AN ABSTRACT OF THE THESIS OF
Lisa Marie Nocile for the degree of Doctor of Philosorhy in Pharmacy presented on
August 06 2002.
Title: Structure Elucidation and Biosynthetic Investigations of Marine
Cyanobacterial Secondary Metabolites
Redacted for Privacy
Abstract approved
William H. Gerwick
This thesis details my investigations of marine cyanobacterial natural
products that resulted in the discovery of thirteen new secondary metabolites, the
isolation of over fifteen previously reported metabolites and the biosynthetic
investigation of two additional cyanobacterial compounds.
Two novel lipopeptides were identified from a Lyngbya majuscula and
Schizothrix sp. assemblage collected in the Fiji Islands. Somamide A is a
depsipeptide consisting of a hexanoate moiety extended by seven amino acids,
including two nonstandard units characteristic of cyanobacterial peptides. In
contrast, somocystinamide A is a unique linear disulfide dimer displaying potent
cytotoxicity against a mammalian neuroblastoma cell line.
The organic extract from a Puerto Rican L. majuscula proved remarkably
rich in chemistry, producing twelve known compounds as well as four new
secondary metabolites. Among these new isolates were the novel sodium channel
blocker antillatoxin B, a new chlorinated quinoline derivative and the new ct-pyrone
malyngamide T.
A collection of L. majuscula from Antany Mora, Madagascar, led to the
isolation of the previously reported antineoplastic agent dolastatin 16 and the
discovery of a new series of lipopeptides, the antanapeptins. These new
molecules are characterized by the presence of the unique 3-hydroxy acid 3hydroxy-2-methyloctynoate, or its reduced double- or single-bond equivalent.
Wewakazole is a novel cyclic dodecapeptide isolated from a Papua New
Guinea collection of L. majuscula. This large molecule contains both a
methyloxazole and two oxazoles, residues rarely observed in marine
cyanobacterial metabolites. Extensive utilization of 1 D and 2D NMR techniques
were required to elucidate the structure of this distinctive peptide.
Biosynthetic investigations of two halogenated cytotoxins were also
conducted on a cultured
L.
majuscula strain originally isolated from Hector Bay,
Jamaica. Stable isotope feeding experiments demonstrated that both jamaicamide
A and hectochlorin derive from mixed PKS and NRPS biosynthetic origins but are
comprised of primary precursors unique to each molecule.
©Copyright by Lisa Marie Nogle
August 06, 2002
All Rights Reserved
Structure Elucidation and Biosynthetic Investigations of Marine Cyanobacterial
Secondary Metabolites
by
Lisa Marie Nogle
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented August 06, 2002
Commencement June 2003
Doctor of Philosophy thesis of Lisa Marie Nogle presented on AuQust 06. 2002.
APPROVED:
Redacted for Privacy
Major Professor, representing Pharmacy
Redacted for Privacy
Dean of'the College of Prmacy
Redacted for Privacy
Dean
of tl4
Graduate School
I understand that my thesis will become part
of
the permanent collection of Oregon
State University libraries. My signature below authorizes release
any reader upon request.
Redacted for Privacy
Lisa Marie Nogle, Author
of
my thesis to
ACKNOWLEDGEMENTS
I would like to express my gratitude to my major advisor, Dr. William
Gerwick, and my committee members, Dr. Mark Zabriskie, Dr. Philip Proteau, Dr.
David Home, and Dr. David Mok for their support and academic guidance
throughout my graduate career at Oregon State University. I would also like to
acknowledge Brian Arbogast and Jeff Morre for their extensive mass spectral
work, and Roger Kohnert for his valuable NMR assistance. Funding for this thesis
research was provided by the National Institute of Cancer, DOW AgroSciences,
and the Marine-Freshwater Biomedical Science Center at Oregon State University.
I am very grateful to my current and former graduate colleagues for their
mentorship, insight, and most importantly, friendship. I would especially like to
acknowledge Brian Marquez, Thomas Williamson and Ana Barrios-Sosa for their
continual inspiration and motivation, Namthip Sitachitta and Lik Tong Tan for their
friendship and encouragement across the miles, and Rebecca Medina, David
Blanchard, Heidi Zhang, Younhi Woo, Andra Vulpanovici and Jane Menino and for
their enduring friendships throughout the years. Completion of this degree would
have been impossible without them all.
