d 12 1.67 (1H, m. b), 31.6 cl 1.70(111, m, a) 13 1.55 (IH, m, a) 34.5 d 14 39.3 s 15 2.46(111. d..7 =- 13.2) 35.2 t 2.89(111, d, J = 13.2) 16 127.8 s 17 7.80 (IH, s) 135.3 d 18 126.9 s 19 7.70 (IH, d, J- 8.3) 130.1 d 20 6.74(111, d, J- 8.3) 115.3 d 21 161.1 s 22 0.89 (311. s) 28.2 q 23 0.88 (3IL s) 15.9 q 24 1.11 (311. s) 25.9 q 25 0.67 (3H, s) 19.5 q 26 1.05 (311. d, .7- 6.7) 15.8 q 27 172.0 s a Multiplicities deteimmed by DEFT 135 WIR expeiimctvts " Signal pattern uncleai due to o\eilapping 247 ëboH HO/,, 248 20 Publication 3 Novel Intracellular Diterpenoids with Biological Activity from the Cyanobacterium Nostoc commune B. Jaki,'" J. Orjala,'^ J. Heilmann. Ï A. LmdetA B. Vogler,1 andO. Sticher: i" Department of Pharmacy, Swiss Federal Institute of Technology (ETH) Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland, Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland, and Department of Chemistry, University of Hohenhcim, Garbenstrasse 30, D-70593 Stu itgart, Get many *To whom correspondence should be addressed. Ici: - Al 1 635 6050: Fax: \ VAX 1 635 6882. E-mail: sticher(Apharma.ethz.ch 1" ETH Zurich ^ University of Zurich *- University of Ilohenheim -'• Current affiliation: AgraQuest Inc.. 1 530 Drew A\enue. Da\is, CA 95616 249 Abstract. Five novel extracellular metabolites with an unprecedented diterpenoid skeleton, 5-[(5-carboxy-2-hydroxy)-benzylj-11 -hydroxymethyJ-2,5,6,8a, 11 -pentamethyl- do-decahydro-cyclopenta 2,5,6.8a, 1 l-pentamethyl-dodeca-hydrocyclopenta-'-(7^-naphtalene (4), and 5T(5~carboxy- 2-hydroxy)-benzylj-1 1 -acctyl-2,5.6,8a-tetramethyldodecahydrocyclo- pcnta All comnostins showed antibacterial acthities. Additionally, cytotoxic and moUuscicidal activities were found for comnostin B. Cyanobacteria arc known to produce a number of interesting secondary metabolites, including peptides, alkaloids, nucleosides and lactones.1 Nevertheless, the occurrence of diterpenoids in cyanobacteria is rather uncommon. To our knowledge there are only two reports. Prinsep et al. described the isolation of tolypodiol, a diterpenoid isolated from the cells of Tolypothrix nodosa.1 Recently, we described the structure of noscomin, the first reported extracellulary diterpenoid from the terrestrial cyanobacterium Nostoc commune Vaucher.3 In continuation of our studies of this cyanobacterial strain, comnostins A-E (1- 5), five biologically active metabolites with an unprecedented diterpenoid skeleton, have been isolated from the culture medium. Results and Discussion Nostoc commune (EAWAG 122b) was cultured in an inorganic medium. The culture medium was separated from the cells and subjected to a solid phase extraction on Amberiite XAD-2 resin. The resin was subsequently eluted with MeOH. 'fhe biologically 250 active MeOFT extract was fractionated on Scphadex LH-20. The final purification steps were performed by reversed-phase HPLC as outlined in the experimental section. The molecular formula of comnostin A (1) was deduced as C27H40O4 from ESI- and ' EIMS in combination with A NMR spectroscopy. The negative ESI mass spectrum gave the [M - H J" ion peak at m/z 427. The IR spectrum revealed absorption bands for hydroxy (3300 cm"'), carboxy (1710 cnT1) and aromatic (1604 cm"1 and 1520 ctiT1) moieties. The *H NMR spectaim contained signals for five methyl groups, four tertiary (5 0.86, 0.94, 0.99, LOO, each s) and one secondary (5 0.98 d, ,A 5.5 FIz) C-atoms as well as signals indicating a 1,2,4-substituted aromatic ring (8 6.93, d, /- 8.4 Hz; S 7.75, br d, /= 8.4 IIz; (5 7.89, br s). Furthermore, the methylene protons of a secondary alcohol group (8 3.28 d, 3.60, each d. ,7 - 10.4 Hz) could be detected in addition to a number of ' aliphatic signals. The A spectrum of 1 showed the presence of one carboxy group (<5C 167.8, s) and the previously deduced secondary alcohol moiety (<5C 69.8, t). The observation of three low field quarternary carbon signals (8C 122.0, 126.2, 161.4, each s) and three metbine carbon signals ( Analysis of the DQF COSY and the TOCSY spectra revealed spin system A (H3-26, H- 12, H2-l 1, Hr10). spin system B (H,-9. HrL H2-2). and spin system C (H-7, H2-6, H2- 5). The carbon skeleton was assembled by using the results of an flMBC experiment. In particular, the spin systems B and O were connected by two fragments. One was determined by the correlations observed between H-7 (8 1.02. m, ß) and C-8 (8C 37.9, s) and between C-9 (8C 59.3. d), C-8 (8C 37.9, s). C-7 (<5C 52.1, d), and Hj-24 (8 LOO, s). In addition the correlations observed between Hr21 (8 0.94, s) to C-5 (Sc 35.4. t), C-4 (Sc 48.2, s) and 0-3 (öc 56,0, s), which in turn showed correlations to H3-22 (8 0.99. s), Tl2- 23 (53.28, 3.60. each d, J- 10.4 Hz), H2-2 (8 1.63. m, /i. Ô 1.80. m, a) and Hrl (8 1.27- 1.43) completed the connectivities between spin system B and C. FIMBC couplings from H2-14 (8 2.66. 2.84, each d. ,7 = 14,0 Hz) to C-7 (Sc 52.1, d), C-13 (8C 42.0. s). C-12 (<5C 37.0, d). C-15 (<5C 126.2. s). C-16 (5C 135.5, s). CAO (Sc 161.4, s) and C-25 (oc 17.7. q) established the connectivity of the aromatic ring via a CfL-group 251 (C-14) to C-13 (5C 42.0, s), which in turn is the connecting carbon between the spin systems A and C. Interactions between FI2-14 and C-20 located the aromatic hydroxy- substituted carbon (5C 161.4, s). Further, the correlations from H-l6 (5 7.89, s) and H-l8 (5 7.75, d, J -- 8.4 Hz) to C-27 (5C 167.8. s) enabled us to position the acid moiety at C- 17. 'The relative stereo con figu rational structure of 1 was determined by a 2D-TROESY experiment. The spectrum exhibited the presence of ROEs indicating that CH3-2I, CH2- 23, CH3-24, CH3-25 and CH3A6 were oriented on the same face of the diterpenoid plane (a), while H-7. H-9, CH2~14 and CH3~22 were in the opposite plane (/?). This deduction was subsequently confirmed by a single-crystal X-ray structure analysis (Fig. 1), Comnostins B-E (2-5) were closely related to comnostin A. The molecular formula was deduced as 027H38O4 for comnostin B (2). CAFLgO.s 'or comnostin C (3), C29H44O5 for comnostin D (4), and as A7H^g04 for comnostin E (5) by ESI- and ETAIS as well as NMR spectroscopy. Comnostin B shows an aldehyde (C-23: 5C 205.2. d, H-23: 5 9.6, s) and comnostin C a carboxy group (5C 181.4, s) instead of the secondary alcohol group in position 23 of comnostin A. Comnostin D reveals an acetal group (C-23: 5C 114.9, d: C-24: 5C 60.1, q, H3-24: 5 3.37, s; C-25: 5C 56.7, q, H2,-25: 5 3.48, s) at position 23. It is possible that the acetal is generated by methylation of the aldehyde group of comnostin B during the extraction procedure with MeOFl. Therefore comnostin D may be an artifact of isolation and not a genuine natural product. Compared with comnostin A. comnostin E lacks the methyl group in position 22. An acetyl group replaces the secondary alcohol group (C-22: 5C 226.7, s; C-23: 5C 13.7. q; H- 23: 5 1.03, s). 252 LC-NMR-experiments. To exclude potential uncertainties from possibly incomplete axenic cultures, the cyanobacterial origin of the presented compounds was proven by conducting LC-NMR-experiments on the DCM-MeOH (2:1) extract of lyophylizcd cyanobacterial cells. The diterpenoid skeleton could be detected as a major compound in the cell material but in a minor concentration in comparison to the extracellular isolates. For the LC-NMR study, the crude DCM-MeOH (2:1) extract was separated into six fractions by VLC over Si gel employing an n-hexanc-FtOAc (80:20 to 20:80) gradient. Fraction 3 (ndiexane LtOAc. 60:40) was investigated by LC-NMR using the on-flow method (200 Lig injection, RP-18, ACN -ILO. 60:40, flow 0.7 ml/min). The diterpenoid skeleton could be identified by 'll NMR spectroscopy. As these medium derived compounds were found to occur in the cell extracts in a minor concentration it is suggested that they are actively released into the culture medium. Bioactivity, Comnostins C and E display a selective potent antibacterial activity. Comnostin C had a MIC value for Escherichia coli equal to tetracycline and comnostin E had a MIC value for Staphylococcus epidermidis equal to chloramphenicol. Moderate antibacterial activity against Bacillus cerens could be detected for comnostins A-E, against Staphylococcus epidermidis for comnostins A-D, and against Escherichia coli for comnostins A. B and D. Comnostin B additionally showed moderate cytotoxic activity in KB- and Caco-2 cell assays and a strong moUuscicidal effect against Biomphalaria glabrata. Details are listed in Table 1. Considering them as aethely released compounds and regarding the results of the biological testing it may be concluded that comnostins A-F play a special role in defense mechanism against enemies or other competitors. 