AN ABSTRACT OF THE THESIS OF
Min Wu for the degree of Master of Science in Pharmacy
presented on September 13, 1996. Title: Novel Bioactive Secondary
Metabolites from the Marine Cyanobacterium Lyngbya majuscula
Abstract
ved:
Redacted for Privacy
William H, Gerwick
Marine algae have been recognized as a rich resource of new
and unusual organic molecules with diverse biological properties.
The current need to develop new antifungal, anticancer, antibiotic
and antiviral drugs has led to an intense research effort into the
discovery, isolation and structure determination of potential
medicinal agents from marine algae.
In the past two years, I have participated in a drug discovery
program designed for antitumor, antifungal and other agents of
potential pharmaceutical utility from the marine cyanobacterium
Lyngbya majuscula. This research utilized modern chromatographic
and spectrochemical techniques including 2D NMR spectroscopy.
Brine shrimp toxicity guided the fractionation that led to the
discovery of the biologically active compound kalkitoxin from a
Curacao Lyngbya majuscula extract. The structure of this new
thiazoline ring-containing lipid was determined spectroscopically
by interpretation of 2D-NMR experiments, including heteronuclear
multiple quantum coherence (HMQC), heteronuclear multiple-bond
coherence spectroscopy (HMBC) and 'H-'H COSY at room
temperature and elevated temperature. Kalkitoxin shows modest
molluscicidal toxicity, good brine shrimp toxicity and extremely
potent ichthyotoxicity.
From the same extract of Lyngbya majuscula, I also isolated
two other secondary metabolites, malyngamide J and malyngamide
L.
The structures of these new compounds, including
stereochemistry, were determined by spectroscopic techniques
including 2D-NMR experiments and by comparison with other
known malyngamides.
Novel Bioactive Secondary Metabolites from the Marine
Cyanobacterium Lyngbya majuscula
by
Min Wu
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed September 13, 1996
Commencement June 1997
Master of Science thesis of Min Wu presented on September 13,
1996
APPROVED:
Redacted for Privacy
Major Professor, representing Pharmacy
Redacted for Privacy
Dean of College of Pharmacy
Redacted for Privacy
Dean of Grad ate School
I understand that my thesis will become part of the permanent
collection of Oregon State University libraries. My signature below
authorizes release of my thesis to any reader upon request.
Redacted for Privacy
Min Wu, Author
ACKNOWLEDGEMENTS
I am extremely grateful to my advisor Dr. William H. Gerwick
for his patience, guidance, inspiration and support throughout my
graduate studies.
I would like to thank my committee members, Dr. Kevin Gable,
Dr. George H. Constantine and Dr. George S. Bailey for their valuable
advice and assistance.
I acknowledge the following people for their technical
assistance in my graduate research: Brian Arbogast for his
providing the high quality mass spectra and helpful suggestion;
Rodger L. Kohnert for his assistance to set NMR experiments.
Jeannie Lawrence for her generous help in obtaining CD data.
I would like to thank my lab colleagues for their help,
friendship and encouragement. I especially thank Namthip
Sitachitta, Mary Roberts and Brian Marqez for critically reading the
manuscript.
I am deeply grateful to my parents for their love, support and
confidence to me.
TABLE OF CONTENTS
Page
CHAPTER I:
GENERAL INTRODUTION
1
Biomedical Potential Marine Natural Products
2
Marine Toxins
7
Bioactive Natural Products From Marine Cyanobacteria
15
Descriptions of Chapters
17
CHAPTER II:
KALKITOXIN FROM THE CYANOBAC 1 ERIUM
LYNGBYA MAJUSCULA
18
Abstract
18
Introduction
19
Results and Discussion
24
Experimental Methods
47
CHAPTER III:
TWO NEW MALYNGAMIDES FROM THE
CYANOBACTERIUM LYNGBYA MAJUSCULA
51
Abstract
51
Introduction
52
Results and Discussion
56
Experimental
73
CHAPTER IV
CONCLUSION
76
Page
79
BIBLIOGRAPHY
APPENDIX:
Spectral Data
88
LIST OF FIGURES
Page
Figure
I.1
Structures of Several Marine Natural Products and
Derivatives with Biomedical Utility
3
I.2
Structures of Biomedical Potential Marine Natural
Products
4
1.3
Structure of Palytoxin
5
1.4
Structures of Several Marine Toxins
8
1.5
Structure of Maitotoxin
9
I.6
Structures of Brevetoxin-A and Brevetoxin-B
I.7
Structures of Bioactive Natural Products from
10
Cyanobacteria
12
Structures of Bioactive Natural Products from
Cyanobacteria
13
Structures of Bioactive Natural Products from
Cyanobacteria
14
II. 1
Structures of Natural Products from Cyanobacteria
20
11.2
Bioactive Metabolites from Cyanobacteria
22
11.3
Bioactive Metabolites from Lyngbya majuscula
23
11.4
Extraction of Lyngbya majuscula
26
11.5
Bioguided Fractionation of Kalkitoxin
27
II. 6a
Brine Shrimp Assay I for Bioguided Isolation of
1.8
1.9
Kalkitoxin
28
LIST OF FIGURES (Continued)
Page
Figure
II.6b Brine Shrimp Assay II for Bioguided Isolation of
Kalkitoxin
I1.6c
28
Brine Shrimp Assay III for Bioguided Isolation of
Kalkitoxin
II.6d Brine Shrimp Assay IV for Bioguided Isolation of
29
Kalkitoxin
29
11.7
The Overall Planar Structure of Kalkitoxin
31
11.8
Six Partial Structures of Kalkitoxin
31
11.9
'H NMR of Kalkitoxin in D6-DMS0 at 298K
33
II.10 Two slowly interconverting tert-amide isomers present
at room temperature 298K
34
II.11
'H NMR Of Kalkitoxin In D6 -DMSO at 340K
35
11.12
"C NMR Of Kalkitoxin In D6 -DMSO at 340K
36
11.13
"C NMR Of kalkitoxin In D6 -DMSO at 298K
37
11.14
'H -'H COSY Of kalkitoxin In D6 -DMSO at 298K
38
11.15
'H -'H COSY Of kalkitoxin In D6 -DMSO at 340K
39
11.16 HMQC Spectrum Of Kalkitoxin In D6 -DMSO at 298K
40
11.17 HMQC Spectrum Of Kalkitoxin In D6 -DMSO at 340K
41
11.18
Partial Structures of Kalkitoxin Connected by HMBC
42
11.19
'H and '3C NMR Assignment of Kalkitoxin
43
LIST OF FIGURES (Continued)
Page
Figure
11.20 Pure Kalkitoxin in Brine Shrimp Toxicity Assay
45
11.21
Mollucicidal Activity of Kalkitoxin
45
11.22
Ichthyotoxic Effects of Kalkitoxin
47
III. 1
Secondary Metabolites from Different Varieties of
L. majuscula
53
111.2
Structures of Various Malyngamides from L. majuscula
55
111.3
The Isolation of Malyngamide J
57
111.4
The Isolation of Malyngamide L
59
111.5
'H -1H COSY of Malyngamide J
6
111.6
Partial Structures of Malyngamide J by 'H -'H COSY and
1
BETCOR
62
111.7
The Structure of Malyngamide J (29) with 11-1 and '3C
NMR Assignments by HMBC Correlations
64
111.8
'H -'H COSY of Malyngamide L
70
111.9
The Structure of Malyngamide L (30) with 'H and 13C
NMR Data
II1.10 Stereochemistry of Dimethoxylated Xylose Residue in
Malyngamide J
III.1 1 Proposed CD and NOE of Two Malyngamide J
Configurations
7
1
67
68
111.12 Structures of Malyngamides J and L with Stereochemistry 7 2
LIST OF FIGURES (Continued)
Page,
Figure
IV.1
The chemical diversity of Lyngbya majuscula from
Playa Kalki, Curacao
78
LIST OF TABLES
Page
Table
The ratio of N-methyl group resonances in the 'H NMR
spectra of the two conformers of kalkitoxin in various
NMR solvents
34
11.2
'H and "C NMR Data of Kalkitoxin Isolated from
Lyngbya majuscula
44
III. 1
and "C NMR Data of Malyngamide J (29) Isolated
from L. majuscula
63
111.2
'H and "C NMR Data of Malyngamide L (30) from
L. majuscula
65
II.1
LIST OF APPENDIX FIGURES
Page
Figures
A.2
IR Spectrum of 15
89
A.3
HMBC Spectrum of 15 in D6 -DMSO at 298K
90
A.4
41 NMR Spectrum of 15 in C6D6
91
A.5
HMBC Spectrum of 15 in D6-DMS0 at 340K
92
A.6
135 DEPT Spectrum of 15 in D6 -DMSO at 298K
93
A.7
45 DEPT Spectrum of 15 in D6-DMS0 at 298K
94
A.8
135 DEPT Spectrum of 15 in D6 -DMSO at 340K
95
A.9
135 DEPT Spectrum of 15 in D6 -DMSO at 340K
96
A.10
'H Decoupling Spectrum of 15 at 8 5.95 in C6D6
97
A.11
IR Spectrum of 29
98
A.12
LRFAB Mass Spectrum of 29
99
A.13
CD Spectrum of 29
100
A.14
'3C NMR Spectrum of 29
101
A.15
'H NMR Spectrum of 29
10 2
A.16 HMBC Spectrum of 29
103
A.17
HETCOR Spectrum of 29
104
A.18
DEPT (135 and 90) Spectrum of 29
105
LIST OF APPENDIX FIGURES (Continued)
Page
Figure
A.19
NOE Difference Spectrum-1 of 29
106
A.20
NOE Difference Spectrum-2 of 29
107
A.21
NOE Difference Spectrum-3 of 29
108
A.22
NOE Difference Spectrum-4 of 29
1 09
A.23
NOESY of 29
110
A.24
13C NMR Spectrum of 30
111
A.25
1H NMR Spectrum of 30
112
A.26
114-1H COSY of 30
113
A.27
HETCOR Spectrum of 30
114
A.28
LRFAB Mass Spectrum of 30
115
LIST OF ABBREVIATIONS
COSY
'H -'H Chemical Shift Correlation Spectrometry
CD
Circular Dichroic Spectroscopy
DEPT
Distortion less Enhancement by Polarization Transfer
DMSO
Dimethylsulfoxide
EIMS
Electron Impact Mass Spectrometry
EtOAc
Ethyl Acetate
FABMS
Fast Atom Bombardment Mass Spectrometry
FT
Fourier Transform
FTIR
Fourier Transformed Infrared Spectroscopy
HETCOR
Heteronuclaer Correlation Spectroscopy
HMBC
Heteronuclear Multiple Bond Correlation Spectroscopy
HMQC
Heteronuclear Multiple Quantum Coherence
HPLC
High-Performance Liquid Chromatography
HRMS
High Resolution Mass Spectrometry
IPA
Isopropyl Alcohol
IR
Infrared or Infrared Spectroscopy
MS
Mass Spectrometry
NCI
National Cancer Institute
NMR
Nuclear Magnetic Resonance
NOE
Nuclear Overhauser Effect
TLC
Thin Layer Chromatography
TMS
Tetramethylsilane
Ultravioler or Ultraviolet Spectrometry
UV
NOVEL BIOACTIVE SECONDARY METABOLITES FROM THE
MARINE
CYANOBACTERIUM LYNGBYA MAJUSCULA
CHAPTER I. GENERAL INTRODUCTION
Terrestrial plants and animals represent biologically important
entities and have been utilized to treat human diseases since
plants
antiquity. Studies of the secondary metabolites of terrestrial
and animals were begun in the 1800's.' However, until the last
three decades of this century, there have been increasingly
intensive efforts toward exploring the marine environment for
useful biomedicinal agents.' Especially over the last decade,
advances in diving technology have opened up vast areas of
unexplored marine environments and habitats to marine natural
product scientists. The marine environment is an exceptional
reservoir of bioactive natural products, many of which exhibit
structural features not found in terrestrial natural products. In
addition, many marine compounds have been found to be useful as
biochemical tools for exploring cellular processes at the molecular
level.