Finally, I would like to express my gratitude to my wonderful family,
especially Keith Mayhugh, for their support and encouragement. I am forever
indebted to them for their patience and love through these challenging years.
CONTRIBUTION OF AUTHORS
Dr. Thomas Williamson and Dr. Brian Marquez ran the 2D NMR
experiments discussed in chapters two and three, respectively. Dr. Marquez also
performed the extensive HMBC experiments discussed in chapter five and
consulted on the biosynthetic studies described in chapter six. Brian Arbogast ran
all mass spectra except for the desmethoxymajusculamide C spectra found in
appendix A, which were run by Jeff Morre.
Both Gloria Zeller and Christina Jamros assisted in the antimicrobial
assays used for testing in chapter four. Dr. Tatsufumi Okino performed the
ichthyotoxicity assay described in chapter four. Additionally, Dr. Okino ran the
neuroblastoma assay used in chapters two, three and four. Dr. Doug Goeger also
conducted this assay for the compounds in appendices A and B.
TABLE OF CONTENTS
PAGE
CHAPTER ONE: GENERAL INTRODUCTION
GENERAL THESIS CONTENTS
1
HIGHLIGHTS OF MARINE NATURAL PRODUCTS
3
REFERENCES
19
CHAPTER TWO: NOVEL BIOACTIVE LIPOPEPTIDES FROM A
FIJIAN MARINE CYANOBACTERIAL MIXED ASSEMBLAGE
ABSTRACT
25
INTRODUCTION
26
RESULTS AND DISCUSSION
29
EXPERIMENTAL
57
REFERENCES
61
CHAPTER THREE: NEW AND DIVERSE SECONDARY METABOLITES
FROM A PUERTO RICAN COLLECTION OF LYNGBYA MAJUSCULA
ABSTRACT
63
INTRODUCTION
64
RESULTS AND DISCUSSION
67
TABLE OF CONTENTS (Continued)
PAGE
EXPERIMENTAL
88
REFERENCES
91
CHAPTER FOUR: THE ANTANAPEPTINS, NEW CYCLIC
DEPSIPEPTIDES FROM A MADAGASCAN LYNGBYA MAJUSCULA
ABSTRACT
93
INTRODUCTION
94
RESULTS AND DISCUSSION
97
EXPERIMENTAL
112
REFERENCES
115
CHAPTER FIVE: WEWAKAZOLE, A NOVEL CYCLIC DODECAPEPTIDE
FROM A PAPUA NEW GUINEA COLLECTION OF LYNGBYA MAJUSCULA
ABSTRACT
117
INTRODUCTION
118
RESULTS AND DISCUSSION
120
EXPERIMENTAL
134
REFERENCES
137
TABLE OF CONTENTS
(Continued)
PAGE
CHAPTER SIX: BIOSYNTHETIC STUDIES OF HECTOCHLORIN
AND THE JAMAICAMIDES
ABSTRACT
138
INTRODUCTION
139
RESULTS AND DISCUSSION
143
EXPERIMENTAL
162
REFERENCES
167
CHAPTER SEVEN: CONCLUSION
169
BIBLIOGRAPHY
172
APPENDICES
184
LIST OF FIGURES
FIGURE
PAGE
11.1
Bioactive metabolites from marine microalgae.
26
11.2
Isolation and purification of compounds 12 and 15.
29
11.3
1H and 13C NMR spectra of somamide A (12).
32
11.4
HSQC spectrum of somamide A (12).
33
11.5
HSQC-TOCSY spectrum of somamide A (12).
34
11.6
ROESY spectrum of somamide A (12).
35
11.7
HMBC spectrum of somamide A (12).
36
11.8
Partial sequencing of 12 by HMBC and ROESY correlations.
37
11.9
Low resolution positive FABMS of somamide A (12).
38
11.10
Amino acid sequencing of 12 by mass spectral
fragmentation analysis.
38
11.11
1H and 13C NMR spectra ofsomocystinamideA(15).
41
11.12
COSY spectrum of somocystinamide A (15).
43
11.13
1H and 13C NMR spectra of intermediate product mixture.
44
LIST OF FIGURES
(Continued)
FIGURE
PAGE
11.14
1H and 13C NMR spectra of degradation product (16).
45
11.15
1H NMR spectral comparison of the somocystinamide
decomposition process.
46
11.16
HSQC spectrum of degradation product (16).
48
11.17
HSQC-TOCSY spectrum of degradation product (16).
49
11.18
HMBC spectrum of degradation product (16).