253 Experimental section General Experimental Procedures. Optical rotation was recorded with a Perkin Elmer 242 Polarimeter using MeOH as solvent. Fhe IR spectra were measured on a Perkin Elmer system 2000 FT-IR infrared spectrometer as a pressed KBr disc. The UV spectra were recorded in MeOH using an UVIKON 930 spectrophotometer. ESIMS spectra were measured on a Finnigan ESQ 7000 mass spectrometer. EIMS spectra on a Hitachi-Perkin Elmer-RMUGM mass spectrometer at 70 eV. lH and 13C NMR spectra were recorded with a Braker AMX-300 spectrometer operating at a basic frequency of 300 MHz, using the solvent signal (CD3OD, 'H 5 3.31, 13C 5 49.0 or (CD3)2CO, 'H 5 2.05, 13C 5 29.8 and 206.0) as a reference. HPLC separations were performed with a Waters model 590 pump connected to a Rhcodyne HPLC injector, a Knauer variable wavelength detector, and a Knauer HPLC column (Hypersil ODS, 3 /an. 250 x 16 mm). LC-NMR experiments were outlined on a Varian 500MHz spectrometer, which was connected online to a Varian HPLC system with an analytic RP-18 column (Grom ODS, 5 /mi, 250 x 4 mm). ' MeOD as solvent was used as a reference (MeOD, 11 5 3.31). Sephadcx LH-20 (Pharmacia) was used for open column chromatography (column 4 x 100 cm). For TLC controls, RP-18 F254 precoated sheets (0.25 mm, Merck) were used. All solvents used were HPLC grade. Organisms and Culture Conditions. Nostoc commune Vaucher, designated strain EAWAG 122b. was isolated from a sample collected at Mellingen, Switzerland, 1965. The culture is deposited in the Culture Collection of Algae at the Swiss Federal Institute for Water Resources and Water Pollution Control (FAWAG), Dübendorf. Switzerland. The cyanophyte was cultivated in 10-L glass bottles containing a modified inorganic culture medium (Z).4 "fhe cultures were illuminated continuously with fluorescent lamps (Philips TLM/33 Rs 40 \V) at 29 //mol/s/m2, aerated with a mixture of 2% C02 in air and incubated at a temperature of 24 + 1 °C. The cyanobacterial cultures were harvested after 25-30 days. The supernatant was separated from the cells by filtration and adsorbed on a column filled with 250 g Amberiite XAD-2 resin (non-polar, surface area 330 m2/g). 254 Subsequently, the column was washed with MeOH. This MeOH extract was dried in vacuo at temperatures below 35 °C. Crystallographic Analysis of l5. A crystal with the composition C27H40O4TI2O obtained from MeOFI, was used for an X-ray structure determination. All measurements were made on a Rigaku AFC5R difffaetometer using graphitc-monochromatcd CuKa radiation (A ^ 1.54178 A) and a 12 kW rotating anode generator. The intensities were collected using co/20 scans, and three frequently measured standard reflections remained stable throughout the data collection. The data collection included the measurement of the Friedel opposites of all symmetry-unique reflections. The intensities were corrected for Lorentz and polarization effects, but not for absorption, fhe space group was determined from packing considerations, a statistical analysis of intensity distribution and the successful solution and refinement of the structure, Friedel pairs were not merged. The structure was solved by direct methods using SHELXS866, which revealed (he positions of all non-hydrogen atoms. The asymmetric unit contains two comnostin A molecules and two water molecules. The comnostin A molecules are of the same absolute configuration, and differ principally only in the orientation of the hydroxymethyl substituents. The difference in torsion angles about the C(3)-C(23) bond in molecule A and C(43)-C(63) bond in molecule B is about 103°. The non-hydrogen atoms were refined anisotropically. All of the H-atoms, except those of the hydroxy groups and water molecules, were fixed in geometrically calculated positions \d{C-K) - 0.95 A]. The hydroxy and water H-atoms were fixed in the positions indicated by a difference electron density map. The distances and angles, but not the orientation, of the hydroxy H-atoms were then geometrically optimised. Each H-atom was assigned a fixed isotropic displacement parameter with a value equal to 1.2Ceq of the atom to which it was bonded. Refinement of the structure was carried out on F using full-matrix least-squares procedures, which minimised the function Sw(\F0\ - \FC\)~. where w ^ |rj2(F0) t- (0.005Ao)2|A A correction for secondary extinction was applied. Data collection and refinement parameters are given in Table 4. A \iew of the molecule is shown m Figure 1. All calculations were performed using the TEXSAN crystallographic software package.7 The absolute configuration has not been determined. The cnanhomorph used in the 255 refinement was chosen arbitrarily. Isolation of comnostins A-E. The MeOH extract (1.2 g) obtained from 85 L microscopically cell-free culture medium was applied to a gel-filtration column (4 x 100 cm, Sephadex LH-20, Pharmacia). Elution was carried out with 1.5 L MeOHALO (1:1), 1 L of MeOHAAQ (75:25). 1 L of MeOH, 500 ml of MeOH-Me2CO (1:1) and 200 mL acetone 100% to obtain 16 fractions (20-40 mg). Bioactive fraction 9 (79 mg, eluted with MeOHALO, 75:25) was subjected to reversed-phase HPLC using McOI-I-McCN -TT?0 (63:25:12) as an eluent to yield comnostin C as a pure compound (3.5 mg) in addition to fractions 1-3. Of these fractions, one (fraction 2) was rechromatographed on reversed- phase material with MeCNAAO (80:20) as eluent to give comnostin A (8 mg) and comnostin B (9 mg). Purification of the bioactive fraction 7 (50 mg, eluted with MeOH-H20, LJ) on reverse phase HPLC using MeOH-ILO (40:60) as an eluent, gave comnostin D (3.3 mg) and comnostin E (4.0 mg). Biological Testing Antibacterial Assay. The MIC determinations for comnostins A-E were performed as previously described8. Test organisms were Bacillus cerens (ATCC 10702, Gram- positive), Staphylococcus epidermidis (ATCC 12228. Gram-positive), and Escherichia coli (ATCC 25922. Gram-negative). Cytotoxic Assay. The EIAq values9 for comnostin B were assessed using a KB cell line (ATCC CCL 17) as well as a Caco-2 cell line (ATCC HTB-37). The volumes were modified for cultivation in 24-well plates with Eagles Minimum Essential Medium (21090-022; Gibco. LifeTechnologies. Switzerland) for KB cells and Dulbccco's Modified Eagles Medium (31966-021. Gibco, Life Technologies. Switzerland) for Caco- 2 cells. 256 ÎVlolluscicidal Assay. The MIC determination for comnostin B was performed with freshwater snails of the species Biomphalaria glabrata.10 Physical and spectroscopic data Comnostin A (1): white, amorphous solid (8.2 mg); m p. 255 °C; [afD5 IA4° (c 0. t, MeOH); IR (KBr) Vmax 300. 1710, 1604, 1520. 1130 em4; UV A ^H252 nm; !H NMR (300 MHz, (CTA)2CO) and nC NMR (75.5 Milz. (CD3)2CO), see Table 2 and 3. EIMS - m/z (rel. int.) [M][ absent, 411 [M GYVf C 1 )• 382 [VI - COOH - H]h 0 1), 367 [M - - C02 All* AD, 277 [M C8II70^]u (100). 259 [M CgII703 - WyCif (65). 151 |M - C^IL^O]1 (50), 107 [M C19H (100). Comnostin B (2): white, amorphous solid (9.1 mg); m.p. 258 °C; [cc]DA45°(c 0.1, MeOII); IR(KBr) vmax 3283. 1704. 1602, 1512 cm4; UVA^a°H251 nm; lH NMR (300 MHz, (CD3)20O) and nC NMR (75.5 MHz. (CD,)2CO). see Table 2 and 3; EIMS m/z (rel. int.) [Mj1 absent. 409 |M - OHf (A). 381 [VI COOHj1 (A), 275 [C8I170314 - (100), 247 [M C8H70; COP (51). 151 [M - Cj^Of (64), 107 [M - C1QFl31OJ+ (24); ESIMS m 'z (rel. int.) [M - Hj~ 425 (100). Comnostin C (3): white, amorphous solid (3.5 mg); m.p. 252 °C; [afp ' 37°(c 0.1, MeOH); TR(KBr) Vmax 3350. 1715. 1620, 1525 cm"1; UVA^M25l nm; 'll NMR (300 MHz, CD3OD) and nC NMR (75.5 MHz. CD,OD). ->ee Table 2 and 3; EIMS m/z (rel. int.) [M|* absent. 425 [M OHJ (A). 396 [M COOH-H] A< 0. 381 [M- C02 - - OHf A 1), 291 [M C8HA),] (2), 274 [M C8H70, - OHjJ (81), 273 |M - 08H7O3 - - H201H (8), 274 lM G8HAA OHj4 (273). 151 [M - C,9H3J02]4 (22), 107 LM - - C]9H3102 C02]H (48): FS1MS m z (rel. hit ) [M - H]A41 (100). ->57 Comnostin D (4): yellow, amorphous solid (3.3 mg); m.p. 251 °C; [a]'D+2\°(c 0.1, MeOH); IR (KBr) Vmax 3350, 1710, 1622, 1525 cm4; UVA^ea°H246 nm; lH NMR (300 MHz, CD3OD) and nC NMR (75.5 MHz, CD3OD), see Table 2 and 3; EIMS m/z (rel. int.) [Mf absent. 429 [M C02 - Ilf (A), 321 |M - C8H70^r AD, 151 fM - C21H37021H (4), 107 [M-AAjH^AV C02J (19); ESIMS m/z (rel int.) [M-HV471.3 (100). Comnostin E (5): yellow amorphous solid (4.0 mg): m.p. 256 °C; [af£ 1 15°(e 0.1, MeOH); IR (KBr) Vmax 3345, 1722, 1623, 1525 cm4: UVA^fH247 nm; Hl NMR (300 MFIz, CD3OD) and nC NMR (75.5 MHz. CD3OD), see Fable 2 and 3; EIMS m/z (rel. int.) [M]"1 absent, 409 [M - OH|' (<1), 382 [M - CAf (< I), 381 [M - COOTT|+ A 0, ' ' 380 [M - COOH H] (A). 365 |M - C02 -- OH] (A). 275 [M - C8H705]h (36), 257 [M- CgH703 - FLO]1 (6).151 [M - Cl9ll310] (6). 107 [M C19H>,0 - C02]h(54); ESIMS (MeOH) m/z (rel. int.) [M - FI]A25 (100). Acknowledgments We thank Dr. Hans-Rudolf Bürgi, Dr. Marianne Bosli, and Mr. Frank Sunder (FAWAG, Dübcndorf/Switzerland) for providing and cultivating the cyanobacterial material, Dr. Oliver Zerbe (ETH Pharmacy Department) for assistance in NMR measurements, Dr. Engelbert Zass (ETH Chemistry Department) for performing literature searches, Mr. Oswald Greter and Dr. Walter Amrein (ETH Chemistry Department, Mass Spectral Service) for recording mass spectra as well as Mr. Michael Wasescha for technical assistance and performing KB-cell assays. References and Notes (I) Cannell, R. J. P. Pesticide Science. 