The discovery and isolation of potentially useful bioactive
natural products leads to the next stage of drug development:
structural characterization and analysis. The area of molecular
characterization has benefited tremendously from advances in
digital electronics and modern analysis instruments of incredible
2
power.
In particular, nuclear magnetic resonance spectroscopy
(NMR) is able to completely define the three-dimensional structure
of molecules with only micrograms of material. Currently, highfield and microprobe NMR technologies provide a means to
investigate the structure of trace quantities of natural products.'"
Biomedical Potential of Marine Natural Products
The exploitation of resources from the sea has taken place over
thousands of years. For centuries the people of China and Japan
have prepared an extract from the boiled fresh blades of the red
alga Digenea simplex to use as a remedy for intestinal parasite
infections of children." The consequent isolation and structure
determination of the biologically active constituent gave the
unusual amino acid, a-kainic acid (1, Figure I.1).12 The origins of
the antiviral drug Ara-A (2), which is used in combination with
other agents to treat leukemia, can be traced back to the
serendipitous isolation of the arabinosyl nucleosides
spongothymidine (3) and spongouridine (4) from the sponge
Cryptotethia cryta. The observation of beach flies killed by contact
with the marine annelid Lumbriconereis heteropoda led to the
discovery of an unusual sulfur containing toxic amine, nereistoxin
(5) which served as a lead compound for the synthesis of a new
insecticide, PADEN (6).13.14
3
0
COOH
N
HN
I
......
COOH
HOH2C
0
114))
1
Kainic acid
OH
2
Ara-A
0
HN
H
0
HOH2C
0
HO
OH
3
OH
Spongothymidine
4
Spongouridine
H3C
H3C
GS
/
H3C
5
Nereistoxin
./
SCONH2
HC(
N
SCONH2
6
PADEN
Figure I.1 Structures of Several Marine Natural Products and
Derivatives with Biomedical Utility.
4
e
0
HO
0
411
CI
Br
8
Halomon
7
Stypoldione
N
H2N
0
10
Cribrostatin
1
12
Dolasatin 10
13
D idemn in -B
Figure 1.2
OMe
Structures of Biomedical Potential Marine
Natural Products.
OH
.OH
OH
OH
OH
:
OH
OH
0
0
)1-,,
HO ..,,.,/.
H
Me
OH Me
N
H
,OH
HO,,
OH
'
"OH
OH
0
0
HO
M
/*
OH
s'OH
OH
a
Me
OH
"OH
'OH
E
OH
OH
11
Figure 1.3
Structure of Palytoxin
(11
6
Throughout the 1980s, biomedical investigations of marine
natural products focused on ion channel effectors and central
nervous system membrane-active toxins, anticancer and antiviral
agents, tumor promoters, and anti-inflammatory agents.'
Stypoldione (7, Figure I. 2) , a potent fish toxin, was isolated
from the tropical brown algae Stypopdium zonale. It was found to
be cytotoxic by inhibiting mitotic spindle formation via inhibition of
microtubule polymerization, the mechanism of action for several
17
Halomon (8) from a
clinically useful anticancer agents.16.
Philippine collection of the red alga Portieria hornemannii has been
selected by the NCI Decision Network Committee for preclinical drug
development due to its extreme selectivity to brain, renal and colon
tumor cell lines." The deep-water sponge Dercitus sp. collected in
the Bahamas yielded the novel aminoacridine alkaloid dercitin (9),
which possesses in vitro cytotoxic activities in the low nanomolar
Cribrostatin 1 (10) from deep-blue
specimens of Cribrochaline sp. shows selective activity against all
concentration range.19
nine human melanoma cell lines in the NCI's panel.'
Palytoxin (11, Figure I. 3) has not only been found in Palythoa
soft corals but also in wide variety of other organisms,' such as the
seaweed Chondria armata, crabs belonging to be the genera
Demania and Lophozozyinus, a triggerfish Melichtys vidua, and a
file-fish Alutera scripta.22-24 More recently, polytoxin analogs have
been isolated from the dinoflagellate Ostreopsis siamensis, calling
into question the biogenetic origin of palytoxin.25 It stimulates
arachidonic acid metabolism and regulates the response to
epidermal growth factor by activating a sodium pump in a signal
7
transduction pathway using sodium as a secondary messenger.
These activities make palytoxin a useful tool for probing cellular
recognition processes!'
The sea hares are herbivorous opisthobranch molluscs that
concentrate and store selected algal metabolites from their diet.
Dolabella auricularia, collected in the Indian Ocean, has been the
source of more than 15 cytotoxic peptides, the dolastatins. The
most active metabolite, dolastatin 10 (12, Figure 1.2) is one of the
most potent antineoplastic substances known.' Didemnin-B (13)
extracted from the Caribbean tunicate, Trididemnum solidum,
exhibits an impressive array of in vivo antitumor, antiviral, and
immunosuppressant activities and became the first marine
compound to enter human cancer clinical trials as a purified natural
product.n. 28
Marine Toxins
Marine toxin research has become a crucial part of marine
natural products studies due to their involvement in human
intoxication, animal poisonings and economic impact brought on by
these types of incidents. In general, marine toxins are highly
targeted to specific biomolecular receptors and have unique
structural features not found in terrestrial compounds. For
example, one of the best known toxins, tetradotoxin (14, Figure 1.4),
isolated from the pufferfish and finally traced to symbiotic bacteria
(e.g., Shewanella alga), together with saxitoxin (15) produced by a
8
0"
+
NH2
HO
14
Tetradotoxin
15
Saxitodn
HO
16
Okadaic acid
Me
H
OH
O
.o
%
Me
**OH
s0i
H
H
H
.t.
H
0
o
R2
17
Ciguatoxin R1 = CH(OH)CH2OH; R2 = OH
Figure 1.4 Structures of Several Marine Toxins.
OH
H3C
H3C 0
CH3
OH
0 CH3
OH
OH
OH
0
0
0
OH CH3 OSO3Na OHO
OH
18
Figure 1.5
Structure of Maitotoxin
OH
OH
O
0
011 0
OH
OH OH
HO
OH
10
CHO
19A
Brevetoxin-A
CHO
19B
Brevetoxin-B
Figure 1.6
Structures of Brevetoxin-A And Brevetoxin-B
11
number of dinoflagellate species (e.g., Alexandrium spp and
Gymnodinium catenatum) as well as some strains of the fresh water
cyano-bacterium Aphanizomenon flos-aquae,'" are involved
frequently in fatal poisoning due to blocking the sodium channel in
excitable membranes.32-34
Another class of shellfish toxins, well
known as diarrhetic shellfish poisons [e.g., okadaic acid (16) found
in the dinoflagellate Prorocentrum lima 35], have been shown to be a
Ciguatoxin (17),
completely new type of phosphatase inhibitor.36,
a polyether compound that causes seafood poisoning in humans by
ingestion of coral reef fish that become toxic through their diet was
37
found to be produced by the epiphytic dinoflagellate Gambierdiscus
toxicus.38 Maitotoxin (18, Figure I. 5) with the largest molecular
weight (3422Da) of any non-biopolymer natural product and its
extremely potent bioactivity, has attracted much attention in the
scientific community. Maitotoxin's lethality against mice (LD50 = 50
ng/ml) indicated that it might be the most potent nonproteineceous
toxin in nature." The total structure of maitotoxin has recently
been proposed on the basis of extensive spectroscopic analysis
including high-field multi-dimensional NMR methods."
Along the Florida coast, the dinoflagellate Gymnodinium breve
causes massive fish mortality.' Brevetoxin A (19, Figure I. 6) is
the most potent ichthyotoxin among the suite of toxins produced by
the microalga (its lethality against zebrafish is reportedly 3 ppb42'
43). These toxins have proved to be valuable tools as activators of
sodium channels."
As these previous studies show, many of the marine toxins
with unique structures and pharmacological properties have been
12
20
Motuporin
21
Microcystin-LR
OH
OH
22
Aeruginopepsin
Figure 1.7
Structures of Bioactive Natural Products From
Cyanobacteria.
13
OMe
IOII
N
0
0
OMe
OH
0
H
OMe
23
Tolytoxin
11
NH
HNC
0
O
24
Westiellamide
25
Tantazole B
26
Tolyporpin
Figure 1.8 Structures of Bioactive Natural Products from
Cyanobacteria.
14
27
6-cyano-5-methoxy-12-methylindolo(2,3-a)carbazole
28
Microcolin A
CH3
H2 C
OCH3
29
Curacin A
Figure 1.9 Structures of Bioactive Natural Products from
Cyanobacteria.
15
found to be very useful tools for biological, medical, pharmacological
and ecological studie,'." as well as attractive targets for chemical
modification followed by structure-activity relationship studies.',
as
$ioactive Natural Products From Marine Cyanobacteria
The cyanobacteria, or blue-green algae, are prokaryotic
photosynthetic organisms which have been identified as a rich
source of unusual toxins". 51 and biologically active lead
compounds.' An early survey of marine cyanobacteria crude
extracts showed 6% to be cytotoxic to KB cancer cells with MICs < 30
ug/ml, 9% to possess antifungal activity, and > 5% to possess
antiviral activity to Herpes simplex type II." Reports of the potent
toxicity associated with cyanobacteria growing in nature suggest
that cyanobacteria serve as promising sources of cytotoxic,
fungicidal, antiviral and antimicrobial active compounds.5"4
Motuporin (20, Figure I. 7), which is derived from symbiotic
cyanobacteria, was isolated from the sponge Theonella swinhoei.55
Microcystin-LR (21) is the most common microcystin cyclic
heptapeptide from Microcystis, Anabaena, Oscillatoria, and Nostoc
species.