50
11.19
Low resolution positive FABMS of degradation product (16).
51
11.20
HSQC spectrum of somocystinamide A (15).
54
11.21
HMBC spectrum of somocystinamide A (15).
55
11.22
Low resolution positive FABMS of somocystinamide A (15).
56
Previously reported non-toxic metabolites from collection
PRLP-1 6ISept/97-03.
66
1H NMR spectral comparison of (A) antillatoxin (4) and
(B) antillatoxin B (15).
67
111.3
HSQC spectrum of antillatoxin B (15).
71
111.4
TOCSY spectrum of antillatoxin B (15).
72
111.1
111.2
LIST OF FIGURES (Continued)
FIGURE
PAGE
111.5
HMBC spectrum of antillatoxin B (15).
73
111.6
1H NMR spectrum of malyngamide T (16).
75
111.7
HSQC spectrum of malyngamide 1 (16).
76
111.8
HMBC spectrum of malyngamide T (16).
77
111.9
1H NMR spectrum of quinoline 17.
79
111.10
HSQC spectrum of quinoline 17.
80
111.11
HSQMBC spectrum of quinoline 17.
81
111.12
Key coupling constants and HSQMBC correlations for 17.
82
111.13
1H NMR spectrum of tryptophan derivative 18.
83
111.14
HMBC spectrum of tryptophan derivative 18.
85
111.15
Proposed biogenic relationships between secondary
metabolites from Lyngbya majuscula.
87
IV.1
Dolastatins isolated from both marine invertebrate and
cyanobacterial sources.
94
IV.2
Bioactive peptides from Lyngbya majuscula.
95
LIST OF FIGURES
(Continued)
FIGURE
PAGE
IV.3
Isolation scheme for the antanapeptins.
97
IV.4
1H NMR spectrum of antanapeptin A (9).
98
IV.5
HSQC spectrum of antanapeptin A (9).
99
IV.6
HSQC-TOCSY spectrum of antanapeptin A (9).
100
IV.7
HMBC spectrum of antanapeptin A (9).
101
IV.8
Cyanobacterial metabolites containing C8 alkynoate moieties.
103
IV.9
Partial structure sequencing of 9 by HMBC correlations.
104
IV.1O
1H NMR spectral comparison of the antanapeptins.
106
IV.11
Low resolution positive FABMS of antanapeptins A-D.
108
IV.12 Marine invertebrate metabolites with structural homology
to the antanapeptins.
111
V.1
1H and 13C NMR spectra of wewakazole (5).
121
V.2
HSQC spectrum of wewakazole (5).
122
V.3
TOCSY spectrum of wewakazole (5).
123
LIST OF FIGURES (Continued)
FIGURE
PAGE
V.4
HMBC spectrum of wewakazole (5).
124
V.5
Oxazole and methyloxazole containing metabolites
from marine macroorganisms.
126
V.6
Partial amino acid sequencing by an 8 Hz optimized HMBC.
128
V.7
MS/MS fragmentation of the [M+H] parent ion m/z 1142.
128
V.8
Remaining structure fragments of 5, with arrows indicating
the expected ring closing linkages.
129
V.9
1 D HMBC of 5.
131
V.10
Possible orientations for the MeOxz-Phe-Oxz fragment in 5
based on either a (A) 3J coupling or (B) a 4J coupling from
H 3.29/3.17 to & 163.6.
131
Vl.1
Bioactive halogenated metabolites of marine origin.
139
Vl.2
Summary of the biosynthetic units forming barbamide (6)
and curacin A (7).
140
V13
Proposed biosynthetic composition of jamaicamide A (12).
143
VI.4
Pyrrolidone-containing metabolites from marine sources.
144
LIST OF FIGURES
(Continued)
FIGURE
PAGE
13C NMR spectra of (A) natural abundance jamaicamide A,
(B) jamaicamide A isolated from cultures provided with
[1 -1C]acetate and (C) jamaicamide A isolated from cultures
provided with [2-13C]acetate.
146
13C NMR spectrum of jamaicamide A (12) isolated from
cultures provided with [1,2-13C2]acetate.
147
13C NMR spectrum of jamaicamide A isolated from cultures
provided with S-[methyl-13Cjmethionine.
148
13C NMR spectra of A) jamaicamide A isolated from cultures
provided with S- [11 C]alanine and (B) jamaicamide A isolated
from cultures provided with S-[3-13C]alanine.