1993. 39, 147 153. 258 (2) Prinsep, M. R.; Thomson, R. A.; West, M. L.; Wylie, B. L. /. Nat. Prod. 1996, 59, 786A88. (3) Jaki, B.; Orjala, L; Stichcr, O. J. Nat. Prod. 1999. 62, 502-503. (4) Hughes, E. O.; Gorham, P. R; Zehnder. A. Can. J. Microbiol. 1958, 4, 225 A36. (5) Crystallographic data (excluding structure factors) for the structure reported in this paper have been deposited with the Cambridge Crystallographic Data Center. Copies of the data can be obtained, free of charge, on application to the Director, CCDC. 12 Union Road. Cambridge CB2 1EZ, LK. (fax: +44-(0) 1223-336033 or e-mail: [email protected]). (6) Sheldrick, G. M. SHELXS-86, Acta Crvstallogr 1990, A46, 467-473. (7) TEXSAN. Single Crystal Structure Analysis Software, Version 5.0. Molecular Structure Cooperation, The Woodlands: Texas, 1989, (8) Rios, J. 1.; Recio. M. C; Villar, A. .7 Ethnopharmacol 1988. 23. Ill 449. (9) Swanson. S.M.: Pezzuto. J.M., In Drug Bioscrecning: Drug Evaluation Techniques in Pharmacolog]- Thompson. F.B. Ed.: VHC: New York, Weinheim, Basel, Cambridge, 1990. (10) Hostettmann, K.; Kizu. IF; Tomimori, T. H. Planta Med. 1998. 44, 34- 35. 259 Table 1. BiologicalActivities of the isolates Antibacterial Compound activity[MIC] MoUuscicidal Cytotoxicity activity[MIC] [ED50] B c S.e. E.c. B glahraia KB cells Caco-2 cells 32 comnostin A (1) ppm 16 ppm 128 ppm comnostin B (2) 32 ppm 16 ppm 128 ppm 20 ppm 0.40 ppm 0.18 ppm comnostin C (3) 32 ppm 16 ppm 64 ppm comnostin D (4) 16 ppm 32 ppm 128 ppm comnostin E (5) 128 ppm 4 ppm chl oramphenicol 8 ppm 4 ppm tetracycline 64 ppm CuS04 100 ppm podopliyllotoxin 0.01 ppm 0.02 ppm ~ Bacillus ejndumtdis B c cereus S e Staplnloc occ us 12 c Lsiluiuhia coli B glahiala Biomphalaria glabrata Tabic 2. lR NMR SpectralData of 1-5 (300 MHz, S ppm. J Hz) H ja 2a 3b 4b 5' 1 1.27-1.43c 1.37-1.49c 1.42-1-46' 1.34-1-45'' 1.75-1.82e ß) .ß) 2 1.63(111. m. 1.64(111. m 1.27(111. m /A 1.16 (ill.m. A 2.03 (111.m, ß) 1.80(111.m. a) 1.73 (IH, m, a) 2.48(1H. in a) 1.68 (IH. m. a) 2.45 (IH, m. co 3 1.81-1.86e A) 5 1.14 (1H. m. 0) 1.08 (IH. ni 1.79(1 H. m A 1.52 (III,m. A 1.20 (IH, m. 0). 1.43 (IH, m, a) 1.15 (ill.m, a) 1.84 (IH. m a) 1.63 (IH. m, a) 1,33 (IH. m. a) 6 1.63 (IH. m. a) 1.66 (III.m, a) 1.63(1H, m o). 1.48(111.in. a), 1.60 (IH. m. a). ß) ß) 2.05(111. m. 2.13 (111.m./3) 2,15 (111. m ß) 2.01 (III.m. 2.13(111. m.,ß) 7 1 02 (HI. m. ß) 1,04-1,08' 0 96 (111. m ß) 0 86-0.87' 0.89-0 90' ßj 9 1 1.38 1 45 (lH. m. /J) (IH, m. 40(lll.m ß) 1.30(1 H. m, ß) 1.43 H H. m./?) 10 1.24-1 32r 1.28-1.34'' 1.33-1.38' 1.22-1 29' 1.57-1,61' 11 1.16-1.33' 1.17-1.39' 1.29-1.31' 1.17-1.38' 1.27-1.32' 12 1 27' 1.25' 1.25' 1.22' 1.33' 14 2 2.62d(139 . 2.61 66(1(14.0) 2.65(1(14.1) (1(14.1)5 2.65d(14.0). 2.84 d (14.0) 2.88d(14.1) 2.81 d (13.9 2.86(1(14.1) 2.80(1(14.0) 16 7.89 br s 7.99 br s 7.84 br s 7.77 br s 7.83 br s 18 7 75brd(8.4) 7 76brd(8.4j 7.70 brd (8.3) 7.65 br d (8.4) 7.50 brd (8.4) 19 6,93 d (8.4) 6.95 d (8.4) 6.78 d (8,3) 6.72 d (8.4) 6.79 d (8.4) 21 0 94 s 0.98 s 1,16s 1.16 s 0.96 s 22 0 99 s 0.92 s 0 94 s 0.84 s 23 3 28 d (104) 9 60 s 4.15 s 1.03 s 3.60 d (10.4) 24 1.00 s 0.95 s 0.93 s 3.37 s 0.93 s 25 0.86 s 0.87 s 0.85 s 3.48 s 0.86 s 26 0.98 d (5.5) 1.02 d (6.2) 1.01 d(6 0) 0.88 s 1.07 d (7 5) 27 0.80 s 28 0.96d(5.5) 'measured in (CD3)2CO.'measured in MeOD. ^Multiplicityand intensityof the signalsare unclear due to overlapping. Table 3. 5C NMR Spectral Data of 1-5 (75.5 MHz, 5 ppm)/ C b ,b 1 3e 5e î 20.6 t 20.4 t 20.0 t 20.5 t 20 l t 2 34.5 t 43.2 t 32.7 t 34.5 t 37 61 3 56 Os 59.1 s 57.7 s 52.9 s 51 6 s 4 48 2s 46.9 s 46.6 s 47.0 s 40 7 s 5 35 4 t 36.9 t 34.4 t 35.5 t •37 2 t 6 20 71 20.4 t 20.9 t 20.8 t 22 9t 7 52 1 d 51.8 d 52.3 d 52.5 d 51 6d 8 37 9s 38.0 s 38.3 s 38.3 s 38 7 s 9 59 3d 58.8 d 58.3 d 58.9 d 51 6d 10 40 7t 40.4 t 40.8 t 41.lt 40 5 t 11 28 Ot 29.0 t 28.4 t 28.4 t 28 51 12 37 Od 37.0 d 37.3 d 37.5 d 37 Od 13 42 Os 42.0 s 42.4 s 42.4 s 42 7s 14 37 1 t 37.0 t 37.4 t 37.41 37 9t 15 126 2s 126.1 s 126.6 s 126.6 s 127 8s 16 135 5d 135.4 d 135.9 d 135.8 s 136 Od 17 122 0 s 121.9 s 121.8 s 122.6 s 122 2 s 18 130 1 d 130.1 d 130.4 d 130.3 d 130 4d 19 115 6d 115.7 d 1 15.6 d 115.5 d 115 5d 20 161 4 s 161.5 s 162.5 s 162.8 s 162 l s 21 18 3q 20.2 q 17.2 q 17.8 q 24 0q 22 23 5 q 19.7 q 23.7 q 16.9 q 226 7 s 23 69 81 205.2 s 181.4 s 114.9 d 13 7 q 24 16 7q 16.6 q 19.3 q 60.1 q 16 9q 25 17 7 q 17.8 q 18.3 q 56.7 q 18 2q 26 17 18.3 8q q 17.9 q 17.9 q 18 3q 27 167 8s 167.8 s 170.5 s 18.3 q 171 2s 28 17.5 q 29 171.1 s a '' Multiplicity by DEPT Measured 111 (CDfbCO,L mea>uied m MeOD Table 4. Crystallographic Data for (1) Empirical formula C27H40O4.H2O Formula weight 446.62 Crystal colour, habit colourless, tablet dimensions 0.15 0.40 0.40 Crystal [mm] _ _ Temperature [K] 297(1) Crystal system triclinic Space group P\ Z 2 Reflections for cell determination 25 20 range for cell determination [°] 93- 96 11.3260(8) b\A] 16.293(1) c[k] 6.625 (2) a[°] 90.04(1) ß[°] 92.28(1) y[°] 84.489(5) v[te\ 1215.8(3) F(000) 488 Dx [g cm-3] 1.220 p(MoKa) [mm-1] 0.655 20(max) [°] 120 Reflection ranges -12- h^ 12: -18 -=- k< 18; -7 < / <7 Total reflections measured 7684 Symmetry independent reflections 7684 Reflections used [l>2o(f)] 7322 Parameters refuted 575 R 0.0620 Rw 0.0654 Goodness of fit 4.815 extinction coefficient 10~' Secondary 5.6(6) _ Final Ampler 0.002 Ap (max; min) [c Â"3] 0.44: -0.34 264 l7ACM AN* Of.0 Figure 1. of the Ortep drawing two independent molecules of 1 at 297K, with 50% probability ellipsoids H atoms are represented by circles of aibitraiy radius 265 23 R> 111,-ClLOH 2 R, = CHO 3 R, - COOH v 7 14 OH 23 " v° \ 5 H^corAOC IE 24 25 IHC 266 21 Publication 4 New Antibacterial Metabolites from the Cyanobacterium Nostoc commune (FAWAG 122b) B. Jaki, J. Heilmann, and O. Sticher Department ofApplied BioSiences, Institute of Pharmaceutical Sciences, At iss Federal Institute of Technology (ETLI) Zurich, 8057 Zurich, Switzerland To whom correspondence should be addressed. Tel.: H-41 1 635 6050. FAX: 1 635 6882. E-mail: sticherApharma-c^lz-ch 267 Abstract. Two new compounds, a diterpenoid and an anthraquinone as well as an indan- derivative, which is reported as natural product for the first time, have been isolated from the cell material of the cultured cyanobacterium Nostoc commune Vaucher (EAWAG 122b) by means of bioguided isolation. The structures were determined with spectroscopic methods, mainly NMR, infrared spectroscopy and mass spectrometry. All isolates exhibit antibacterial activity. Terrestrial and marine blue-green algae have been proven to be an extremely valuable source of no\cl bioactive agents.1 We recently reported the structures of noscomin2 and comnostins A-E3. These bioactive compounds were isolated from the culture medium of the cyanobacterium Nostoc commune Vaucher (EAWAG 122b). In a continuing investigation of this cyanobacterium, we report here the isolation, structure elucidation, and biological activity of three further compounds, 8-[(5-carboxy~2,9-epoxy)-benzyl]- 2,5-dihydroxy-1.1,4a,7,8-pentamethy 1-1,2.3,4.4a,6,7.8,9.10,1 Oa-dodecahydro-phenan- threne (1), 1.8-dihydroxy-4-methyl-anthraquinone (2) and 4-hydroxy-7-mcthyl-indan-J- on (3) from the cell material. ESIMS of (1) gave the [M - H] ion peak at m z 439,2 and EIMS the LMf ton peak at m/z 440.1 analyzing for C-7HUlO,. The IR spectrum revealed bonds for hydroxyl (3416 cm '), carboxyl (1686 cm"1) and aromatic (1603 and 1546 cm ') groups. The 'H NMR spectrum contained signals for five methyl groups, four tertiary (8 0.87, 1.08, 1.11, 1.20, each s), one secondary (<5 1.14, d, J -- 6.9) as well as signals indicative of a 1,2,4-substitutcd aromatic ring (8 6.9, d, ,7 8.2 Ha 8 7.73. d. J - 1.9 Hz, 8 7.77, dd. /-= 1.9, 8.2 Hz). Further three protons attached to an oxygenated carbon (8 3.21, dd, 5.0. 1 1.2; 4.76, m; 4.25 ni) could be detected, m addition to a number of aliphatic signals (see 'fable I). The nC NMR spectral data of 1 showed the presence of one carboxy group (A 170.2. s), three tertiary (8C 64.8, 77.1, 79.5, each d) aliphatic ox\gen substituted G atoms, and one C=C double bond (8L 138.5, 146.9, each s). Further the observation of three low-field quaternary carbon signals (8{ 126.9, 127.0, 163.8. each s) and three methine carbon signals (A 121.6, 130.6. 133.6, each d) confirmed the presence of a tnsubstituted aromatic ring. 268 Analysis of the DQF-COSY and the TOCSY spectra revealed spin system A (H-,-26, H- 13, FI2-12, and H-l 1), spin system B (H,-l, H2-2, H-3), and spin system C (H-7, FL-6, FL 5). The structure was assembled by an LIMBC experiment. In particular, the spin systems B and C were connected by two fragments. One was determined by the correlations between H-7 (5 4.76, m) and C-8 (5( 138.5. s) as well as C-9 (8, 146.9, s) and between C- 1 (5C 36.2, t), C-5 (A 45.6, d), C-9 (5C 146.9. s), C-10 ( The second is generated on the basis of interactions from the quaternary carbon C-4 (5C 39.9, s) to TL-22 (5 0.87. s) and H,-23 (5 1.11. s). which both further correlated to C-3 (5C 79.5, d), and C-5 (5( 45.6. d). fhe spin systems A and C were connected by a fragment determined due to the correlations H-7 (5 4.76. m) to C-8 (5C 138.5, s) and C-14 (8 41.4, s) as well as H-13 (5 2.01. m) to C-8 (5 138.5 s). The ring skeleton of the diterpenoid was completed by the correlations between H-l 1 (5 4.25. m) and H2-12 (5 1.44, dd J -- 2.9. 