It displays an LDso of 50 µg /m1 in mice.'' 57 From recent
reports, Microcystis aeruginosa has been shown to simultaneously
produce a series of depsipeptides called aeruginopepsins (i.e.
aeruginopepsin 917S-A, 22), which appear to enhance the toxic
effects of the microcystins.58
16
Tolypothrix, Scytonema, and Cylindrospermum species have
been shown to contain cytostatic and antimitotic metabolites called
sytophycins. Tolytoxin (23, Figure I. 7), as one member of them,
exerts a selective inhibitory effect on certain human tissue/tumor
types in comparative bioassays performed at the U. S. National
Cancer Institute." Westiellamide (24), a bistratamide-related cyclic
peptide isolated from Westiellopsis prolifica, was cytotoxic against
Tantazole B (25) from
the KB and LoVo cell lines at 2
Scytonema mirabile is cytotoxic to KB cells at doses ranging from
0.01 to 10 pig/m1,61 while tolyporphin (26) produced by Tolypothrix
ondosa potentiates the cytotoxicity of adriamycin or vinblastine in a
vinblastine-resistant tumor cell line (SK-VLB) at doses as low as
4g/mi.62
1
Cyanobacteria also appear to be a rich source of new
antiviral compounds.
For example, the Nostoc sphaericum
metabolite, 6-cyano-5-methoxy-12-methylindolo[2, 3-cdcarbazole
(27) displays anti-HSV-2 activity."
Two additional classes of potentially important marine natural
products were isolated from Caribbean collections of Lyngbya
majuscula.
Microcolin A (28) suppresses the two-way murine
mixed lymphocyte reaction at low nanomolar concentrations."
Curacin A (29), a unique thiazoline-containing lipid isolated from a
Curacao collection of Lyngbya majuscula, is an exceptionally potent
antiproliferative agent that shows some selectivity for colon, renal,
and breast cancer-derived cell lines.'
17
Description of Chapters
This thesis consists of a total of four chapters and an appendix.
Following this general introduction, chapter II describes the
discovery of a potent new toxic natural product named kalkitoxin.
Determination of the chemical structure of kalkitoxin was made
possible through the extensive investigation by NMR spectroscopy
including 2D-NMR at two different temperatures. Chapter II also
details the methods for the brine shrimp toxicity guided
fractionation and bioactivity evaluation of kalkitoxin.
The third chapter describes the isolation and structural
elucidation of two new malyngamides from the Curacao Lyngbya
majuscula. The structure of these two molecules were determined
by using 2D-NMR methodology together with comparisons of other
malyngamides. The stereochemistry of malyngamide J was
investigated by using NMR and circular dichroic spectroscopic
techniques.
Chapter IV give a conclusion about this investigation of Curacao
Lyngbya majuscula and perspective of these three novel bioactive
metabolites.
18
CHAPTER II
KALKITOXIN FROM THE CYANOBACIERIUM LYNGBYA MAJUSCULA
Abstract
Lyngbya majuscula, a chemically and biologically rich strain of
cyanobacteria, has been intensively researched for its biomedical
Brine shrimp toxicity assay-guided fractionation of an
organic extract of Lyngbya majuscula from a Curacao collection led
potential.
to the isolation of a potent brine shrimp toxic compound. The
structure of this new thiazoline containing lipid, named kalkitoxin,
was deduced from extensive spectroscopic investigation. Other
biological properties of kalkitoxin such as ichthyotoxicity and
molluscicidal activity have also been investigated.
19
Introduction
Cyanobacteria, or blue-green algae, are found almost
everywhere that light and water are available. In freshwater
environments, blooms of toxic cyanobacteria can pose serious
dangers for wildlife and livestock." A large number of interesting
metabolites have been isolated from cyanobacteria. Their unique
structures and biological activities have received increasing
attention from chemists and pharmacologists."'
Most of the investigated cyanobacteria are freshwater or
Only a limited number of marine cyanobacteria
have been searched for secondary metabolites. Nevertheless, there
terrestrial in origin.
are a number of indications that cyanobacteria are important
players in the production of interesting compounds that are found
From the brackish water species,
Nodularia spumigena, the cyclic pentapeptide nodularin (1, Figure
widely in marine environments.
II. 1) which had caused problems in the Baltic Sea and New Zealand,
was isolated.' This compound is closely related to microcystin (2),
a potent hepatotoxin and protein phosphatase 2 and 2A inhibitor
from the fresh-water cyanobacterium Microcystis aeruginosa.68
A marine strain of Nostoc linckia produced the potent cytotoxin
borophycin (3), the structure of which was determined by X-ray
crystallography.' Tjipanazole Al (4), a N-glycoside of indolo-[2,3c]carbazole isolated from Tolypothrix tjipanasensis, exhibited
appreciable fungicidal activity in tests against phytopathogenic
fungi." From an Australian Cylindrospemopsis raciborskii culture,
20
0
OH 00.0 0
43 H COOH CH3
II 3 C4
CH3
N
H
.,OCH3
H3C' H CH3
0 O %OHO
H
0
0
0
H COOH
Na+
-o
0
3
Borophycin
NH
1
HN NH2
Nodularin
Cl
COON CH30
HNI%-/%11N-Tr)1.NH
OCH3
H3C
0 CH
0
H3
NH H CH3 H HN
...
4
0
Tjipanazole Al
CH3
NvAirIiiA'
NCH 3
ei H3 CH3
0
C0011
HN
HN3' NH2
OH
63so
2
Microcystin-LR
H3C
N
NH
NH HN
0
N
H
5
Cylindrospermopsin
Figure II.1 Structures of Natural Products from Cyanobacteria.
21
which was obtained following an outbreak of hepatoenteritis on
Palm Island in northern Queensland, cylindrospermopsin (5), a
potent hepatotoxin, has been isolated.'
of the most important cyanobacterial natural products
have been isolated from Lyngbya majuscula, a marine filamentous
form. Lyngbyatoxin A (6, Figure 11.2), together with
debromoaplysiatoxin (7), were isolated from a toxic strain of
Lyngbya majuscula collected in Hawaii and shown to be the cause of
the severe contact dermatitis known as swimmer's itch.' Both
classes of compound have played a significant role in the study of
protein kinase C (PKC) due to their binding to the phorbol ester
receptor on PKC.72." Majusculamide A (8), an unusual fatty acid
amide derivative, was reported from a deep water Lyngbya
majuscula."
Our investigation of marine algae for anticancer drug leads has
resulted in the identification of several structurally diverse
cytotoxic metabolites from microalgal sources. Hormothamnin A
(9), isolated from the northern Puerto Rican Hormothamnion
enteromorphoides, was found to be potently antimicrobial to
Bacillus subtilus and cytotoxic to several cancer cell lines.' Isolated
from the Caribbean cyanobacterium Lyngbya majuscula, barbamide
(10, Figure 11.3), a chlorinated metabolite with molluscicidal
activity, together with antillatoxin (11) which exhibits extremely
potent ichthyotoxicity, were reported recently:16a' Utilizing toxicity
to brine shrimp as a bioassay, a unique thiazoline containing lipid,
curacin A (12), was discovered from the organic extract of a
Curacao collection of Lyngbya majuscula.' Curacin A is a potent
22
OH
7
Debromoaplysiatoxin
6
Lyngbyatoxin A
N1-12
OCH3
8
Majusculamide A
9
Hormothamnin A
Figure 11.2 Bioactive Metabolites from Cyanobacteria
23
Cl
CI
Cl
OCH3 CH3
N
H
10
Barbamide
11
Antillatoxin
OH
0
r''ri
0
Il
N.'''''N
CH3 0 A CH3 0
H
N
E
1
1
13
Microcolin A
CH3
H3C
CH3O
CH
H3C
A
H3C
N
N
I
I
CH3 CH3 CH3 0 A CH3 0
H3C
OH
14
Microcolin C
Figure 11.3 Bioactive Metabolites from Lyngbya majuscula.
24
antimitotic agent that inhibits microtubule assembly and colchicine
binding to tubulin (Figure 11.3).78.79 A Venezuelan collection of
Lyngbya majuscula yielded an immunosuppressive linear peptide
microcolin A (13),64 while a Curacao collection provided microcolin C
(14).81 These lipopeptides exhibited potent cytotoxic activity
against several cell lines in vitro such as A 549 (79% inhibition),
SW-480 (97% inhibition), and HMEC (90% inhibition) performed by
the Sandoz Corp.
Results and Discussion
Lyngbya majuscula from Playa Kalki, Curacao was collected at a
depth of 20 ft. It was preserved in isopropanol and stored at -20
°C.
The organic extract was produced from the homogenized alga in
CH2C12/Me0H (2:1, v/v), allowed to soak around 30 minutes, and
filtered through cheese cloth.
The algal material was placed in fresh
solvent (CH2C12 /MeOH, 2:1 v/v), heated to a gentle boil up to 20
minutes, and filtered. This process was repeated two times. The
filtrate was partitioned between CH2C12 and water, and the organic
fraction was reduced in vacuo and stored in Et20 (4.32 g, dark green
oil).
The isopropyl alcohol preserved sample (1L) was filtered prior
to extraction. This alcohol was removed from the filtrate by
evaporation in vacuo and added to the aqueous extract. The
CH2Cl2 /MeOH extracted algal residue was soaked in Me0H/H20 (3:1,
v/v) overnight, filtered, and the alcohol was removed in vacuo. The
25
aqueous extract was partitioned between sec-butanol and water.
The sec-butanol fraction (0.6 g, dark green oil) was reduced in
vacuo and stored in Me0H (Figure 11.4).
This investigation focused on the organic extract. Twodimensional TLC analysis (EtOAc /hexane 1:1; CHC13/Me0H 9:1) of the
organic extract suggested the presence of several UV-active
secondary metabolites. The crude extract was highly toxic to brine
shrimp (100% killed at 10 ggim1).82 Hence, the brine shrimp
toxicity assay was utilized to guide each fractionation of this extract
(Figure 11.5). The first fractionation step utilized gradient vacuum
chromatography (4%-40% EtOAC/hexane) to give seven fractions
after recombination. Among them, fraction C gave the highest
toxicity against brine shrimp (100% killed at 1 µg /ml, Figure II.6a).
Further flash chromatography (4%-40% EtOAC/hexane) was applied
to fraction C to give a total of ten fractions. Brine shrimp toxicity
assay II exhibited a selective toxicity in fraction C-4 (Figure II.6b).