150
VI.9
Metabolic pathways of alanine and pyruvate.
151
VI.1O
Expansions of the 13C NMR spectrum of jamaicamide A
isolated from cultures provided with [13C3,15N]13-alanine.
152
Qualitative HPLC comparison of jamaicamide production in
(A) 100-fold bromide ion media, (B) normal bromide ion
control and (C) bromide ion depleted media.
153
VI.12
Proposed biosynthetic composition of hectochlorin (8).
154
V113
13C NMR spectra of (A) natural abundance hectochiorin,
(B) hectochlorin isolated from cultures provided with
[1-'3C]acetate and (C) hectochtorin isolated from cultures
provided with [2-13C]acetate.
155
V15
VI.6
Vl.7
Vl.8
VI.11
LIST OF FIGURES
(Continued)
FIGURE
PAGE
V114 13C NMR spectrum of hectochlorin (8) isolated from cultures
Vl.15
VI.16
provided with [1,2-13C2]acetate.
156
13C NMR spectrum of hectochiorin isolated from cultures
provided with S-[methyl-13C}methionine.
158
13C NMR spectra of (A) hectochiorin isolated from cultures
provided with [1-13C}pyruvate, (B) hectochiorin isolated from
cultures provided with S- [1-13C]alanine and (C) hectochlorin
isolated from cultures provided with S-[3-13C]alanine.
160
LIST OF TABLES
PAGE
TABLE
11.1
NMR spectral data for somamide A (12) in DMSO-d6.
31
11.2
NMR spectral data for compound 16 in 1:1 CDCI3/CD3OD.
47
11.3
NMR spectral data for somocystinamide A (15) in CDCI3.
52
111.1
NMR spectral data for antillatoxin B (15) in CDCI3.
70
111.2
NMR spectral data for malyngamide T (16) in CDCI3.
75
1II.3
NMR spectral data for quinoline alkaloid 17 in C6D6.
82
111.4
NMR spectral data for compound 18 in CDCI3.
84
IV.1
NMR spectral data for antanapeptin A (9) in CDCI3.
102
IV.2
1H and 13C data for antanapeptins B (10), C (11) and
D (12) in CDCI3.
107
V.1
NMR spectral data for wewakazole (5) in CDCI3.
125
Vl.1
Relative enhancements of carbon resonances from
jamaicamide A biosynthetic feeding experiments.
149
Relative enhancements of carbon resonances from
hectochiorin biosynthetic feeding experiments.
157
VI.2
LIST OF APPENDICES
APPENDIX
A
B
C
D
E
PAGE
Isolation and structure elucidation of desmethoxymajusculamide C from a Fijian Lyngbya majuscula
185
Isolation and structure elucidation of jamaicamide C
from a cultured Jamaican Lyngbya majuscuia
192
Isolation of known metabolites from a Puerto Rican
collection of Lyngbya majuscula
195
Isolation of curacin D from a Fijian assemblage of
marine cyanobacteria
199
Isolation of malyngolide from a Papua New Guinea
collection of Lyngbya majuscula
200
LIST OF APPENDIX TABLES
PAGE
TABLE
A.1
NMR spectral data for desmethoxymajusculamide C (1)
187
in CDCI3.
B.1
1H and 13C NMR comparison of jamaicamides C (3) and B (2).