5.8) to C-9 (Sc 146.9, s). Couplings from FL-15 (5 2.62; 2.77, each d, ./=- 14.1 Fl/) to C-8 (5C 138.5, s), C-13 (5C 34.4. d). C-14 (5C 41.4. s), C-16 (5t, 127.0. s). C-21 (5C 163.8, s), and CH,-25 (5C 26.4, q) established that the aromatic ring was attached via a CFL-group (<5( 38.2, t). Correlations between H-7 (5 4.76. m) and C-21 (5C 163.8, s) determined the ether connection between the aromatic ring system and the diterpenoid skeleton. Further correlations from C-27 (8C 170.2. s) to H-17 (5 7. A, d, ,7 -= 1.9 1 Iz) and FL 19 (S 7.77, dd, J= 1.9. 8.2 FIz) enabled us to position the carboxy group at C-18 (5C 126.9, s). Interactions between HA 5, H-7, and C-21 located the aromatic hydroxy-substituted carbon at C-21. The relative stereochemistry of 1 was determined by a 2D TROESY experiment. The TROESY spectrum exhibited the presence of ROEs indicating that H-13, Hr23, Hr24, and Hr25 were oriented on the same face of the diterpenoid plane (a), while H-3, FI-5, and H-7, 11,-2-- were in ß-position. Comprising the same diterpenoid skeleton with an additional ether ring connection, compound 1 is structurally related to noscomin/ ^ EIMS of (2) ga\e the [M H] ion peak at m z 255.2 analyzing for CA1AV fhe 'H NMR spectrum of (2) contained signals for one methyl group attached to an aromatic ring (8 2.60, s). as well as two aromatic doublets (5 7.21, d,./ - 8.7 Hz; 7.49, d. / - 8.7 Hz) 269 and three aromatic double doublets (5 6.70, dd, J = 2.7, 8.0 Hz; 6.74, br d, J -= 8.0 Hz; 6.76, dd, .7-2.7, 8.0 Hz), (see Table 1). In the nC NMR spectrum a total of 15 carbon signals could be observed. The spectrum shows the presence of one methyl group (5C 22.5 q), two carbonyl groups (5C 191.2. 184.8, each s), and two aromatic oxygen substituted carbons (5t 149.3, 161.1, each s). Additional, five low-field mcthine carbon signals (5C 116.5, 117.8, 119.1. 125.9, 139.4, each d) and four low-field quaternary carbon signals (SL 115.2, 120.9, 134.4, 150.5. each s) could be detected (sec Table 2). The structure was assembled by an FIMBC experiment. Ring A was established by the couplings from HA I (8 2,60, s) to C-4a (5C A4.4. s), 0-4 (5C 128.3, s), and C~3 (5C 139.4, d); from H-3 (5 7.49. d, ,7-8.7 Flz) to C-1 (5f 161.1, s). C-4a (t% 134.4 s), and C-4 (5C 128.3, s), as well as from H-2 (5 7.21, d, .7 8.7 Hz) to C-9a (5C 115.2. s). Correlations observed from H-5 (5 6.76, dd,./ - 2.7, 8.0 Hz) to C-lOa (5( 150.5, s), C-6 (5C 117.8, d), and C-7 (5( 116.5. d), from H-6 (5 6.74. br d, .7- 8.0 Hz) to C-7 (Sc 116.5. d), C-8 (5C 149.3. s), and C-10a (5( 150.5. s). and from H-7 (8 6.70, dd, J - 2.7, 8.0 Hz) to C-6 (5C 117.8, d), C-5 (c\ 119.1, d), C-8 (5, 149.3. s), and C-8a (5t 120.9, s) generated ring C. An additional correlation could be observed between H-5 (5 6.76, dd, J 2.7, 8.0 Hz)andCM0(5( 184.8, s). The 1 hydroxy - 4 methyl substitution pattern of ring A instead of a 1 methyl - 4 hydroxy was proven by infrared spectroscopy, which showed two separated carbonyl bonds at ' ' 1608.0 cm belonging to the chelated carbonyl group C-9 and 1740.0 cm belonging to the free carbonyl group at C-10. To our knowledge compound 2 is the first anthraquinone isolated from a cyanobacterium. EIMS of (3) gave the [M I II] ion peak at m/z 163.1 analyzing for CAAA- r^ie Hi NMR of 3 contained signals for one methyl group (5 2.3S, s) attached to an aromatic ring as well as two aromatic (8 6.81; 7.38 each d, J - 8.5 Hz) and two aliphatic doublets (8 2.52, 3.15, dd, ,7- 5.5, 18.1 Flz: see Table 3). The nC NMR data were assigned indirectly by analysing HMQC and HMBC spectra (see Table 2). These data comprise typical shifts for five low-field quaternary (8( 207.4, 162.3, 129.2, 143.2, 117.6 each s) and two low- field tertiary (5C 118.7, 136.2. each d) carbons, two aliphatic CH, moieties (Sc 31.3, 33.4, each t), and one methyl group (5( 18.1, q). 270 An HMBC experiment established Ring B by the couplings from H-5 (5 7.38, d, J= 8.5 Hz) to C-4 (5C 162.3, s), C-6 (5C 136.2, d), and C-7 (5C129.2, s), from Hr8 (5 2.38, s) to C-7 (5C 129.2, s), C-7a (5C 143.2, s), and C-6 (5C 136.2, d), and from H-6 (5 6.81, à,J = 8.5 Hz) to C-7a (5C 143.2, s). Correlation observed from H-2 (5 2.52, 3.15, dd, .7- 5.5. 18.1 Hz, each) to C-3 (5t 31.3, t). C-3a (5C 117.6. s), and C-1 (<5C 207.4, s), and from H-3 (5 1.63, 2.66) to C-4 (5C 162.3, s), C-2 (5t 33.4, t). and C-7a (50 143.2, s) generated ring A. Although, it is known as a synthetic compound', this is the first report of 3 as a natural product. Compound 1 displays a selective potent antibacterial activity against Staphylococcus epi¬ dermidis equal to chloramphenicol. Moderate antibacterial activity against Staphylococcus epidermidis could be detected for compounds 2 and 3, and against Bacillus cereus for compounds 1, 2, and 3. Data arc listed in Table 3. Experimental Section General Experimental Procedures. Optical Rotation was recorded with Perkin Elmer 242 Polarimeter using McOH as solvent. The IR spectra were measured on a Perkin Elmer 2000 System FT-IR infrared spectrometer as liquid films on a pressed KBr disc. The UV spectra were recorded in MeOH using an H vi con 930 spectrophotometer. ESIMS spectra were measured on a Finnigan TSQ 7000 mass spectrometer, EIMS spectra on a Hitachi-Perkin Elmer-RMUGM mass spectrometer at 70 eV. 'FI and nC NMR spectra were recorded with a Bruker AMX-300 or a Bruker AMX-500 spectrometer operating at a basic frequency of 300 or 500 MHz. respectively, using solvent 'H 5 ' (CIL,OFI. 3.31. A 5 49.0) as a reference. HPLC separations were performed with a Merck-Hitachi pump connected to a Rheodyne HPLC injector, a Merck variable monitor and a wavelength Knauer HPLC column (Hypersil ODS, 5 (im, 250 x 16 mm). Si gel was used for open column chromatography and VLC. For TLC controls, RP-18 sheets F254 precoated (0.25 mm, Merck) were used. All solvents were HPLC" grade. 271 Organisms and Culture Conditions. Nostoc commune Vaucher, designated strain EAWAG 122b, was isolated from a sample collected at Mellingen, Switzerland, 1965. The culture is deposited in the Culture Collection of Algae at the Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG, Dübendorf, Switzerland). The cyanophytc was cultivated in 10-L glass bottles containing a modified inorganic culture medium (Z)4. The cultures were illuminated continuously with fluorescent lamps (Philips TLM/.33 Rs W) at 29 umoLs/nr, aerated with a mixture of 2% C02 in air and incubated at a temperature of 24 ± 1 °C. The cyanobacterial cultures were harvested after 25-30 days, filtrated and lyophylized. Isolation. The MeOH/lLO 70:30 extract obtained from 43 g lyophylized cell material was applied to a Si gel vacuum liquid chromatography. Elution was carried out with a step gradient hexane/EtOAc (90:10 to 10:90) mixture to obtain 11 fractions. Fractions 5 and 6 exhibited antibacterial activity. Bioactive fraction 6 (80 mg, eluted with hexane/EtOAc 1:1) was subjected to a Si gel open column chromatography using CHCyMeOH 30:70 as eluent to yield 5 fractions. Of these fractions, one (fraction 4) was purified by means of reversed phase chromatography (MeCN/FLO 60:40) and gave I (4 mg). Bioactive fraction 5 was subjected to a Si gel open column chromatography using CHCyMeOH 45:55 as eluent to yield 8 fractions. Of these fractions, one (fraction 5) was purified by means of reversed phase chromatography (MeOH/ILO 65:35) and gave 2 (3 mg) and 3 (3 mg). Antibacterial Assay. The MIC determination for 1, 2 and 3 was performed, as previously described." Test organisms were Bacillus cerens (ATCC 10702) and Staphylococcus epidermidis (ATCC 12228). Physical and spectroscopic data Compound 1: white amorphous solid (4 mg): [a]"- A 5° (c 0.1, McOH); YNXM^U 280 nm; !H NMR (500 MHz. MeOD) and nC NMR (125 MHz. VIcOD) see Table 1 and 2. EIMS (MeOH) m z (rel. int.) 440 [M] ( 1), 422 PM-MLO] (< 1), 389 [M 3 OFF] (< i), - - 361 [M 2 OH COOH] (< 1). 289 [M CsH-0 ] « 1), 271 [M - CALOALOf A), 272 - 163 [M C18FL90,f (< O, 119 [M - ClgH„A - C02]' (1); ESIMS (MeOH) m/z (rel. int) [M-H]' 439.2(100). Compound 2: red oil (3 mg); R (KBr disc) vmax 3387, 1740, 1608 cm"1; UV^H 276 nm; 'H NMR (300 MHz. McOD) and nC NMR (75 MHz, MeOD) see Table l and 2; - - EIMS 254 [M]+ (6). 239 [M CIL] (28). 225 [M H - CO| (7), 207 [M - FI - CO - H,0]v (9). 