The continued fractionation monitored with the brine shrimp
toxicity assay (column chromatography, 40% EtOAC/hexane) led me
to focus on fraction C-4-G (Figure II.6c). The final purification was
performed by HPLC (40% EtOAC/hexane) to yield 12.8 mg of
compound 15, named kalkitoxin (Figure II.6d, Figure 11.7).
Kalkitoxin showed [a],), = +16° (c = 0.07, CHC13). High resolution EIMS
(70 eV) gave a major [M]+ ion at m/z 366.2696 analyzing for
C21H381\120S (-0.8 mamu dev.). The formula C211438N2OS indicated that
kalkitoxin possessed four degrees of unsaturation.
From "C NMR
analysis, two of these degrees were due to double bonds, one due to
carbonyl group, and the remaining one to a ring system.
Lyngbya majuscula
(in 1 L)
partially defrost
2. filter off preservation alcohol
1 .
3. extract cold in 2:1 CH2Cl2 /MeOH
4. gently heat it in 2:1 CH2Cl2 /MeOH
reduce
aqueous
for 30 min, cool, filter
5. repeat #4 twice
partition
between
CH2Cl2 / H2O
residue I
soak in 3:1 Me0H/H20
overnight, then filter
add
organic solvent layer
H20
(discard)
1
aqueous layer)
reduce at 40 °C,
partition with
sec-butanol
cJ2Cl2 layer
algal
material
i'
sec-butanol
i'
filter particulates,
reduce on rotovap at 40 °C
H20
Organic extract
(4.32 g)
"Aqueous extract"
(0.66 g)
Figure 11.4 Extraction of Lyngbya majuscula.
Organic Extract of Lyngbya majuscula
(4.32g)
Gradient Vacuum Chromatography
EtOAc / Hexane 4%-100%
Brine Shrimp
E-1C2
Brine Shrimp
Assay II
C1
Gradient Flash Chromatography
EtOAc / Hexane 4%--100%
I
C3
G
F
E
D
(420 mg)
I-1
I
I
Assay I
C6
C5
C4
I
I
I
1
I
C8 C9 C10
C7
(101.2 mg)
Column Chromatography
EtOAc / Hexane 40%
Brine Shrimp
Assay III
I
C4A
C4B
C4C
C4D
C4E
I
C4F
1
C4H
C4G
C41
(26.3mg)
HPLC 40% EtOAc / Hexane
Brine shrimp
Assay IV
1
3
LKalkitoxin
(12.8 mg)
Figure 11.5
Bioguided Fractionation of Kalkitoxin
4
C4..)
28
120%
--o 10 vg/m1 each fraction
g 100%
io
80%
0 1 µg /ml each
60%
fraction
40%
20%
ft
*
0%
4
3
2
1
7
6
5
TLC Fraction number
Figure II.6a Brine Shrimp Assay I for Bioguided Isolation of
Kalkitoxin.
120%
c 100%
vs°
0-0.5 ug/m1 each fraction
80%
0--0.1 ug/ml each fraction
60%
a,
40%
2 20%
0%
1
2
3
4
5
6
7
8
9
10
TLC Fraction number
Figure II.6b Brine Shrimp Assay II for Bioguided Isolation of
Kalkitoxin.
29
120%
,s 100%
0
co
0 0.1 µg /ml each
80%
fraction
0-- 0.01 µg /ml each fraction
60%
c 40%
4' 20%
0%
2
1
4
3
5
6
8
7
9
10
11
TLC Fraction number
Figure II.6c
Brine Shrimp Assay III for Bioguided Isolation of
Kalkitoxin.
120%
c 100%
0
4J
80%
0-- 0.1 µg/m1 each fraction
60%
0-- 0.01 µg /ml each fraction
u_
40%
32
20%
0%
1
2
4
3
HPLC Fraction number
of
Figure II.6d Brine Shrimp Assay IV for Bioguided Isolation
Kalkitoxin.
30
The structure elucidation of compound 15 was mostly based on
extensive 1D and 2D NMR experiments. A complication of this
structure elucidation was that at room temperature (298K) many of
NMR signal were twinned in a 3:2 ratio (in D6-DMSO, Figure 11.9).
That was due to the presence of two slowly interconverting amide
conformers (Figure II.10). The fact that these twinned proton peaks
changed ratios in different NMR solvents confirmed that they arose
from two conformations of this molecule (table II.1). However, at
high temperature (340K) these peaks coalesced to singlets (Figure
While the high temperature NMR experiments provided
valuable information for this structure elucidation, there was a
II.11).
complicating factor in that at the high temperature some of '3C NMR
signals were missing and some '11-'3C correlations were lost (Figure
Therefore, the structure elucidation was based on the
combination of the two sets of NMR experiments at 298K and 340K.
11.12-13).
Data from 'H -'H COSY, HMQC, DEPT and 'H NMR decoupling
experiments (Figure 11.14-17) allowed deduction of six partial
structures (a-f, Figure 11.8) for compound 15. Partial structure b
contained a tertiary amide group which caused the two
conformations in this molecule.
Partial structure f possessed a
terminal olefin adjacent to a thiazoline ring.
This partial structure
assignment was also based on analysis of EIMS data and
comparisons of '3C NMR chemical shifts with model compounds.'' 85,
86 Partial structure e possessed a CH3-CH-CH2 grouping while partial
structure a contained a high field C4 group with two methyls. The
partial structure c had a high field C4 group with one methyl group.
31
2
16
4'
1
12
15
14
15
Figure 11.7 The Overall Planar Structure of Kalkitoxin.
c
a
CH3
CH3
f
e
d
/
CH3
CHCH2
i
H
sS5
H
-->----Ths
NZ(
Pr
Figure 11.8
Six Partial Structures of Kalkitoxin.
32
HMBC data were used to connect these six partial structures to
give the full structural assignment of kalkitoxin (Figure 11.18-19).
Notably, the proton at 54.90 in partial structure f and the
methylene protons at 62.23 and 62.45 in partial structure e were
both correlated to the quaternary carbon at 6168 of the thiazoline
ring.
Similarly, partial structures b and c were readily connected
by observing long range coupling between the methyl protons
62.95/2.80 (at 298K) of amide b and the methylene carbon
647/44.8 of c as well as the carbonyl of the amide at 8174.8 and the
methylene protons at 63.32 of c. In partial structure d, the
methylene protons at 61.10 showed clear correlations to the
methine carbon 636.7 of partial structure e and the methine at
627.5 of partial structure c. Table 11.2 displays '11 and 13C NMR data
and coupling constants of kalkitoxin.
Kalkitoxin was found to possess highly potent brine shrimp
toxicity (LC50 = 28 ng/ml, Figure 11.20). This rapid in-house screen
has been shown to have a good correlation (p = 0.036) with the 9KB
cytotoxicity assay. 88,89 Also, kalkitoxin displayed modest
molluscicidal activity to Biomphalaria glambrata (LC100 = 22 µg /ml,
Figure II.21).84 This freshwater mollusc is an intermediate host for
three species of parasitic trematode blood flukes (shistosomes).
Over 200 million people in over 75 countries are infected with
schistosomiasis (bilharzia).87
Chronic infection may result in
disturbances of the central nervous system, liver fibrosis, portal
hypertension, and possibly liver cancer.
The difficulty and costs
18
1
14
4
15
16
1
2
48
3
4b
\12
P
0
0,
6.0
5.5
5. 0
4'5
4.0
5
PPM
0
2.5
2.0
,
1.5
298K.
Figure 11.9 11-1 NMR Of Kalkitoxin InD6-DMSO At Room Temperature
34
Table II.1 The ratio of N-methyl group resonances in the 'H NMR
spectra of the two conformers of kalkitoxin in various NMR solvents
solvent
chemical shift
ratio
CDC13
2.95/2.90
1.27
: 1
C6D6
2.79/2.43
0.5
:1
D6-acetone
3.05/2.85
1.58
: 1
D4-Methanol
2.95/2.80
1.2
:
D6-DMS0
2.95/2.80
1.5
:1
D4-Me0D/CDC13
3.06/2.94
1.22: 1
1
:
1
1
* at room temperature 298K.
0-
02.80
H3C.
CH3
(in D6 -DMSO)
2.95
Figure II.10
Two slowly intercoverting tert-amide isomers
present at room temperature 298K.
5
6
14
6I0
5.5
5.0
15
4.5
4.0
35
PPM
Figure II.11 1H NMR Of Kalkitoxin In D6 -DMSO At 340K.
2'
6
16
18
47
14
3'
13
11
I
170
160
"
I
150
140
130
120
110
I-
T
100
90
80
70
60
50
PPM
Figure 11.12 13C NMR Of Kalkitoxin In D6 -DMSO At 298K.
40
10
8
30
5'
20
4'
10
170
160
150
140
130
120
110
100
90
80
70
60
PPM
Figure 11.13 "C NMR Of KaLkitoxin In D6 -DMSO At 340K.
50
40
30
20
10
ai
6.0
5.5
5.0
4.5
4.0
3.5
4.k___AJAieil
3.0
2.5
2.0
1.5
1.0
P PM
Figure II.15 11-I-1H COSY Of Kalkitoxin In D6 -DMSO At 340K.
_
16
1.0
!N.
1.5
8
2.0
0
e
2.5
I
lot
c.)
3.0
ct,P
3.5
4.0
_
4.5
.a
O
0
_
5,0
co o
Pa
-
030 El
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
PPM
In D6-DMS0 At 298K.
Figure 11.14 '11-11-1 COSY Of Kalkitoin
5.5
6.0
8
a
8.0
O.
PPM
Figure 11.16 HMQC Spectrum Of Kalkitoxin In D6 -DMSO At 298K.
2
1
4
16
13
PH p1 '11
1A1
101
.00
i°
did
1
16
6,0
5.5
5.0
4.5
4.0
3.0
3.5
2.5
2.0
1.5
1.0
PPM
Figure 11.17 HMQC Spectrum Of Kalkitoxin In D6-DMS0 At 340K.
Partial structures deduced from combination of
1H-1H COSY, HMQC, DEPT and1H decoupling data
r) HMBC (Selected,1H --0-13C )
Figure 11.18 Partial Structures of Kalkitoxin Connected by HMBC.
43
2.95/2.80
1.45
1.25
1.45
0.97
0.84
2.61
3.32
I
11..5257
0
0.83
1H chemical shifts
114.6
137.4
34.7/33.0
N<7.46.1
34.9
27.5
17.1/17.7
47/44.8
11.7
26.8
13C chemical shifts
Figure 11.19 1H and 13C NMR Assignment of Kalkitoxin (298K).