193
LIST OF ABBREVIATIONS
Abu
2-Amino-2-butenoic acid
Ahp
3-Amino-6-hydroxy-2-piperidone
Ala
Alanine
Ara-A
Adenine arabinoside
Ara-C
Cytosine arabinoside
br
Broad
Cl
Chemical Ionization
COSY
Correlated Spectroscopy
d
Doublet
DHiv
Dihydroxyisovaleric acid
DMSO
Dimethylsulfoxide
EC
Effective Concentration
EtOAc
Ethyl Acetate
FAB
Fast Atom Bombardment
FDAA
Nct-(2,4-dinitro-5-fluorophenyl)-L-alaninamide
GC
Gas Chromatography
Gly
Glycine
Hex
Hexanoic acid
Hiv
2-Hydroxyisovaleric acid
HMBC
Heteronuclear Multiple Bond Correlation
Hmoya
3-Hydroxy-2-methyl-oct-7-ynoic acid
HPLC
High Performance Liquid Chromatography
HSQC
Heteronuclear Single Quantum Correlation
HSQMBC
Heteronuclear Single Quantum Multiple Bond Correlation
IC
inhibitory Concentration
lie
isoleucine
lR
Infrared Spectroscopy
LD
Lethal Dose
m
Multiplet
LIST OF ABBREVIATIONS (Continued)
Me
Methyl
MeOH
Methanol
MeOxz
Methyloxazole
MetSO
Methionine sulfoxide
MS
Mass Spectrometry
NMR
Nuclear Magnetic Resonance
NOE
Nuclear Overhauser Exchange
NRPS
Non-Ribosomal Peptide Synthetase
Oxz
Oxazole
PFPA
Pentafluoropropanoic acid
Phe
Phenylalanine
PKC
Protein Kinase C
PKS
Polyketide Synthase
Pro
Proline
q
Quartet
ROESY
Rotating Frame Overhauser Exchange Spectroscopy
s
Singlet
SAM
S-Adenosyl Methionine
SPE
Solid Phase Extraction
t
Triplet
TFA
Trifluoroacetic acid
Thr
Threonine
TOCSY
Total Correlation Spectroscopy
tR
Retention Time
Tyr
Tyrosine
UV
Ultraviolet Spectroscopy
Val
Valine
VLC
Vacuum Liquid Chromatography
STRUCTURE ELUCIDATION AND BIOSYNTHETIC INVESTIGATIONS OF
MARINE CYANOBACTERIAL SECONDARY METABOLITES
CHAPTER ONE
GENERAL INTRODUCTION
GENERAL THESIS CONTENTS
Investigation of marine cyanobacteria for their production of novel and
bioactive chemistry is the focus of our laboratory and of the research presented
within this Ph.D. thesis. Collections of these amazing organisms have been
acquired from exotic locations around the globe, as demonstrated by the various
projects described in the subsequent chapters. The chemistry of marine
microalgae, particularly that of the cyanobacterium Lyngbya majuscula, is
extremely rich and diverse. As such, the organization of the thesis chapters is
based on collection site rather than structural theme.
Following the general introduction to marine natural products included in
this first chapter, the second chapter describes the structure determination and
biological activities of two novel and chemically distinct Iipopeptides, somamide A
and somocystinamide A, isolated from a mixed cyanobacterial assemblage of
Lyngbya majuscula and Schizothrix sp. collected in 1997 from Somo Somo, Fiji.
Next, chapter three details the extensive chemical exploration of a 1997 Puerto
Rican L. majuscula that led to the isolation of several known and bioactive
compounds as well as four new metabolites, including a potent sodium channel
activating neurotoxin of unique structure. The fourth chapter discusses the
detection of a previously described marine molluscan anti-neoplastic agent,
dolastatin 16, from a cyanobacterial collection acquired in Antany Mora,
Madagascar, 2000. In addition to this biosynthetically and medicinally significant
2
finding, the antanapeptins, a new structural series that possesses high homology
to several other molluscan compounds, are also reported from this collection.
Chapter five then describes the challenging structure and stereochemical
determination of wewakazole, an oxazole-containing cyclic dodecapeptide isolated
from a L. majuscula collected in Wewak Bay, Papua New Guinea, in 1998. This
complex molecule required detailed analyses of NMR and mass spectral data and
the application of a 1 D HMBC experiment to aid in its characterization.
Chapter six details the biosynthetic investigations of two chemically diverse
classes of secondary metabolites isolated from a laboratory cultured Jamaican
Lyngbya majuscula. Stable isotope-labeled precursors and altered culturing
conditions were utilized in this work to gain insight into the biosynthetic building
blocks and enzymatic processes involved in metabolite assembly.
The closing chapter, seven, concludes this thesis with a brief summary of
the projects described within the text. General thoughts on the future direction of
natural products and the pursuit of the marine environment as a viable source for
novel chemistry of medicinal value are also considered.