189 [M H - CO - 2FLOr (9). Compound 3: yellow oil (3 mg): >H NMR (300 MHz, MeOD) and nC NMR (75 MHz, * MeOD) see Table 1 and 2; EIMS 163 [M Fl] (42), 147 [M + II - OAff (26), 135 [M + II- COP (93). Acknowledgment. The authors thank Dr. Hans-Rudolf Bürgi, Dr. Marianne Bosli and Frank Sunder (EAWAG, DübendorfAwitzerland) for providing and cultivating the cyanobacterial material, Dr. Oliver Zerbe (ETH Institute of Pharmaceutical Sciences) for assistance in NMR measurements, Dr. Engelbert Zass (E 1T1 Chemistry Department) for performing literature searches, Mr. Oswald Greter and Dr. Walter Amrein (ETH Chemistry Department, Mass Spectral Service) for recording mass spectra. References (1) Harrigan, G. G.; Yoshida, W. Y.; Moore. R. E.; Nagle, D. G.; Park, P. A; Biggs, L; Paul, V. P.; Mooreberry. S. L.; Corbett, T. H.: Valeriote, E. A. .7. Nat. Prod. 1998,67, 1221-1225. (2) Jakl, B., Orjala. J. E.; Sticher, O. J. Nat. Prod 1999. 62. 502-503. (3) Jaki, B.: Orjala, L; Heilmann, L; Vogler. B: Linden, A.; Sticher, O. J. Nat. Prod., in press (4) fobias, M. A., J. Org. Chem. 1970, 35, 267-269 (5) Hughes. E. O.; Gorham, P. R.; Zehnder, A. Can J. \th robiol. 1958, 7. 225-236. (6) Rios, J. 1,; Recto. M. C; Villar. A. ,7 Ethnopliurmacol 1988, 23, 127-149. 273 Table 1. 'H-NMR spectral data of 1-3 (300" or 5006 MHz, 8 ppm, J Hz, MeOD) r t 3" _h " 1 1.26 (ddd, 4.4, 11.3, 17.5, ß) 2.05 (ddd, 3.2, 12.7, 17.5. a) 2 1.73 (m) 7.21 (d, 8.7) 2.52 (dd, 5.5, 18.1) 3.15 (dd, 5.5, 18.1) 3 3.21 (dd. 5.0, 11.2) 7 49 (d. 8.7) 2.26e 1.63' 3a 4 4a 5 l.68(dd.3.2, 7.3) 6. A (dd. 2.7, 8.0) 7.38 (d, 8.5) 6 1.98 (d, 14.1, a) 6.74 (brd. 8 0) 6.81 (d, 8.5) 2.21 (d, 14.1, ß) 7 4.76 (m) 6.70 (dd. 2.7. 8 0) 7a 8 2.38 (s) 8a 9 9a 10 10a 11 4.25 (m) 2 60 (s) 12 1.44 (dd, 2.9, 5.8) 13 2.01 (m) 14 15 2.62 (d, 14.1) 2.77 (d. 14.1) 16 17 7.73 (d, 1.9) 18 19 7.77 (dd, 1.9,8.2) 20 6.90 (d, 8.2) 21 22 0.87 (s) 23 1.11 (s) 24 1.20 (s) 25 1.08 (s) 26 1.14 (d. 6 9) 27 274 to to to to to to to to -'M'O^OOMvlxlC?! 4^ 4^ U> oO to o -J ff\ U) Ji W KJ — O iooov)o\ui4iwto OOP £> P> p p -J U 4> OO -o. to oo 4^ OJ -J ^D 'Oi o voooo\ O-* —' Oi O^ OO --J oo .— c> 4i. oo -vi --J r ro 4^ 4^ O OO Oh On tO ro - _ ,-, rv CO C> O « Ol O rt 03 -+ r_h "-• kD _D JD ^ » a D. P- Cu Q- O CU Çu r zi p. £. p o o to — K* o to Oh OO ^o to 4i OO to OO to 'Ol CN 4^ «Ol :oi P o o o <-v Of) oo to to vD OO 'Ol oc —* 4^ OO 4s. lo co CO CO co C/j CO d. a C-, CO CO O-, a. to¬ to oco -J 'Ol 'S i— OO oo to w oo 4^ to Oj —> —- oo O OO 'Ol o oo oo 4^ to to to 7i CO CO Q_ Q^ CO to Oh T1 0s Table 3. Biological Activities of the Isolates Compound Antibacterial Activity [MIC] Staphylococcus epidermidis Bacillus cereus 1 4 ppm 128 ppm 2 32 ppm 32 ppm 3 128 ppm 64 ppm chloramphenicol 4 ppm 8 ppm 276 23 22 OH 0 OH j 11 i AS*A-S.>\ c B A a sir T4 0 CH3 11 277 22 Publication 5 Two Novel Cyclic Peptides with Antifungal Activity from the Cyanobacterium Tolypothrix byssoidea (EAWAG 195) B. Jaki, O. Zerbe, J. Heilmann, and O. Sticher* Department ofApplied BioSiences, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETLI) Zurieh, CH-8057 Zürich, Switzerland * To whom the correspondence should be addressed. 'Pel.: 4+41 1 635 6050. FAX: ++41 1 635 6882. E-mail: [email protected] 278 Abstract Two novel cyclic tridecapeptides, tolybyssidins A (1) and B (2), were isolated Prom the culture medium of mass cultured cyanobacterium Tolvpothrix byssoidea (EAWAG 195) by means of bioguided isolation. The gross structures of these peptides were determined by ID and 2D NMR experiments and tandem mass spectrometry. Both peptides contain the non-natural amino acid dchydrohomoalanm (Dhha) as well as proteinogenic amino acids albeit with D- or L-configuration. The compounds exhibit moderate antifungal activity against yeast Candida albicans. 279 Introduction Cyanobacteria have received considerable attention in the past as a source of natural products with unique structures and biological acthities.1 Malyngolide,2 majusculamidc Cf and kawaguchipcptin A and F34 are well-described examples of bioactive agents from this group of ubiquitous prokaiyotic organisms. Herein, we report on the isolation and structure determination of two novel cyclic tridecapeptides with antifungal activity, tolybyssidins A and B, both isolated from the cyanobacterium Tolypothrix byssoidea (Flass.) Kirch. (FAWAG 195). Results and Discussion Isolation Tolypothrix byssoidea (EAWAG 195) was isolated from a sample collected in 1967 in Pokhara (Nepal) from a granite block and was subsequently mass cultured. The following separation from the culture medium included filtration to remove cell mass. Thereafter, the cell-free supernatant was subjected to an amberiite XAD-2 column. The amberiite resin was subsequently eluted firstly with MeOFl followed by DCM. The biological active MeOFI extract was separated by open column chromatography using CFICL, and MeOH as a step gradient mixture. Gross Structure Determination A was isolated as a Tolybyssidin white amorphous powder. The MALDI mass spectrum showed a positive ion peak [M4Nap at 1488 m>'z, indicating a molecular weight of 1465 for 1. 'fhe 'FI NMR spectrum in d3-methanol displayed 13 amide proton signals between 9.50 and 7.23 ppm that completely vanished upon exchange into fully-dcuterated methanol. In addition, signals occurred in the aromatic region coding for one phenyl group and doublets were found between 0.74 and 1.25 corresponding to 17 aliphatic 280 2.03 methyl groups. Surprisingly for the case of a peptide a singulct at ppm appeared indicative of an OCOCFL, group. The distribution of signals with doublets in the amide- proton region and signals covering the complete range between 5 and 0.7 ppm was a clear indication that the investigated compound was a peptide indeed. Further spectroscopic evidence supporting the classification of the compound as a peptide was derived from the HC NMR spectrum, which re\ealed a total of 13 (amide-) carbonyl and DEPT90 signals in the range between 166.1 and 174.6 ppm. By using DEPT135 experiments, 27 mcthine. 10 methylene, and 17 methyl signals were identified. These signals, along with 3 additional quaternary carbon signals at 130.3, 137.9 and 162.2 ppm, indicated that a total of 71 carbons were present in 1. From these data, 1 was undoubtedly identified as a peptide. Since the compound gave a negative response in the nmhydrin test, 1 had to be cither a cyclic peptide or an acyclic peptide with a protected N-terminus, Sequential resonance assignment was performed largely following the protocol developed by Wüthrich and coworkers". In a first step interpretation of the 80ms TOCSY and DQF-COSY spectra led to the classification of a total number of 13 different spin systems corresponding to the following ammo acids: Phe. Val, Thr, Arg, Leu. He, Ac¬ Thr, Pro, and Dhha. However, since the sequential resonance assignment procedure usually requires the primary sequence to be known m advance, the amino acid * identification was mainly based upon complete A assignments of the ammo acids and comparison of these values with the random coil carbon shifts. In almost ail cases very close correspondence was observed with de\iations being smaller than 1 ppm. The 1 J 3 detailed H and C assignment is given in 'fable 1. At* VI These spin systems were subsequently linked sequentially \ia (i,Al) IF.Fl NOE's. NOEs were obsened for Phe NH'Phe a-H, Phe NH lA a-H, Dhha NFFPhe a-H, Val1 NH/Val1 a-H, AcThr Nil AcThr a-H. Thr' NH Thr1 a-H. Ihr1 NH/Acfhr a-H. Val2 NH/VaP a-H. Vaf NH 'Ihr1 a-H. VaP NH Val' a-H, YaP NH VaP a-H, Arg NH/Arg a- H, Arg NH/Pro a-H, Leu NHAeu a-H, Leu NH Arg a-H. Thr2 NFLThA a-H, Thr2 NH/Lcu a-H. lie1 NHTle1 a-H, He1 NILAhr a-H, IA NFLIle2 a-H. He2 NFITlc1 a-Il. 281 Furthermore, amide protons typically displayed intraresidual and intcrcsidual (i,i-l) (FlN,C) HMBC correlations supporting the sequential assignment. Such FIMBC correlations were observed for Phe a-H/Phe C=0, Dhha ß-H/Dhha C=0, VaP a-H/DMia CA), Val' a-FI/VaP CAO. Ac-Thr a-FLAc-Thr C O. Thr1 a-H/Ac-Thr C-O, Thr1 a- FI/Thr1 C-O, VaP a-H/ThtJ CM), VaP a-H VaP C A), VaP a-H/VaP CA), VaP a- H/Vaf CM), Pro a-lLVaP CA). Pro a-H Pro C-O, Arg a-H/Pro CA), Arg a-H/Arg C-O, Ecu a-H/Arg C-O, Leu a-Il Leu C-O. Thr a-H/Lcu (AO, 'Thr a-H/Tlir7 0-0, lie1 a-FI/Thr2 C-0, He" a-ILTle1 A O. lie2 a-H lie1 CA), Ile2 a-H/Ile2 CAO. Phe a-FI/Ile2 CAO. These data, together with a molecular weight of 1465 as determined by MALDI mass spectrometry are consistent with the molecular formula OAAcAAA/- No FIMBC correlations between Ac- fhr a-H and Val1 C--0 as well as no NOE's between Ac-Thr NH and VaP a-H could be found. Flow ever, the ring connection between VaP and Ac-Thr could be confirmed by an NOE observed between VaP H,A and Ac-Thr H-,r6 and with the analysis of the tandem mass spectrum of 1 showing the fragment ion [M+FI- Dhha-Val-AcThr-Thr-Val-Val] at m/z 842. The following fragments that helped to establish the sequence of the molecule 1 were detected in the tandem mass spectrum: m'z (rel. int.) 1466.5 FM+Hp (72), 1423 [M+H- (CH,)AHp(25), 1323 [MAI-AcThr] (73), 1284 | M ALDhha-Valp (7). 1222 [M+H- AcThr-Thrl1 (6), 1123 [1423-H-Val-Val-Thr] (39), 1119 [ l423-(CH3),CPLP1u>Ilef (1), 1024 [M+H-Ilc-Phe-Dhha-Val 1 (100). 907 [1423AI-(CIL)2CH-Phe-Ile-I1e-Thrp (11), 842 LM+H-Dhha-Val-AcThr-Thr-Val-Val| (6). 