44
Table 11.2
C-atom
1
and "C NMR Data* of Kalkitoxin Isolated From
Lyngbya majuscula
"C (D6 -DMSO)
(D6-DMS0)
8
8(mult, J in Hz)
114.6
5.25 d (17.3)
5.10 d (10.6)
5.95 ddd (17.3, 10.6, 6.2)
4 .90 m
3.46 dd (10.8, 8.7)
3.03 dd (11.0, 7.9)
4
137.4
77.6
37.8
5
6
168.0
37.5
7
11
36.7
33.4
39.3
27.5
34.9
12
47/44.8
13
19.0
16.0
16.0
2.23 bdd (9.0, 14.5)
2.45 m
1.84 m
1.57 m
1
1.10 m
1.45 m
1.45 m
1.25 m
3.32 dd (5.6, 12.8)
0.87 s
0.85 s
0.83 s
34.7/33.0
2.97/2.80
2
3
8
9
10
14
15
16
1'
2'
3'
4'
5'
174.8
36.1
26.8
11.7
17.1/17.7
s
2.61 dd (6.7, 13.4)
1.25 m
1.57 m
0.84 d
0.97 d (6.9)
* Data reported at room temperature 298K.
45
120%
100%
80%
60%
40%
20%
0%
01
10
1
100
50
500
1000
Concentration (ng /mI)
Figure 11.20 Pure Kalkitoxin in Brine Shrimp Toxicity Assay.
120%
100%
80%
60%
40%
20%
0%
1
5
10
25
Concentration (ug/ml)
Figure 11.21
Molluscicidal Activity of Kalkitoxin.
100
500
46
120%
100%
80%
60%
40%
20%
0%
0.01
0.1
1
5
10
100
1000
5000
Concentration (ng /mI)
Figure 11.22
Ichthyotoxic Effects of Kalkitoxin.
involved in treating these large groups of people in remote
impoverished areas has prompted a concerted effort by the World
Health Organization (W.H.O.) to look for new means of preventing
infection by controlling the snail vector.
Kalkitoxin showed extremely potent ichthyotoxicity to the
goldfish (LC50 = 5 ng/ml, Figure 11.22)." This is almost the same
potency as the well known and highly potent ichthyotoxic
metabolite, brevetoin A produced by G. breve (although tested
against different fish). 42,43 As has occurred in the past, insect
antifeedants, antitumor agents, plant growth inhibitors, and
insecticides have been isolated from ichthyotoxic plants." These
biological features of kalkitoxin suggest possible ecological
interactions and inspire further investigation.
47
Biosynthetically, kalkitoxin may derive from an acetyl CoA
joined with a decarboxylated cysteine residue and a malonyl CoA to
form a thiazoline ring attached with a terminal olefin.
The middle
polyketide chain of kalkitoxin may derive from the consequent
condensations of methyl malonyl CoA, SAM and acetyl CoA. The
terminal C5 chain including the nitrogen of the amide is possibly
from a decarboxylated isoleucine.
Unfortunately, this research was not able to solve the
stereochemistry of kalkitoxin due to the limited amount of sample.
Experimental Methods
General Methods.
Ultraviolet spectra were recorded on a
Hewlett Packard 8452A diode array spectrophotometer, and
infrared spectra (IR) were recorded on a Nicolet 510P-RHS spectrophotometer.
Nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker AM 400 NMR spectrometer. All
NMR
chemical shifts are reported relative to an internal
tetramethylsilane (TMS) standard and '3C spectra referenced to the
center line D6 -DMSO at 39.25 ppm. Low resolution mass spectra
(LRMS) were obtained on a Varian MAT CH7 spectrometer, while
high resolution mass spectra (HRMS) were obtained on a Kratos MS
50 TC.
High performance liquid chromatography (HPLC) was done
using a M-6000 pump, U6K injector and either R 401 differential
refractometer or a lambda-Max 480 lc spectrophotometer.
TLC-
grade (10-40 ilm) silica gel was used for vacuum chromatography,
48
Kieselgel 60 silica (40-63 1.tm) was used for flash chromatography,
and Merck aluminum-backed TLC sheets (silica gel 60 F254) were
used for thin layer chromatography.
All solvents were distilled
from glass prior to use.
This Lyngbya majuscula was collected from Playa Kalki,
Curacao (20 ft underwater) on 11 August 1994 and preserved in
isopropyl alcohol and frozen until workup.
The defrosted alga was
homogenized in CH2C12 /MeOH (2:1, v/v). The algal residue was
repetitively extracted with CH2C12 /MeOH (2:1, v/v) twice. The
mixture was filtered and the solvents removed under vacuum to
yield a residue which was partitioned between CH2C12 and H2O. The
CH2C12 solution was collected. A total of 4.32 g dark-green oily crude
organic extract was obtained.
The bioguided fractionation of the crude extract was obtained
by silica gel vacuum chromatography, flash chromatography, and
column chromatography.
Finally, a normal phase HPLC was applied
to give kalkitoxin (15 12.8 mg), as a light yellow oil (Figure 11.7).
The 'H and '3C NMR spectra of kalkitoxin show very clean base
lines and The HPLC of kalkitoxin displayed a symmetric peak. These
suggested kalkitoxin great purity (> 95%)
Kalkitoxin(1 5). IR (CHCI3 v 2961, 2928, 2880, 1643, 1464,
1086, 1410, 1380 cm'; UV (MeOH) Xmax 250 nm (E = 2600); Optical
rotation [a]p = +16° (c = 0.07, CHC13); HR EIMS (70 eV) m/z obs. [M]+
366.2696 (16, -0.8 mmu dev.) C21H381\120S, [M-CH3]+ 351.2472 (5.5,
0.2 mmu dev), [M-C2H3]+ 339.2465 (5.7, -0.5 mmu dev), [M-CH2CH3]+
337.2294 (2.5, -1.9 mmu dev), 281.2591 (7, -0.2 mmu dev), [M05H9O]+ 281.2049 (3.9, -0.2 mmu dev), 265.2271 (2.4, -0.8 mmu
49
dev), [M-C61-18NS]+ 240.2329 (25, 0.2 mmu dev), [M-C,H14NO]+
238.1639 (5, 1.0 mmu dev), [M-C8H16NO]+ 224.1470 (1.9, -0.3 mmu
dev), 212.9960 (2, 0.9 mmu dev), 195.1860 (2.3, -0.1 mmu dev),
[M-C111122N0]+ 182.1003 (4.4, -0.05 mmu dev), 156.1750 (5.2, -0.2
mmu dev), [M-C13H26NO]+ 154.0683 (100, -0.8 mmu dev), 149.0244
(1.5, 0.5 mmu dev), 142.1237 (2, 0.5 mmu dev), 140.0531 (2.6, -0.3
mmu dev), 129.1155 (3.8, 0.1 mmu dev), [C6H9NS]+ 127.0459 (26,
0.3 mmu dev); 'I-1 NMR and "C NMR data see Table 11.2.
The brine shrimp toxicity assay was
Brine Shrimp Bioassay.
used to analyze levels of toxicity at different concentrations of
sample and this assay was utilized to guide fractionation of the
sample.
Brine shrimp eggs were incubated in Instant Ocean® water
at 28 °C for 24 hours before the assay to allow the eggs to hatch and
mature. Approximately 90% of the eggs usually hatch after this
incubation period.' A shallow rectangular dish filled with artificial
seawater (Instant Ocean®, Aquarium Systems, Inc.) containing a
plastic divider with several 2 mm holes was used to separate the
unhatched eggs from the nauphali.
The eggs were sprinkled into
one compartment that was kept in the dark for the incubation
period.
After 24 hours, the phototropic nauphali were collected
from the lighted compartment.
Approximately 15 shrimp were
added to each vial containing different concentrations of sample in
4.5 ml artificial seawater.
Appropriate solvent controls were done
in duplicate.
The brine shrimp were added to each vial using a long
stem pipette.
After 24 hours at 28 °C, the shrimp were counted
using a dissecting light microscope.
The percentage of dead shrimp
50
relative to the total number of shrimp was recorded for LC
measurement.
Ichthyotoxicity Assay. A single goldfish Carassius auratus was
added into a 50 ml beaker containing 40 ml of distilled H2O. The
sample was dissolved in 40 gl EtOH. In order to get an even solution
of the test compound, a microliter syringe was used to add the
sample solution at different water levels. The fish was observed for
a one hour period. End points were established as death (lack of
breathing) for LC measurement.
Molluscicidal Assay. The sample was dissolved in 40 tl EtOH
and diluted to 20 ml with distilled water to make several different
concentrations. Two Biomphalaria glambrata were placed in each
beaker and the beakers covered with glass plate for the duration of
the assay.
The condition of the snails was evaluated after 24 hours.
Snail were considered dead when no heart beat could be observed
upon microscopic examination.
Minimum lethal doses required to
kill the snails (LC100) were recorded.
51
CHAPTER III
TWO NEW MALYNGAMIDES PROM THE CYANOBACTERIUM LYNGBYA
MAJUSCULA
Abstract
Two new secondary metabolites, malyngamide J and
malyngamide L, have been isolated from a Curacao collection of the
cyanobacterium Lyngbya majuscula. The structure elucidation of
these new compounds was accomplished by analysis of 1H NMR, "C
NMR and ID NMR, along with comparisons with known natural
products.
Stereochemistry and biological properties have been
investigated.
52
Introduction
Cyanobacteria have been a source of diverse types of
secondary metabolites. Fatty acids are one of the most common
compositions in cyanobacteria, which are normally present as C14 to
C18 components.
Malyngic acid, 9(S), 12(R), 13(S)-
trihydroxyoctadeca-1 0(E),15(Z)-dienoic acid (16, Figure III.1) is a
major fatty acid that occurs in both shallow-water and deep-water
varieties of Lyngbya majuscula,91 while 9-methoxy-9methylhexadeca-4(E),8(E)-dienoic acid (17) is present in moderate
amounts in the deep-water Lyngbya majuscula."
In contrast to the situation in eukaryotic algae, nitrogen.
containing secondary metabolites are common in cyanobacteria.
Shallow-water varieties of Lyngbya majuscula contain amides of the
fatty acid 7(S)-methoxytetradec-4(E)-enoic acid (18), which in free
form displayed antimicrobial activity to the gram positive bacteria
Staphylococcus aureus and Bacillus subtilus. On the other hand,
deep-water collections of Lyngbya majuscula provided amides of 7-
methoxy-9-methylhexadec-4(E)-enoic acid (19)."