HIGHLIGHTS OF MARINE NATURAL PRODUCTS
Introduction
Terrestrial biodiversity has long been recognized and exploited as a source
of valuable bioactive substances, from traditional herbal remedies to modern
pharmaceuticals. In fact, an estimated 57% of the 150 most prescribed drugs are
either natural product derivatives or are synthetic complements provided by natural
product lead structures. Despite the fact that oceans comprise over 70% of our
world's surface and harbor more than 80% of all life on earth, the majority of
reported natural products originate from terrestrial
sources.1
In contrast to the field of terrestrial drug discovery, however, exploration of
marine natural products began just over 50 years ago with the pioneering work of
Bergman and Feeney and their discovery of the nucleosides spongothymidine and
spongouridine from Tethya crypta. Subsequent development of the synthetic
analogues arabinosyl cytosine (Ara-C) and arabinosyl adenine (Ara-A) has
provided clinically relevant agents for the treatments of cancer (acute myelocytic
leukemia, non-Hodgkin's lymphoma) and viral infections (herpes simplex virus,
acute type B hepatitis),
0
HN
ON
HO-i0I
respectively.59
0
HN(
O
HO-10I
NH2
NH2
Nc-N
LNN
HOicj
N
ON
HO-i0I
tc
OH
Spongouridine (1)
OH
Spongothymidine (2)
OH
Ara-A (3)
OH
Ara-C (4)
An avoidance of the marine environment for scientific exploration likely
developed from the difficulties of underwater collection and isolation of materials.
However, with the development of SCUBA diving as a scientific tool in the 1960's,
4
and more recently submersible vehicles, the search for novel chemistry from the
oceans has rapidly advanced. In fact, the marine natural product database
MarinLit currently has recorded over 14,000 references for some 13,800
compounds of marine derivation. 10-12
The oceans are, however, an extreme and harsh living environment, with
high salt concentrations, high pressure, variable temperatures and low nutrient
availability. As such, marine organisms have developed unique biochemical and
physiological strategies for communication, reproduction, and protection in order to
survive this hostile and competitive world. Because conditions in the ocean are so
markedly distinct, the chemistry produced by its inhabitants is also quite varied.
While terrestrial sources of pharmaceuticals and biochemicals have been
considerably explored, less than one percent of marine species have been
examined for production of novel
chemistry.1
Thus, the oceans represent a rich
and still largely untapped resource for biologically active compounds. In addition,
completely unknown biochemical pathways in pathogens or disease may be
discovered and targeted by such unique chemotypes, leading to the development
of novel therapeutics.
AnticancerAgents from Marine Invertebrates
The main focus for marine natural products research has been the pursuit
of new anticancer agents. Indeed an astonishing number of metabolites deriving
from marine organisms display antimitotic activity, with several candidates
currently in clinical or preclinical trials.
Because of their prevalence and ease of collection, sponges have been the
dominant source of isolated marine natural products. Though they are the simplest
of the multicellular animals, appearing on earth more than 500 million years ago,
sponges produce some of the most intriguing and biologically active chemistry.
Examples of such novel metabolites include the cytotoxins (+)-discodermolide (5)
and halichondrin B (6). Discodermolide was isolated from the sponge Discodermia
dissoluta as a potent immunosuppressive agent and inducer of tubulin
polymerization.13
Discovered by researchers at Harbor Branch Oceanographic
istitiute, discodermolide was licensed to Novartis Pharmaceutical Corporation in
5
1998 for further development. Continued research has focused on the preparation
and evaluation of natural and synthetic analogs that may lead to more efficient
syntheses and a better understanding of structure-activity relationships within this
novel class of
antimitotics.14
OH
OH
NH2
Discodermolide (5)
Discodermia dissoluta
Halichondria okadai
Several sponge genera, including the Japanese sponge Halichondria
okadai, are found to produce cytotoxic polyether macrolides known collectively as
the
halichondrins.15'16
Halichondrin B (6), in particular, has been identified as a
potent inhibitor of microtubule assembly, displaying in vivo activity against a range
of cancer cell lines. Although a scarcity of natural product has hampered efforts to
develop halichondrin B as a new anticancer drug, chemical synthesis has allowed
for the design of structurally simpler analogues that retain the remarkable potency
of the parent compound.17 Additionally, the isolation of halichondrins from a
variety of sponge species also suggests that biosynthesis may actually originate in
an associated microorganism, potentially providing a cultivable resource for
halichondrin production.
Advances in both isolation techniques and structure elucidation
instrumentation have also provided natural product chemists with the ability to
discover novel chemistry present in only miniscule quantities. For example, in the
late 1960's extracts of the bryozoan Bugula neritina were found to inhibit
progression of PS leukemia in mice. However, it was not until the mid-i 980s that
the first of now 18 cytotoxic bryostatins was identified.1819 This family of
macrocyclic lactones has exhibited exceptional activity against various cancers
including non-Hodgkin's lymphoma, melanoma and renal carcinoma, with
bryostatin 1 (7) currently in phase II clinical trials.