722 [MAI-Phe-Tlc-Ilc-Thr-Leu-Arg]J (4), 699 [M-Thr-Leu-Arg-Pro-Val-Val-Thr] (5). ToTybyssidin B was obtained as yellow-white solid. MALDI MS and r'C NMR data were consistent with the molecular formula C7JFn,Nlt,01(S. The 'H NMR data indicated 2 to be a peptide as well. Due to the limited quantity of the isolated material carbon signals of 2 had to be exclusnely detected indirectly through HSQC and FIMBC correlations. The 282 detailed H and A assignment is given in Table 2. The NMR spectroscopic identification of 2 was performed analogously to 1. In this process the spin systems of the amino acid residues were again identified by interpretation of the TOCSY and COSY spectra and sequentially linked through NOE's and FIMBC correlations. NOEs were observed for Met NH/Mct a-H. Met NFL Tyr a-H. VaP NFI/VaP a-H, VaP Nil/Met a-H, Thr1 NH/Tln-1 a-H, Thr1 NTLVaP a-H. He NILIle a-H. He NH/Thr1 a-H. Thr' NH/ThP a- H, Thr2 NFl/Ile a-H, VaP NH/VaP a-H, VaP NH/Thr2 a-H, VaP1 NH/VaP a-H. VaP NH/VaP a-H, VaP NlLVaP a-H, VaP NH YaP a-H. Phe NH/Phe a-H, PhcNFI/VaP a-H, VaP NH/Val5 a-11, VaP NH/Phe a-H, Dhha-NH Yak a-Il, Arg NFI/Arg a-FI, Tyr NFl/Tyr a- H,TyrNH/Arga-H. The ring connection between Dhha and Arg could be unambiguously confirmed by analysis of the tandem mass spectrum of 2 with the fragment ion [M-FLPhe-Valp at m/z 1244 and the fragment ion of this fragment [1244-Dhha-Arg] at m/z 1004. Fhe following fragments, supporting the sequence of the molecule 2, were detected in the tandem mass spectrum: m/z (rel. int.) 1473 [M-HO] (3). 1447 [M-H-CH(CH,)2]' (24), 1445 fM-H- CHfOHjCPP]1 (100), 1383 [M-H-OFI(C6H()CH,] (10). 1372 [1473-Thr] (30), 1347 [1447-Val]1 (17), 1310 [MHI-Val-Dhha] (10), 1284 [1383-VaLf (15), 1245 |1445-Val- Thrp (5), 1244 [M-H-Phc-Val] (10). 1217 [1447-Met-Valp (4), 1183 [1284-Thr]' (< 1), 1052L1183-Metp(t). 1004 [1217 'H-Thr-llej (< I), 283 Absolute Stereochemistry Analysis of the acid hydrolysate as Marfey derivatives6 indicated two amino acids (Pro and Leu) of 1 to be D and 10 amino acids (Val1. VaP. VaP, AcThr, Thr', 'Thr, Arg, Ile1, He2, and Phe) to be L. All amino acid residues of 2 were determined to be L. Biological Activity Tolybyssidins A and B inhibit the growth of the yeast Candida albicans at a concentration of 32 /ig/mL for 1 and 64 /./g'mL for 2, respectively. A MIC value of 8 /ig/mL was detected for the reference compound miconazole. Experimental Section General Methods MALDI MS spectra were measured on a Perseptivc Biosystems Voyager Elite spectrometer and tandem VIS spectra on a Finmgan 1 CQ Ion Prep spectrometer. NMR spectra were recorded on a Bruker DRX-500 spectrometer operating at a basic H frequency of 500 MHz at 298K, using the solvent line (LAYOFF 'il 53.31, nC 549.0) for For homonuclear 2D TOCSY referencing. proton experiments (DQF-COSY , and NOESY ) standard experiments were performed with suppression of the methanol OH " line by low-power presaturation. [ H, A"] HSQC and HMBC1 experiments utilised pulsed-field gradients for coherence selection". HPI 0 separations were performed with a Merck-Hitachi pump connected to a Rheodyne HP FC injector, a Merck variable wavelength monitor, and Knauer HPLC columns (Hypersil ODS, 5 |im. 250 \ 16). Si gel (Si gel 60F,5„ 40 - 50 Jim. Merck) was applied for open column chromatography. For TLC controls, RP-18 F>tprecoated sheets (0.25 mm. Merck) were used. All solvents were of HPLC grade. 284 Organisms and Culture Conditions Tolypothrix byssoidea (Hass.) Kirch., designated strain EAWAG 195, was isolated from a sample collected in Nepal, 1967. The culture is deposited at the Culture Collection of Algae at the Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG). Dübendorf. Switzerland. The cvanophyte was cultivated in 10-L bottles containing a modified inorganic culture medium. The cultures were illuminated continuously with fluorescent lamps (Philips TLM/33 Rs 40 W) at 29 pmol/s/nP, aerated with a mixture of 2% CO, in air, and incubated at a temperature of 24 ± 1 °C. The cyanobacterial cultures were harvested after 25 - 30 days. The supernatant was separated from the cells by filtration and adsorbed on a column filled with 250 g of amberiite XAD- 2 resin (nonpolar, surface area 330 nr/g). Subsequently, the column was eluted with MeOH. Isolation of 1 and 2 The McOH extract (0.8 g) obtained from 90 L microscopically cell-free culture medium was applied to an open column (4 x 100 cm, Si Gel). Elution was carried out with CHCp/MeOFl as step gradient to obtain ten fractions (20 100 mg). Bioactive fraction 6 (80 mg, eluted with CHCP-MeOH 25:75) was subjected to reversed phase HPLC (UV detection. 220 nm) using MeCN ILO 1:1 as an eluent to yield four fractions (5 - 30 mg). Bioactive fraction 3 (30 mg) was rechromatographed with reversed phase HPLC with a MeCN/FLO 80:20 eluent to yield t (8 mg) and 2 (3 mg) as pure compounds. HPLC Analysis of the Marfe\ Derivatives of 1 and 2 To the acid hydrolysate (6 N HCl. 16 h. 110 "C) of a 200 /ig portion of 1 and 2, respectively, 100 /A of I-fluoro-2.4-dimtrophenyl-5-f-alanine amide (L-FDAA) in Mc2CO (10 mg/mL) and 200 /A of l M Naff CO, were added, and the reaction mixture was kept at 80 AN for 3 mm. Then 100 /iL of 2N HCl and 400 /A of 50% MeCN were added and analysed by reversed phase HPLC: Lichrosorb RP-18 (250 x 4 mm); gradient 285 elution from MeCN/ILX)/TFA 95:5:0.1 to MeCN/H20/TFA 40:60:0.1 in 60 min; IJV detection 340 nm, flow-rate 1.5 mL/min. Retention times of the standard amino acids are (min): L-Leu (41.42), D-Lcu (45.31), L-Phe (41.76), D-Phe (44.51), L-Ile (40.70), D-Tle (44.94), L-Val (37.07), D-Val (40.56), L-Pro (31.44), D-Pro (32.78), L-Arg (27.82), D- Arg (28.68), I.-Thr (26.84). D-Thr (28.42). L-AcThr (2.5.27). D-AcThr (26.05), L-Met (37.07), D-Met (38.25), L~Tyr (48.56), D-'Tyr (50.96). Retention times of the amino acids of 1: D-Leu (45.31), L-Phe (41.76), L-lle (40.70), L- Val (37.07), D-Pro (32.78), L-Arg (27.82), L-Thr (26.84). L-AcThr (25.27). Retention times of the amino acids of 2: L-Phe (41.98). L-llc (40.96), L-Val (36.86), L- Arg (27.98), L-Thr (26.84), L-Met (37.07), L-Tyr (48.56). Preparation of the Acetyl Derivative of Thr (Acetyl-Threonine, AcThr) Thr (5.6 mg), anhydrous pyridine (0.5 mL) and acetic anhydride (0.5 mL) were kept dark and at room temperature for 18 h. The reaction mixture was diluted with 2 ml, of water and stored at 4 °C for 1 h. Then it was applied to a reversed phase cartridge, which had been washed with 10 mL of water. Pyridine, acetic anhydride and non acetylated Thr were eluted with water, followed by the elution of acetylated compound with CHC1V Evaporation of CHCF under reduced pressure below 30 ÜC yielded 2.5 mg of the acetyl derivative, which was completely dried in a vacuum drying oven at 25 - 28 °C. The identity of AcThr was proven by lH NMR spectroscopy measured in CD,OD. in comparison to the 'H NMR spectrum of non acetylated 'Thr the dcrivatized compound showed the expected additional CfL, signal at 1.80 ppm and a double acctylation was excluded. Antifungal Assay Die MIC determinations for t and 2 were performed as described previously.11 The yeast Candida albicans (ATCC 26790) was applied as a test organism. 286 Acknowledgment We thank Dr. Hans-Rudolf Bürgi, Dr. Marianne Bosli, and Frank Sunder (EAWAG, Dübendorf, Switzerland) for providing and cultivating the cyanobacterial material. Dr. Peter James and Manfredo Quadroni (ETH Biochemstry Department, Mass Spectral Service) as well as Dr. Ernst Schröder (Finnigan MAT. Bremen) for recording mass and tandem mass spectra, and Dr. Engelbert Zass for perfonning literature searches. References (1) Patterson, G. M. L.; Larsen. L. K.; Moore, R. E. ,7. Appl Phycol 1994, 6, 151 A 57. (2) Cardcllina, J. FT; Moore, R. E.; Arnold. E. V.; Clardy, J. T. Org. Chem. 1979, 44, 4039A042. (3) Carter, D. C; Moore. R. IA Mynderse. J. S.; Niemczura, W. P.; Todd, J. S. J. Org. Chem. 1984.79.236 241. (4) Ishida, K.: Matsuda, H.; Murakami, M.: Yamaguchi. K. J. Nat. Prod. 1997, 60, 724A26. (5) Wüthrich, K. NMR of Proteins and Nucleic Acids. Wiley, 1986. (6) Marfey, P. Carlsberg Res. Commun 1984,79, 591-596. (7) Piantini, A; Sorensen. O. W.: Ernst. R. R. J. 4m Chem. Soc 1982,707. 6800 6801. (8) Braunschweiler, L.: Ernst, R. R. J. Magn. Reson. 1983, 53, 521 528. Bax, A.; Davis, D. G.,/. \lagn. Reson. 1985. 65. 355 -360. (9) Teener. J.: Meter. B. H.: Bachmann. P.; Ernst, R, R. J. Chem. Phvs, 1979, 71, 4546. (10) Bodenhausen, G.; Ruben, D, J. Chem. Phvs. Lett. 1980, 69. 1 85-189. A.: (11) Bax, Summers. M. .7 . im. Client. Soc. 1986.108, 2093 A094. (12) Keeler, J.. Clowes, R.1Y Da\is, A. F., Laue. E. D. in Methods in Enzvmol 1994, 279. 145 207. (13) Rios, .1. L.: Recio. M.C.; Villar, A. ,1 Etlmopharmacol 1988. 23, 127 -149. 