Malyngamide A (20) and malyngamide B (21) are two such amides
of 7(S)-methoxytetradec-4(E)-enoic acid (18), which have been
found in several shallow-water varieties of Lyngbya majuscula
from Hawaii.9" Their structures were determined by NMR
analysis and chemical degradation studies. Malyngamide C (22), a
chlorine-containing amide of 7(S)-methoxytetradec-4(E)-enoic acid,
was isolated from a shallow-water Lyngbya majuscula found on the
53
OH
OH
16
OH
17
OH
18
OH
Figure III.1 Secondary Metabolites from Different Varieties of
L. majuscula.
54
reefs of Fanning Island in the Line Islands.' The complete
structure of 22, including absolute stereochemistry, was
determined by spectral and chemical studies. From a deep-water
collection of Lyngbya majuscula found on the pinnacles in Eniwetok
lagoon, malyngamides D (23, Figure 111.2) and E (24), two closely
related amides of 7(S)-methoxy-9-methylhexadec-4(E)-enoic acid
(19) have been found. Detailed spectral analysis and chemical
degradation studies defined the structures and the ring
stereochemistry."
Malyngamide F (25) was reported from shallow water
collections of a Caribbean Lyngbya majuscula.
This secondary
metabolite showed mild cytotoxicity (ID50 <30 µg /ml) against KB
The structure elucidation was based on 2D
cells in tissue culture.
NMR data and chemical interconversion.97 Malyngamide G (26) was
isolated from a cyanobacterium epiphyte on the brown
mediterranean alga Cystoseira crinita.98
Guided by ichthyotoxicity
against goldfish, malyngamide I-1 (27) was isolated from another
Caribbean L. majuscula.
The structure and the absolute
stereochemistry of the cyclohexenone moiety was elucidated by
spectroscopic analysis and exciton chirality in the circular dichroism
spectrum.99
The latest report of malyngamides was of malyngamide
I (28), isolated from a Okinawan shallow water collection of
Lyngbya majuscula.
This compound showed moderate toxicity
towards brine shrimp (LD50 ca. 35 µg /m1) and goldfish (LD50 < 10
µg/m1)."°
55
OCH3
24
OH
OCH3
25
H3C
OCH3
CH3
Cl
26
OCH3
0
0
/\7.\.77./.)(N
CH3
H
27
OCH3
0
CH3
28
Figure 111.2 Structures of Various Malyngamides from
L. majuscula.
56
Results and Discussion
For this work, a collection of Lyngbya majuscula was made
from Playa Kalki, Curacao in about 20 ft of water. The sample was
preserved in isopropanol and stored at -20 °C.
The organic extract
was obtained from the homogenized alga by soaking in CH2C12 /MeOH
(2:1, v/v) for 30 minutes followed by filtration through cheese
cloth. The algal material was placed in fresh solvent (CH2C12/Me011,
2:1 v/v), heated to a gentle boil for 20 minutes, and filtered. This
process was repeated twice.
The filtrate was partitioned between
CH2C12 and water, and the organic fraction was reduced in vacuo and
stored in Et20 (4.32 g, dark green oil).
The isopropyl alcohol preserved sample (1L) was filtered prior
to extraction. This alcohol was removed by evaporation in vacuo
and the residue was added to the aqueous extract. The
CH2C12 /MeOH extracted algal residue was soaked in Me0H/H20 (3:1,
v/v) overnight, filtered, and the alcohol was removed in vacuo. The
"aqueous extract" was partitioned between sec-butanol and water.
The sec-butanol fraction (0.68 g, dark green oil) was reduced in
vacuo and stored in Me0H (Figure 11.4).
Two-dimensional TLC analysis of the organic extract suggested
the presence of several UV-active compounds. The fractionation
utilized gradient vacuum chromatography (EtOAc /hexane, 4 %100 %), gradient flash chromatography (Me0H/CHC13, 0%-2%), RP C-
18 plug chromatography (MeOH/H20, 80%-100%) and HPLC (Al ltech,
Lichrosorb Diol column 10u, Me0H/Et0Ac/hexane 1:3:16) to yield a
colorless lipid (29). This was shown to be a new member of the
57
Organic Extract of Lyngbya majuscula from Curacao
(4.32g)
Gradient Vacuum Chromatography
EtOAc / Hexane 4%--100%
1-1
A
Gradient
B
I
I
I
I
C
I
2
0.5%
1
0%
Reversed-phase RP C-18 chromatography
r-----2
I
4
2%
I
1
3
100%
90%
80%
4
100%
Me0H / EtOAc / Hexane
HPLC
0.5
A
I
3
I
1
Gradient Solvent Me0H / H2O
G
I
Flash Chromatography
Me0H / CHCI3 0%--2%
F
E
(70 mg)
D
:
1.5
:
8
B
malyngamide J
14.5mg
Figure 111.3
The Isolation of Malyngamide J (29).
58
malyngamide family, and was therefore named malyngamide J
(Figure 111.3). Another less polar fraction was subjected to silica gel
column chromatography (EtOAc /hexane, 85%), gradient flash
chromatography (Me0H/ CHC13, 0.1%-10% v/v) and HPLC (Alltech,
Lichrosorb Diol column 10u, Me0H/Et0Ac/hexane 1.5:9:120 v/v) to
give a light-yellow oil (30, malyngamide L, Figure 111.4).
Compound 29 showed optical rotation [43 +64° (c = 0.15,
CHC13).
High resolution FAB MS (positive ion, 3-nitrobenzyl alcohol)
gave a major [M+H]+ ion at m/z 608.3798 (0.0 mmu dev.) which was
consistent with a molecular formula of C33H53N09. This molecular
formula indicated that compound 29 possessed eight degrees of
unsaturation.
Analysis of UV (MeOH, Xma, 240 nm c = 5600), 111 NMR
and "C NMR data (Table III.1) revealed the presence of three
double bonds, an amide carbonyl group, an a, 0-unsaturated
carbonyl, and three rings.
By using
COSY and HETCOR spectral data (Figure 111.5),
several distinct spin systems could be assembled (Figure 111.6).
Fragment a possessed a 7-methoxytetradec-4(E)-enonate moiety,
which is a common feature of malyngamides. The partial structure
b placed two consecutive methylene groups contiguous to an amide
N atom by observation of HMBC correlations between the methylene
protons Ha,b-1 (83.43 , 83.37) and the carbonyl carbon C-1' (8172.7).
The deshielded methylene singlets Ha,,,-4 (85.22, 85.28) showed no
correlation by '11-11-1 COSY to any other group and suggested a
terminal olefin with a quaternary carbon in partial structure c.
111-'14 COSY correlations gave the partial structure d with a
59
Extract of Lyngbya majuscula from Curacao
(4.32g)
Gradient Vacuum Chromatography
EtOAc / Hexane 4%--100%
4%
50%
25%
25%
100%
75%
50%
(100 mg)
Column Chromatography
EtOAc / Hexane 85%
Gradient Flash Chromatography
Me0H / CHC13 0.1% -- 10%
I
I
1
2
1
I
3
4
0 5%
0.2%
0.1%
HpLc
10%
Me011 / EtOAc / Hexane
1.5
Fl
1
10
1
4
2
I
3
I
5
:
9
:
120
I-1
6
7
Malyngamide L
6.6 mg
Figure 111.4
The Isolation of Malyngamide L (30).
60
deshielded methylene contiguous to four consecutive deshielded
methines. The more deshielded methine C-12 (1H 54.48, "C 5103) at
the end of this chain suggested an acetal carbon.
In partial
structure e, a long range coupling was clearly observed by 11-1-111
COSY between the deshielded methyl 51.88 (C-11) and the olefin
proton 5 6.42 (H-8). This placed the methyl group on the quaternary
carbon (C-7) of the olefin, next to which were two sequential
deshielded methine groups 54.68 (I1-9), 53.70 (H-10). The "C
chemical shift of 563.8 for the C-10 methine CH 53.70) and a
quaternary carbon at 562.3 (C-5) suggested the presence of an
epoxide ring.
The HMBC correlation between the 11-10 proton
(53.70) and the C-5 quaternary carbon (562.3) confirmed this
assignment.
These partial structures were connected by interpretation of longrange correlations observed in the HMBC spectrum (Figure 111.7).
The correlation cross-peak observed from Hab-1 to C-1' secured the
amide linkage between partial structures a and b.
The correlation
seen from 11.,b-4 to C-5 and C-2 confirmed the connections between
partial structure c, b and e of the cyclohexenone. The correlations
between Ham-16 and C-12 completed a six-membered heterocyclic
ring. The correlation observed from H-12 to C-9 and from H-9 to
C-12 confirmed the linkage between the cyclohexenone and
dimethylated pentose sugar. The correlations seen from the H3-17
methoxyl group (53.6) to C-13 at 582.8 and from the H3-18
methoxyl to C-15 (578.9) placed these methoxyl groups on the
methine carbons C-13 and C-15.
From these data, a full assignment
18 16'
5'
4'
11
MAO
12
8
17
10
9'
18A
13
16b
HO
14
.5
0
43.1
_
1.0
_
1.5
9
0
2.0
O
0
2.5
O
3.0
0
3.5
O
-
4.0
-
4.5
5.0
09
A
0
6.5
5.5
_
6.0
_
6.5
s-00.
0
0
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
PPM
Figure 111.5 1H-1H COSY of Malyngamide J (29).
1.0
.5
PPM
62
H3C
a
C
b
d
e
8 3.60
au-13
8 3.50
ocH3
5 3.32
ocH3
Figure 111.6 Partial Structures of Malyngamide J
by 1H-1H COSY and HETCOR.
63
Table 11.1
'H , '3C and HMBC NMR Data Of Malyngamide J (29)
C-atom
'3C (CDC13)
1
8 for 1
37.5
2
33.0
3
139.9
117.6
4
5
6
7
8
9
10
62.3
194.2
11
16.5
12
13
14
15
16
103
82.8
75.5
78.9
63.4
17
18
58.9
61.0
1'
172.7
2'
3'
4'
5'
6'
7'
8'
9'
36.8
28.9
131.2
127.6
36.6
81.0
33.4
32.0
28.9
29.5
30.0
22.9
10'
11'
12'
13'
14'
15'
NH
OH
136.1
136.9
70.4
63.8
14.3
56.7
'H (CDC13) for 1
8 muti J in Hz
HMBC
3.37 m
3.43 m
2.44 m
2.35 m
1', 3, 2
--
3, 4, 5
5.22 s
5.28 s
5,2
5, 2, 3
6.42 bs
4.68 bd (4.4)
3.7 bs
1.88 s
4.48 d (7.4)
3.02 dd (9.2, 7.4)
3.53 dd (10.6, 8.9)
3.25 dddm (5.0, 9.1, 8.9)
3.15 bdd (8.9, 11.5)
4.04 dd (5.0, 11.5)
3.6 s
3.5 s
11, 10
7, 12, 5, 10
8, 5, 9
6, 7, 8
9, 17
17, 14, 12
2.28 m
2.35 m
5.48 m
5.48 m
2.18 m
3.15 m
1.43 m
1.28 m
1.28 m
1.28 m
1.28 m
1.28 m
0.89 t (6.7)
3.32 s
6.07 m
2.78 bs
1', 3' 4'
4' 5'
3', 6'
3', 6'
5', 7'
6', 15'
-
13, 15
18, 14
14, 15, 12
14, 12
13
15, 14
13', 12'
7'
1'
14, 15, 13
64
14'
O
17
12
H,co
O
13
14
16
15
HO
5.25
3.32
OCH3
1.28
1.28
5.48
1.28
2.35
2.1:
N
3 15
0.89
1.28
1.28
3.37
3.43
2.28
5.48
1.43
H
6.07
1.88
CH3
2A4
2.35
6.42
3.70
.68
3.6
Selected HMBC
4A8
H3CO
3.02
3.53
1H NMR
325
4.04
HO
2.78
OCH3
3.50
117.6
56.7
O
OCH3
22.9
127.6
32.0
29.5
30.0
28.9
33.4
9\2
36.6
172.7 N
14.3
16.5
37.5
28.9
81
131.2
3 6.8
33.0
62.3
CH3
136.1
;Z:g\,.
1
136.9
70.4
0
13C NMR
63.4
OCH3
61
Figure 111.7 The Structure of Malyngamide J (29) with 1H and
13C NMR Assignments by HMBC Correlations.
65
Table 111.2
C-atom
1
2
3
4
'1-1 and 13C NMR Date of Malyngamide L (30) from L. majuscula
13C (CDC13)
5
44.0
136.7
119.7
136.5
198.6
5
6
7
8
38.6
22.8
26.3
9
151.5
172.5
1'
2'
3'
4'
5'
6'
7'
8'
9'
1C'
11'
12'
13'
14'
15'
NH
36.6
29.5
130.9
127.9
36.6
80.9
33.6
32.0
25.5
29.9
30.0
22.8
14.3
56.7
1H (CDC13)
5 muti, J in Hz
3.94 d (6.1)
6.2 bs
2.54 ddd (6.5, 13.5)
2.08 ddd (6.5, 13.2, 10.3)
2.50 ddd (10.2, 4.3)
6.95 t (4.1)
2.24 dd (5.8, 7.5)
2.32 m
5.47 m
5.47 m
2.20 m
3.15 dddd (5.8, 11.4)
1.43 m
1.28 m
1.28 m
1.28 m
1.28 m
1.28 m
0.88 mt
3.34
6.10 m
of the planar structure of malyngamide J (29) was established.
This is the first malyngamide-type natural product to be found
containing a sugar in the molecule.
The relative stereochemistry of the dimethoxylated pentose
sugar in compound 29 was established through the analysis of the
proton coupling constants and nOe observations. The coupling
constant between H-13 (53.02) and H-14 (53.53) was 9.2 Hz which
indicated these two protons were diaxial (Figure III.10). Similarly,
the coupling constants 8.9 Hz between H-15 and H-16a (53.15); 10.6
Hz between H-14 and H-15 (53.25) gave their diaxial orientation,
66
while J15,16b = 5.0 Hz suggested that H-16b (84.04) is in the equatorial
orientation.
A cross peak between H-13 and H-15 in the NOESY
confirmed their axial orientations.
Although the observed coupling
constant between H-12 and H-13 was 7.4 Hz, a nOe correlation
between H-16a, H-14 and H-12 (84.48) provided unambiguous
evidence that H-12 was axial. Thus, an a-dimethoxylated xylose
structure can be assigned.
The absolute stereochemistry of the
sugar was not investigated, but may be D based on the previously
isolation of D-xylose from L. majuscula.
A small coupling constant between H-9 and H-10 (1.7 Hz)
required a dihedral angle between these protons to be around 60 °70°
In combination with dihedral angle calculations from molecular
modeling (Chem3D-Plus), two of the stereomeric models (A and B,
Figure III.11) with dihedral angle 65° and 71° between 11-9 and H-
10 were best matched to the coupling constants based on Karplus's
equations.
Thus, a trans relationship between H-9 and H-10 was
established.
The absolute stereochemistry of the epoxycyclohexenone ring
was determined by the observation of nOe between H-10 and H-4a,
H-9 combined with the CD analysis.' Both diastereomers (A' and
B', Figure 111.11) could reasonably display nOe between H-10 and
H-4a. The negative first Cotton effect (MeOH Aa247 max = -12.5) in
the CD spectrum can only be explained by conformation B', thus
establishing the absolute stereochemistry of C-9, C-10 and C-5 to be
R, S and S, respectively.
Figure 111.12 shows the structures with
stereochemistry of malyngamide J (29).
67
Dimethoxylated pentose ring of malyngamide J (29)
0A
3.6
H3co
3.02
4.48
12
O
3.15
4.04
3.53
HO
2.78
3.25
OCH3
3.50
Coupling constants
Figure Ill.10 Stereochemistry of Dimethoxylated Xylose Residue
in Malyngamide J (29).
68
H
H
4
H
H
B
A
Figure III.11
H
H
Proposed CD and NOE of Two Malyngamide J
Configurations.
69
Compound 30 showed optical rotation [cc]D = -8.4° (c = 0.28,
CHC13). HRFAB MS (positive ion, 3-nitrobenzyl alcohol) gave a
[M+H]+ ion at m/z 424.2618 (0.0 mmu dev.) which was consistent
with the molecular formula C24H38NO3C1.
This molecular formula
indicated six degrees of unsaturation. Examination of the UV (?max
223 nm, E = 8900), and "C NMR indicated the presence of an a, (3-
unsaturated carbonyl (8198.6), an amide carbonyl (8172.5) and six
olefinic carbons forming three double bonds (Table 111.2).
this compound was monocyclic.
Hence,
Furthermore, the 'H NMR spectrum
showed characteristics for a methoxylated fatty acid, a common
feature of malyngamides. Analysis of the 'H -'H COSY (Figure 111.8)
and 'I-1-13C HETCOR data and comparison with the spectral data of
malyngamide F (25)' gave a full structural assignment of
compound 30 (Figure 111.9).
The previous studies of malyngamides from L. majuscula have
shown that the stereochemistry of malyngamides at C-7' are always
of th S configuration." From the same extract, the known
metabolite malyngamide H (27) was isolated," which also contains
a S configuration at C-7'. Biosynthetic considerations suggest that
the 7-methoxytetradec-4-enoate moiety in malyngamide J (29) and
malyngamide L (30) have the same S configuration at position C-7'.
Malyngamide J showed brine shrimp toxicity with LC50 = 18
gg/m1 (ca.) and ichthyotoxicity with LC50 = 40 µg /ml, while
malyngamide L showed brine shrimp toxicity with LC50 = 6 µg /m1
and ichthyotoxicity with LC50 = 7 µg /ml.
14'
4.0
5.0
6.0
7.9
I
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
'
3.0
2.5
2.0
1.5
PPM
Figure 111.8 1H-1H COSY of Malyngmide L (30).
1.0
.5
0 !O
PPM
71
15'
9'
11'
13'
14'
10'
12'
8
3.34
0.88
56.7
OCH3
22.8
14.3
30.0
127.9 29.5
36.6 N....
32.0
29.9
25.5
33.6
80.9
0
0
130.9
2.24
172.5
N 3.94
136.7
198.6
38.6
H
119.7
13C NMR
22.8
CI
26.3
Figure 111.9 The Structure of Malyngamide L (30) with 1H and
13C Data.
ders
15(:)
73
Experimental
General Methods.
Ultraviolet spectra were recorded on a
Hewlett Packard 8452A diode array spectrophotometer, and
infrared spectra (IR) were recorded on a Nicolet 510P-RHS
spectrophotometer. Nuclear magnetic resonance (NMR) spectra
were recorded on either Bruker AM 400 or Bruker AM 300 NMR
NMR chemical shifts are reported relative to
spectrometer. All
an internal tetramethylsilane (TMS) standard and "C spectra
referenced to the center line CDC13 at 77.25 ppm. CD measurements
were obtained on a Jasco 41A spectropolarimeter. Low resolution
mass spectra (LRMS) were obtained on a Varian MAT CH7
spectrometer, while high resolution mass spectra (HRMS were
obtained on a Kratos MS 50 TC. High performance liquid
chromatography (HPLC) was performed using a M-6000 pump, U6K
injector and either R 401 differential refractometer or a lambdaMax 480 lc spectrophotometer. TLC-grade (10-40 gm) silica gel was
used for vacuum chromatography and Kieselgel 60 silica (40-63 gm)
was used for flash chromatography, and Merck aluminum-backed
TLC sheets (silica gel 60 F254) were used for thin layer
chromatography. All solvents were distilled from glass prior to use.
Isolation of Malyngamide J (2 9). The fractionation of the crude
extract was achieved by silica gel vacuum chromatography using a
stepwise gradient from 4% to 100% (v/v) EtOAc in hexane (Figure
111.3).
Two-dimensional TLC (EtOAc /hexane 1:1; CHC13/MeOH 9:1)
suggested the presence of several UV-active secondary metabolites
in fraction E, and its 'H NMR spectrum showed interesting structural
74
features.
Further fractionation was applied by gradient flash
chromatography (MeOH /CHC13, 0%-2%) and continued with
reversed-phase RP C-18 chromatography to remove most of the
pigments.
The final purification was completed by HPLC (10-p.m
Phenomenex Maxial Diol column, Me0H/Et0Ac/hexane 1/3/16) to
yield malyngamide J (29, 14.5 mg, Figure 111.3).
Isolation of Malyngamide L (3 0). The fractionation of the
crude extract was followed by silica gel vacuum chromatography
(EtOAc/hexane, 4%-100%, v/v).
Two-dimensional TLC
(EtOAc /hexane 1:1; CHC13/MeOH 9:1) suggested the presence of
several UV-active compounds in fraction D (Figure 111.4). Further
fractionation was provided by silica gel chromatography (85%
EtOAc /hexane) followed by gradient flash chromatography
(MeOH /CHC13, 0%-10%). The natural product was then isolated by
using HPLC (10-pm Phenomenex Maxail Diol column,
Me0H/Et0Ac/hexane 1.5/9/120) to yield a UV-active natural
product, named malyngamide L(30, 6.6 mg, Figure 111.4).