287 Table 1. lB and 13C NMR Data of 1 in CD.OH Amino acid 'H 7 (Hz) 'V Amino acid 'H 7 (Hz) nn Phe 174.6 (s) Pro 1 173.7 (s) ~> 4.55 (m) 57,6 (d) 2 4.39 (dd.6.2 each) 62.2 (d) 3 2.99 (dd. 7.6,13.3) 37.3 (t) 1.94 (m) 30.6 (t) 3.19 (dd, 7.6,13.2) 2.21 (m) 26.1 p) A -T 137.9 (s) 4 2.07 (ni) 49.2 it) 5.9 7.29" 130.5(d) 5 3.64 (m) 6.8 7.30" 129.7 (d) 3.92 (m) 7 7.23" 128.0 (d) NH 8.75 (d,9.7) Dhha 1 166.1 (s) Ail 1 174.6 (s) io oo ~> cc 130.3 (s) 2 4.31 On) 54.2(d) 3 6.61 (q,7.0) 134.1 (d) 1.65 (m) 30.3 (t) 4 1.31 (d,7.0) 13 3 (q) 4 1.51 (m) 27.7 (t) Ml 9,50 (s) 5 3.09 (m) 40.5 (t) 6 162.2 (s) NH 8.23 (d,8.0) Val 171.0 (s) Leu 1 174.5 (s) 2 4.37" 61.5(d) 2 4.43 (m) 53.4 (d) -, 3 1.85 (nq 31.6(d) 1.67 (m) 41.8 (t) 4 0.97" 19.9 (q) 4 1.59 fin) 25.9 (d) y 0.95 (d,7.1) 19.4 (q) 5 0.92" 23.1 (q) NH 8.14 (d,7,7) 5' 0.91" 22.8 (q) 8.0 (d 7.4) Acetyl-Thr I 172.9(5) 172.4 (s) 2 4.39 (dd,7.2 58.0 each) (d) 4.65 (dd,8.3 each.) 59.9 (d) 3 4.23 (m) 71.6(d) 5.15 Cm) 68.1(d) 4 1.15 (d,6,3) 20.0 (q) 1.21 (d,6.2) 17.7 (q) 5 173.6 (s) 8.44 id,8.3) 6 2.03 (p 22.8 (q) Nil 7.82 (d,7.2) Ihr 1 171.0 (s) 173,3 (s) T 4.05 (dd. 7.2 60.9 each) (d) 4.43 (dd,9.7 each) 59.0 (d) 3 4.32" 68.7 (d) 1.83 (m) 39.6(d) 4 1.25 (d. 6.3) 20.4 (q) l.iO(m) 27.5 (t) to oo NH 8.29 (d,7.2) 1.36 (m) 0.88(t,7.1) 15.1 (q) 0.82 (d. 5.6) 12.1 (q) 8.55 (d. 9.7) Val 173.3 (s) 173.2 (s) 2 4.46" 60.1 (dj 4.10 (dd,6.2 each) 58.1(d) 3 32.2 2.12(m) (d) 1.85 (m) 40.4 (d) 4 0,97' 20.2 (q) 1.01 (m) 4' 0.77 (d. 6.6) 18.5 (q) 1.34 (m) 27.6 (t) NH 7.82 (d, 7.2) 0.82" 14.7 (q) 0.74 (d,6.9) 12.2 (q) 7.23 (6.2) 172.3 (s) 2 4 s 28(dd. 6.6,7.7 58.7 (d) 3 2 13 (m) 31.4(d) 4 0 92' 19.9 (q; X' 0 91' 19.4(a) NH 7 90id, 6 6) ' Multiplicityof the signalsis uncloai due to oveilapping ro o Table 2. 'H and 13C NMR Data of 2 in CD,OH Amino acid LI 7 (Hz) 'C Amino acid 'H 7 (Hz) DC Val1 173.9 Phe 1 (s) 173.3 (s) 2 4.43" 59.2(d) 2 4.54 (m) 56.9 (d) 3 1.81 (m) 32.6 (d) 3 2.58 (dd,8.7,16.2) 40.6 (t) 4 0.84 {cl,57) 19.9 (q) 3.17 (dd,8.9. 13,8) 4' 0.84 (d. 5 7) 15.2 (q) 4 138.8 (s) NH 8.58 (d,9.6) 5.9 7.20" 130.5 (d) 6.8 7.30 131.3(d) 7 7.29 (m) 129.8(d) NH 8.78 (d.8.7) 175.0 (s) VaP 1 to 173.8 (s) i 4.40" 61.2(d) 2 3.97 (dd, 5.9. 8 5) 61.3 (d) 4.03 (m) 68.8 (d) 3 2.1 (m) 32.2 (d) 4 1.22 kl. 5.8) 14.3 (q) 4 0.94' 19.4 (q) NH 8 30 (d.7.4) 4" 0.94" 14.6 (q) Nil 7.76 (d. 8.5) l 175.9 1 (s) Dhha 166.2 (s) i 4.41" 59.2 (d) 2 130.9 (s) 3 1.64 32.8 (m) (d) 6.64 (q,8.3) 134.0 (d) 4 1.16 (m) 27.8 (t) 4 1.29 (d. 7.3) 13.3 (q) 1.49 (m) NH 9.51 (st 5 0.92 (f,7.6) 19.9 (q) 6 0.87 (d,7.1) 12.2 (q; NH 8.04(d,9.1) TIA 1 173.9 1 (s) Arg 175.1 (s) 2 4.62 (dd,8.8 each) 61.2(d) 2 4.06 (dd,8.7 each) 56.6 (d) > j 4.00 (m) 66.5 -, (d) 3 1.66 (m) 32.7 (t) 4 1.20 (d,5.8) 15.9(g) 1.85(m) NH 8.48 (d,8.8) 4 1.53 (m) 27.7 (t) 5 2.80 (dd. 6.0. 13.0) 40.4 (t) 6 159.9 (s) NH 7.39 (d,8.7) Val 175.9 1 (s) Tyr 174.3 (s) 3 4.40" 59.7 (d) 2 4.05 (m) 58.2 (d) -, 3 1.91 (m) 30.4 (d) 2.76 3 (dd.7.6,13.2) 38.1 (t) to 4 0.88 (d. 7. i to ) 18.5(q) 3.28" 4' 0.94" 15.1 (q) 4 129.8 (s) NH 8.12 {d, 8.0) 5 7.08 (d,8.3) 130.6(d) 6 6.80 (d. 8.5) 116.3(d) 7 157.1 (s) 8 6.72 (d,8.3) 115.3(d) 9 7.02 (d,8.4) 130,6 (d) NH 8.93 (d,8.5) Val 173.8 1 (s) Met 172.4 (s) 4 23" 62.3 2 (d) 4.71° 58.2 (d) 2.12 (m) 3 1.81 31.6(d) (m) 34.8 (t) 0.95° 19.5 (q) 2.04 (m) 4' 0.95" 14.6 (q) 2.55" 32.3 (t) NH 7.92 (d,7.6) 2.60" 5 2.10 (s) 15.8 (q) NH 8.99 (d,8.4) 176.2 (s) 2 4.10 (dd,7.5 each) 61.6(d) 3 1.64 (m) 30.3 (d) 4 0.80(c),6.1) 19.4 (q) 4' 0.76 (d.6.4) 14.7 (q) NH 7.25 (d, 7.5) " Multiplicityof the signalsis unclear due to overlapping too OJ o < X o CO > 7j CO 0=0 Ü Ü X Ü « X X / o- -ÜI 0-—-ox o--o I 0 X X L // -Ü- -Ü- -Ü- z-—o-c/ X I x/x co z 1 o o ZX ü OJ CO m a 0=0 X > XZ p-, IÜ--Cl¬-ü Jj -C -j; X 1 Q 0_-Q—=0 o=o eo zi X ZI Ü co_ CO CO 0—-O--ü _J X X ZI 0=0 CO Ü—-o—U—ÜI Ü CO X zx 0=0 est p> X lü—ü O ü- ~Z C'¬ X o o o=p C\I .5- X X X -Ü- -Ü- -ü- -o- -z \ X Ü o I X I C\J IÜ—o ÜX CO X Ü Iü- -ü CO X o _J 3 0) Q 294 X cö -C ü > h- \ _j _j CO CO CO \cM X X X ü ü ü 1 1 CO X X X X o o--ü ü--o \ X 1 x X ü--ü-—z—-o--o--z--o--o-—z X 1 1 X 1 1 X 1 1 i ! \ CSI / o o o X X X = xz ü--ü--o-—ü _J Ü=0 o=c Ü ZI X X x: IÜ- -Ci- -o h" ZX o=o 0=0 x «N z CM CM Cs O) A X X < o-— "Z.—-ü--o--o -Ol _J / 00 z > XÜ— ü- -c CM zx o=o o o=o CO CÖ CO x; X X ZX J= ü~-o :C Q X lü—o- -o XZ o. *o ü o •ü CO O' \ X X O--z -o- -ü—- CO X co X I o--o ü- -ü X X "cö Ü > o co > 295 23 Discussion 23.1 Selection of Cyan abacterial Strains Forty-three different cyanobacterial strains, originating from Switzerland, Nepal and Austria were chosen for cultivation. They belong to five different families and therefore provide a broad spectrum of individual samples. The selected genera are all known to be sources of phytochemicai interesting compounds, however, most of them are not well documented in literature. 23.2 Cultivation Except of the field-collected samples the selected strains as well as the isolates resulting from field collected samples were cultivated in a suitable inorganic media. The medium was chosen with the 'selective media method' (see Chapter 6.2.1). The composition of the different media provides a spectrum of nutrients that fits for nearly every freshwater or terrestrial cyanobacterium. Large-scale cultivation with inorganic media is a technology, which makes it possible to cultivate cyanobacteria cheaply and easily in sufficient biomass under controlled conditions. Nevertheless, the cultnation of cyanobacteria has not yet reached the desired scale of mass cultivation in large tanks as achieved for other bacteria. Efforts to improve the cultivation technology with respect to the production of biomass arc of great importance. For this purpose conditions of growth and secondary metabolite production must be optimized. One problem in the eulthation procedure of cyanobacteria is to get axenic cultures. It is very difficult to separate the c> anobactcrial strains from accompanying bacteria because most of the antibiotics against bacteria would kill the cyanobacterial population as well. Therefore a regularly microscopically check of the cultures is necessary to make sure that the cyanobacterial cell mass is more or less free from accompanying bacterial ceils and do not become overgrown. 296 23.3 Biological Testing Biological evaluations of extracts are usually difficult because of cooccurrence of several classes of compounds, which can cause synergistic, antagonistic or other unpredictable modulations on the bioactivity. Furthermore, interesting lead compounds, which are present in minor quantities in the extracts, can simply be missed. Therefore, it is desirable to have high sensitive assays in order to detect even low amounts of any potent biological active constituent. High selectivity of a test system is required to limit the number of active extracts for further evaluation. Keeping these facts in mind, the cell extracts of the cyanobacterial cells were administered to different biological test systems as well as the extracts of the cell mass of the large-scale cultured strains and their culture media. In the same manner, fractions obtained during the separation process were investigated for their biological activities. In the end the resulting pure compounds were tested in accurate doses in the same bioassays used for the original screening. All investigated bacteria in the biological screening are able to induce a response in at least one of the test systems applied. The two strains selected for large-scale cultivation exhibit several biological activities, which led to the isolation of the presented biological active isolates by means of a bioassay guided isolation procedure. 23.4 Isolation Isolation of the pure compounds of the two large-scale cultured strains Nostoc commune (EAWAG 122b) and Tolypothrix byssoidea (EAWAG 195). yielded from both the cell mass and the culture medium, involved tedious procedures of multistep chromatography. In order to avoid or minimize artifact forming, fast methods of isolation have been preferred. Vacuum liquid chromatography (VLC) and open column chromatography were rapid and effective methods for preliminary fractionation of crude extracts and prepuriftcation of the fractions prior to HPLC separation. Further fractionation and purification, to provide compounds of suitable purity for structural analysis, were achieved by semipreparativc HPLC. Reproducibility and rapidity of the HPLC 297 chromatograms were essential for checking the stability of the components of a fraction. UV detection with suitable wavelengths was successively used for isocratic HPLC separations for all types of metabolites. Isolation procedure was guided by using TLC. !H NMR and bioactivity. !H NMR was found to be a sure method of monitoring and also for checking the stability of metabolites. The diterpenoids could be recognized on TLC by spraying vamllin/H,SOt reagent (stable pink colour) and the cyclic peptides by their UV2,4 quenching activity. 