Malyngamide J (2 9). UV (MeOH) 'max 240 nm (E = 5600);
optical rotation [a]E, = +64° (c = 0.15, CHC13); CD (MeOH): DE = -12.5,
+9.2 (Xmax 247, 217 nm); HR FABMS (positive ion, 3-nitrobenzyl
alcohol) in/z obs. [M+H]+ 608.3798 (C33H54N09, 0.0 mmu dev.); LR
FABMS nilz obs. 608 (28), 448 (100), 416 (26), 192 (17), 175 (25),
163 (15), 136 (13), 101 (24), 95 (13), 87 (24), 81 (26), 75 (27), 69
(42), 59 (12), 55 (31), 45 (33); '11 NMR and 13C NMR data see Table
75
Malyngamide L (3 0). UV (MeOH) Xmax 223 nm (c = 8900);
optical rotation [oc]D = -8.4° (c = 0.28, CHC13); HR FABMS (positive
ion, 3-nitrobenzyl alcohol) m/z obs. [M+H]+ 424.2618 (C24H39NO3C1,
0.0 mmu dev.); LR FABMS m/z obs. 424 (78), 426 (32), 394 (20),
392 (57), 388 (8), 307 (17), 289 (15), 281 (14), 220 (15), 186 (10),
171(18), 169 (51), 154 (100), 150 (17), 138(30), 136 (82), 133 (10);
NMR and "C NMR data see Table 111.2.
Brine Shrimp Bioassay.
See Chapter II. experimental.
Ichthyotoxicity Assay.
See Chapter II. experimental.
76
CHAPTER IV. CONCLUSION
Lyngbya majuscula has been recognized as a chemically and
biologically rich strain. This investigation gave a good
representation showing the diversity of organic molecules produced
by Lyngbya majuscula. From a single collection at Playa Kalki,
Curacao (11 August 1994), we have isolated three novel bioactive
secondary metabolites (Figure. IV). Although not investigated in
this work, these metabolites may play an important ecological role
in the survival of this species.
Kalkitoxin has potent biological activities which inspire us to
further investigation of its biological properties and mechanism of
toxicities.
Kalkitoxin also possesses a unique structure with
relatively simple chemical characterics and small molecular weight.
These features would be advantages for kalkitoxin to serve as a
lead compound in drug development and as biochemical probes for
the discovery of pharmacological and biochemical processes.
The malyngamide class represents a rich family of compounds,
which offer a great deal of investigative possibilities in areas such
as algal physiology, chemo-defense, chemo-communication, and
chemical ecology. These new malyngamides display very
interesting chemical features. In particular, malyngamide J
possesses a pentose ring with a highly electro dense
epoxycyclohexenone ring at one end, and a lipid portion at another
end. A nitrogen-containing unit connects these two parts into the
molecule. These features impressed us based on our knowledge and
77
experience about known drugs. These exciting features also
motivated us to search and develop novel and sensitive techniques
to best detect and uncover their full biological properties.
Extract of Lyngbya majuscula from Playa Kalki, Curacao
4.32g
Gradient Vacuum Chromatography
EtOAc / Hexane 4%--100%
A
B
F
C
S
NS
0
kalkitoxin
12.9 mg
O
0
malyngamide L
6.6mg
Figure IV.1
The Chemical Diversity of Lyngbya majuscula from Play Kalki, Curacao.
79
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88
APPENDIX
RESULT
102.41
100.0--
95.0vxu
J
80.0 --
75.33
i
r
t
4000
i
i
i
3500
3000
i
i
i
II
i
2500
2000
WAVENUMBERS
Figure A.2 IR Spectrum of 15
1
15100
1000
700.0
IN ONO 206K HNSC
cz. o
as.
20
CZ
0.
lea
6°
coo
40
60
00
0.
111
6,3
60
1:1114
100
0=1.0
0
120
ad
0
f40
EX)
160
4
0100
I
'16
cO (:)0
lop
0
0
ED
...
PPM
6.0
5.5
5.0
4.5
4.0
3.0
3.5
2.5
2.0
PPM
Figure A.3 HMBC Spectrum of 15 in D6 -DMSO at 298K
160
Figure A.4 111 NMR Spectrum of 15 in C6D6
IN OMSO 340K
20
40
60
I
80
100
Is
*41
120
140
160
si
'
11!"?
°eel
1
st1
6.0
5.5
5.0
4.5
4.0
3.5
180
°C;)
I
3.0
2.5
2.0
'
I
1.5
PPM
Figure A.S. HMBC Spectrum of 15 in D6 -DMSO at 340K
1.0
PPM
170
160
150
140
Figure A.6
130
120
110
100
90
80
70
60
50
40
30
PPM
DEPT (135) Spectrum of 15 in D6 -DMSO at 298K
20
MNI1213F3 IN DMSO 2116K
1
7
170
'
r
160
1
150
140
130
120
110
100
PPM60
60
70
60
50
40
30
Figure A.7 DEPT (90) Spectrum of 15 in D6 -DMSO at 298K
20
10
IN OMSO 340K
170
160
150
140
130
120
110
100
90
00
70
60
b0
40
30
PPM
Figure A.8 DEPT (135) Spectrum of 15 in D6-DMS0 at 340K
20
10
II
i
6.5
6.0
5!5
5.0
4.5
4.0
3.5
3.0
2.5
PPM
Figure A.9 'H Decoupling Spectrum-1 of 15 in C6D6
di
3.5
3.0
2.5
2.0
Figure A.10 'H Decoupling Spectrum-2 of 15 in C6D6
RESULT
103.01
100.0--
co
CD
63.0-
80.0
76.30
i
4000
3500
I
I T i
3000
i
t
t
i
F
f
I
I
2500
20.00
WAVENUMBERS
Figure A.11 IR Spectrum of 29
1500
I
I
1000
700.0
100_
ea_
69
40_
45
175
101
416
55
87
75 81
0
11111,1,1
192
163
9
S9
136
147
I
11111111i11,111,111tiliniiillhillitdirlITA,J111111111,
11111 111,
1.1
IR
100
50
tilb-rdi l'ir'll'Iir"Thrt'tli"r -r let Y.1' t'. ( .,,-, i t
,
I
,
4
,
,
)1 ,
, -1 Y r
11'1
448
I00
ea_
40
608
20
434
0
111'11
464
I
to
450
476
ti 'It
I
576
II
500
550'
592
t.
seo
"
624
650
Figure A.12 LRFAB Mass Spectrum of 29
I
7e0
rIlijk I.
400
350
300
250
200
.
,
''
.
,
15
10-
5-
-5-10--
-15
200
iiiiiiiiiiIIIIIIIIIiiiiiiIIII
210
220
230
240
250
260
270
280
290
300
310
Figure A.13 CD Spectrum of 29
320
330
340
350
11
200
190
180
170
160
150
140
9
130
9
120
91
9
100
110
90
80
70
PPM
Figure A.14 13C NMR Spectrum of 29
60
50
40
30
20
10
MW//59FC.101
DATE 29-3-96
400.134
SY 133.0
6400.000
01
SF
16384
TO 16384
SW
6024.96
.735
HZ/PT
SI
3.0
2.000
1.360
PW
RD
AO
AG
20
NS
32
298
TE
FM
7600
02
DP
63L PO
0.0
L8
GB
CX
CY
F1
0.0
0.0
35.00
17.00
7.000P
F5
HZ /CM
74..3104 0P
.186
PPM/CM
4390.25
SR
Jif
f'
)
2
5.5
6.0
5.5
5.0
4.5
3.5
4.0
30
2.5
PPM
Figure A.15 'H NMR Spectrum of 29
2.0
1.5
1.0
20
O
9
0
40
60
60
100
I
'.0
o
,..
120
140
180
0
160
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
PPM
Figure A.16 HMBC Spectrum of 29
1.5
1.0
PPM
iil
II
II
I
1.0
ill
I
a
1.5
2.0
2.5
I
3.0
14*
I
.
0
4
3.5
1
4.0
a
4.5
5.0
5.5
it
6.0
6.5
200
180
160
140
120
100
80
60
PPI4
Figure A.17 BETCOR Spectrum of 29
40
2'0
PPI4
11
200
190
180
170
160
150
140
130
120
100
110
90
80
70
60
50
PPM
Figure A.18 DEPT (135 and 90) Spectrum of 2 9
40
30
20
10
)
4.5
4.0
3.5
30
2.5
2.0
PPM
Figure A.19 NOE Difference Spectrum-1 of 29
1.5
1.0
1
1
1
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
I
2.5
2.0
PPM
Figure A.20 NOE Difference Spectrum-2 of 29
1.5
1.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
PPM
Figure A.21 NOE Difference Spectrum-3 of 29
1.5
1.0
.5
1
4!5
4.0
3.5
3.0
2.5
2.1
0
PPM
Figure A.22 NOE Difference Spectrum-4 of 29
-
1.0
-
1.5
2.0
2.5
3.0
_
3.5
-
4.0
4.5
1
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
PPM
Figure A.23 NOESY of 29
1.5
1.0
-
5.0
-
5.5
-
6.0
-
8.5
PPM
200
190
1B 1 0
170
160
150
140
130
120
110
100
90
60
1
1
70
60
PPM
Figure A.24 13C NMR Spectrum of 3 0
1
50
40
1
1
30
2
20
10
'
MWII70F3.201
DATE 26-4-96
400.134
SY 133.0
6400.000
01
SF
16384
16384
6024.96
SW
HZ/PT
.7035
SI
TD
PM
RD
AG
AG
NS
TE
3.0
2.000
1,360
80
32
290
FM
7600
02
DP
63L PO
LB
GB
CX
CY
FI
F2
0.0
0.0
0.0
35.00
18,00
7,492P
-.152P
HZ/CM 87.382
.218
PPM/CM
SR
4394.37
r
7.0
6.5
6.0
5.5
5.0
4.5
3.5
4.0
3.0
2.5
2.0
PPM
Figure A25 1H NMR Spectrum of 30
1.5
1.0
.5
0.0
70
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
PPM
Figure A26 1H-'H COSY of 30
2.0
1
-
1.0
-
2.0
_
3.0
_
4.0
..
5.0
_
6.0
-
7.0
o
o
4
I
t
1
200
180
160
1140
120
100
80
60
PPM
Figure A.27 HETCOR Spectrum of 30
40
20
; PPM
154
135
169
186
143
150
337
71
133
II j11
1111111
)11111i
1
lill'itillAINIP1'1'1101.111WIli't 111.1'k ,
I ir r
r 1+11'1'1 ,.1.1111
-,1% I
r11'ti it Nkk, Y
320
320
283
260
240
220
200
183
160
140
1
289
281
192
165
,
,
340
424
392
426
394
446
388
356
363
ft
360
371
II
1111,-1,
r f17 P
380
I
4°2
1 rt1
,t11-tt
400
8
II
420
461
ih(111?(11(111(11
440
460
1,11111111111111111111
483
500
Figure A.28 LRFAB Mass Spectrum of 3 0
20
540
560