23.5 Structure Elucidation The structure determination of the isolated compounds mainly based on spectral methods. By means of UV spectroscopy, conjugated sv stems were established. General correlations concerning hydroxy 1 and carbonyl groups were recognized by their IR absorptions. Routine onc-dimensional NMR experiments provided the identification of the structural class of the isolates, their functional groups as well as the number of each nucleus. nC NMR data together with the information gained from the MS spectra allowed establishing the molecular formula, the degree of unsaturation and the number of rings present m the molecule. Using these informations together with those obtained from the two-dimensional NMR experiments, the planar structure of the metabolites could be determined. Carbon atoms were assigned with help of the FÏMQC correlations. After identification of the different spins systems by analyzing the TOCSY spectra the proton coupling network was peaced together with help of COSY and HMBC correlations. The Nuclear Overhauser experiments (NOESY. ROESY and TROESY) were necessary to determine the relative stereochemical stracture of the isolates and to assign the sequence of cyclic peptides, fhe primary structure of the cyclic peptides could be confirmed by tandem mass spectrometry. The relative stereochemistry of comnostin B (NC-3) was unclear by analysing the Nuclear Overhauser data due to overlapping peaks and therefore was confirmed by the results of an X-ray crystallographic analysis. The absolute configuration of the amino acid residues could de assigned by HPLC analysis of the Marfey's derivatives. 298 23.6 Biological A ctive Secondary Metabolites Cyanobacteria are known to be a rich source of secondary metabolites with a wide variety of biological activities. The majority of these metabolites have been found in association with the cyanobacterial cells. The occurrence of terpenoids in cyanobacteria is rather uncommon. The presented work describes the isolation of NC-1 NC-7, seven novel metabolites comprising two different unprecedented diterpenoid skeletons, from a cultured Nostoc commune strain. NC-5. NC-6 and NC-7 as well as NC-1 and NC-2 display a selective potent antibacterial activity. NC-5 has a MIC value for Escherichia coli equal to tetracycline, NC-7 as well as NC-1 and NC-2 have MIC values for Staphylococcus epidermidis in the range of chloramphenicol. Moderate antibacterial activity against Bacillus cereus could be detected for NC-1 - NC-5 and NC-7. and against Escherichia coli for NC-1, NC-3, NC-4. and NC-6. Additionally, NC-4 shows moderate cytotoxic activity in KB- and Caco-2 cell assays and a strong moUuscicidal effect against Biomphalaria glabrata. As these medium derived compounds were found to occur m the cell extracts in a minor concentration in comparison to their extracellular concentration it is suggested that they are actively released into the culture medium. Regarding the results of the biological testing it may be concluded that they play a special role m defence mechanism against enemies or other competitors. Two additional new compounds with unusual substitution patterns, an anthraquinone (NC-8) and an indan-dertvativ e (NC-9) were isolated from the MeOH/fFO (7:3) extract of the lyophylized cell material of the same Nostoc commune strain. NC-9 is known as synthetic substance, but it is new as naturally derived compound. NC-8 is a new anthraquinone comprising an unusual o/f/?o-substitution that is not explainable with the classical biosynthetic pathway of anthraquinones. The substitution pattern must have been changed afterwards. Both compounds show moderate antibacterial activity against Bacillus cereus and Staphylococcus epidermidis. 299 Cyanobacteria have received considerable attention as a source of cyclic peptides with different biological activities. The phytochemicai investigation of the culture medium of the large-scale cultured cyanobacterium Tolypothrix byssoidea yielded two novel extracellular cyclic tridecapeptides, designated as TB-1 and TB-2. Both compounds exhibit moderate antifungal activity against the yeast Candida albicans. 300 24 Conclusions Cyanobacteria are regarded as a source of physiologically potent and chemical interesting substances. The results of this investigation verify that cultured cyanobacteria are capable to accumulate a large number of interesting secondary metabolites in the cell mass as well in the culture medium and therefore give promise for further chemical and biological studies on cycanobacteria. For fttrther research the inclusion of following points would be of interest: • Great abundance of extracellular biological active secondary metabolites and the suggestion that they are actively released compounds may point out their ecological significance. Thus, it would be interesting to investigate their hazardous effects and ecological role. • Comparison of the phytochemicai spectrum of re-collected organisms with long-term cultured cyanobacterial strains may reveal the environmental influences on these organisms. • The presented diterpenoids (NC-1 - NC-7). the anthraquinone (NC-8) as well as the indan-derivative (NC-9) are unusual for cyanobacteria and it would be of interest to investigate their biosynthetic pathways in detail. • The influence of the composition of the culture medium and other culture conditions on the growth behaviour, biomass. and chemical drug production should be evaluated. These may enable further chemical and biological investigations of the minor derivatives of the described secondary metabolites. Regarding that most of the presented compounds are isolated from the culture medium of the cyanobacterial cultures it is prospective to optimize the culture conditions in regard to an enhancement of the release of these compounds in the culture medium. 301 • All isolated compounds show biological activity in at least one of the applied test systems. Therefore, further biological activity of the isolates on a broad basis should be evaluated. 302 Curriculum Vitae DATE OF BIRTH May 4th, 1970, Karlsruhe, Germany EDUCATION Humboldt Gymnasium, Karlsruhe, Germany (1980 - 1989) • Abitur, 05/89 Ruprecht Karls Universitiy. Heidelberg, Germany (1989 1994) • Pharmaceutical Science. Certification as Pharmacist, 1995 Swiss Federal Institute of Technology (ETH), Zürich, Switzerland • Ph.D. study under the supervision of Prof. O. Sticher, section Pharmacognosy and Phytochemistry, Institute of Pharma¬ ceutical Science, Department of Applied BioScienccs, since 1996 • Final examination to obtain the degree of Doctor of Natural Sciences, 01'2000 PRACTICAL WORK I,abor Limbach, Heidelberg, Germany • Laboratory assistant at a laboratory of medicinal microbiology (04'92 - 03/93) Ciba-Geigy GmbH, Wehr. Germany • Internship, Section Pharma, Analytical Control Laboratory (11794 -04/95) Fortuna Apotheke, Karlsruhe. Germany • Internship at a pharmacy (05/95 - 10/95) TEACHING Swiss Federal Institute of Technology (F'fH), Zürich, Switzerland • Tutor of the practical course work in phytochemistry and pharmacognosy for students of pharmaceutical science List of Publications B. Jaki, J. Orjala and O. Sticher, A Novel Extracellular Diterpenoid with Antibacterial Activity from the Cyanobacterium Nostoc commune, J. Nat. Prod., 62, 502-503 (1999) B. Jaki, J. Orjala, H.-R. Bürgi and O. Sticher, Biological Screening of Cyanobacteria for Antimicrobial and MoUuscicidal Activity, Brine Shrimp Lethality and Cytotoxicity, Pharm. Biol, 37, 138 - 143 (1999) B. Jaki, J. Orjala. J. Heilmann, B. Vogler, A. Linden and O. Sticher, Novel Extracellular Diterpenoids with Biological Activity from the Cyanobac¬ terium Nostoc commune, J. Nat. Prod., in press B. Jaki, O. Zerbe, J. Heilmann and 0. Sticher. Two Novel Extracellular Cyclic Peptides with Antifungal Activity from the Cyanobacterium Tolypothrix byssoidea (EAWAG 195),./. Nat. Pro., submitted B. Jaki. .1. Heilmann and O. Sticher, New Biological Active Compounds from the Cyanobacterium Nostoc commune (FAWAG 122 b), J. Nat. Prod., submitted B. Frei, VF Heinrich. P. M. Bork, D. Herrmann. B. Jaki, T. Kato. M. Kuhnt. J. Schmitt, W. Schühly and O. Sticher. Multiple Screening of Medicinal Plants from Oaxaca. Mexico: Ethnobotany and Bioassays as a Basis for Phytochemicai Investigation. Phvtomedicine, 5, 177-186 (1997) List of Poster Presentations B. Jaki, J. Orjala. H.-R. Bürgi and O. Sticher. Biological Activity of Cyanobacteria: Evaluation of Extracts and Isolates, 45th Annual Congress of The Society of Medicinal Plant Research, September 7* - P"1, 1997, Regensburg, Germany B. Jaki, J. Orjala and O. Sticher, Novel Extracellular Diterpenoids with Antibacterial Activity from the Cyanobacterium Nostoc commune, 46th Annual Congress of the Society of Medicinal Plant Research, August 31"'- September 4'\ 1998, Vienna, Austria B. Jaki. O. Zerbe. J. Heilmann and O. Sticher, A Novel Extracellular Cyclic Peptide with Antifungal Activity from the Cyanobacterium Tolvpothrix byssoidea, Joint Meeting of the American Society of Pharmacognosy. Ihc Society of Medicinal Plant Research, and the Phytochemicai Society of Europe, 1999, July 26th - 30th, Amsterdam, Ihc Netherlands Oral Presentations Biological Screening of Cyanobacteria and Phytochemicai Investigation of Nostoc commune. Annual Meeting of Ph D. students of the Swiss Federal Institute of Technology (ETH). October 21st, 1997, Zürich, Switzerland