Though their exact mechanism of action is unknown, bryostatins exhibit
potent binding to PKC isozymes, effectively outcompeting phorbol esters but not
acting as tumor promoters
themselves.20
Recent results, however, suggest that
the therapeutic effects of bryostatin I may not involve alterations in levels and
distribution of PKC but rather may directly upregulate baxlbcl-2 ratios
independently of p53.21
Bryostatin 1 (7)
Bugula neritina
As the scarcity of bryostatins from natural sources could prevent their
advancement (14 tons of harvested Bugula produce only half an ounce of
7
bryostatin), both synthetic and aquacultural efforts are currently in development to
meet future supply demands for this promising drug
candidate.22'23
Ascidians, commonly referred to as tunicates or sea squirts, have been
another tremendous source of novel anticancer agents.
In the early 1980's,
extracts of the marine tunicate Trididemnum solidum were found to possess
cytotoxic properties and led to discovery of the didemnins, a novel family of cyclic
depsipeptides that exhibit a remarkable array of antitumor, antiviral and
immunosuppressive activities.24 Large-scale collections of T. solidum were
required to provide sufficient quantities of the potent didemnin B for preclinical and
clinical testing, giving rise to the first human clinical trial in the United States of a
marine natural product against
Additional trials of the second-generation
cancer.25
didemnin, aplidine (eg. dehydrodidemnin B, 8), are currently in progress with 8
displaying both greater efficacy and less cardiotoxicity than its parent compound.26
Studies thus far have indicated that didemnins may exert their antitumor effects by
interfering with protein synthesis through a GTP dependent inhibition of the protein
translation elongation factor
1-alpha.27
OHO
o0
00
0
NH
0
0
OCH3
Aplidine (8)
Aplidium albicans
NH
[]
A second family of tunicate-derived antitumor agents is the ecteinascidins
produced by Ecteinascidia turbinata. While the cytotoxic activity of E. turbinata
extracts was first described in the late 1960's, the minute quantity of these
alkaloids present in tunicate tissue prevented their isolation and structure
elucidation for nearly twenty years.28 Ecteinascidin 743 (ET-743, 9) is currently in
phase II clinical evaluation for treatment of various cancers including soft tissue
sarcoma and advanced uterine carcinoma.
Though recognized as a DNA-interacting
drug,29
researchers believe that
the ET-743 molecule binds to the minor groove of DNA with only two of its three
domains making close contact with the double helix. The third domain of ET-743
is positioned outside the complex, exposed to the cellular environment where it is
available for protein interactions. This feature may contribute to ET-743's second
mode of action, an apparent reversal of multidrug resistance. While the underlying
mechanism is yet unknown, ET-743 does compete for binding of the transcriptional
factor NF-Y to its target sequence on the MDR1 gene which encodes for a Pglycoprotein-mediated pump often involved in tumor cell
resistance.3°
Although total synthesis of ET-743 has been achieved, its structural
complexity has prompted the design of less complicated analogues of equal
potency and greater stability (phthalascidin,
1O).31
OCH
H3C
H
Ecteinascidin 743 (9)
Ecteinascidia turbinata
Phthalascidin (10)
F!]
The sea hare Dolabella auricularia, an herbivorous mollusc from the
Indian Ocean, has been the source of more than 20 cytotoxic peptides, collectively
referred to as the
dolastatins.32
The most active of these, dolastatin 10(11), was
reported in 1987 after a 15-year effort to determine the structure of this powerful
antineoplastic
agent.33
Originally isolated in only minute amounts from D.
auricularia (approximately I mg of 11 per 100 kg of mollusc), subsequent
reisolation of 11 from the marine cyanobacterium Symploca sp. has unequivocally
demonstrated the true origin of this bioactive metabolite.
Dolastatin 10 is
currently under assessment in several phase II clinical studies including advanced
renal carcinoma, indolent lymphoma and chronic lymphocytic
0
OCHP
OCH
leukemia.35
y1
Dolastatin 10 (11)
Dolabella aurIcularia
First reported in 1993, the kahalalides are another collection of cytotoxic
peptides deriving from a Hawaiian mollusc, Elysia
rufescens.36
Interestingly,
during collection of this herbivorous mollusc for chemical evalution, E. rufescens
was observed feeding on the green alga Bryopsis sp. Following collection of the
alga and purification of its active constituents, the Biyopsis sp. yielded the identical
chemistry, including kahalalide F, to that isolated from E. rufescens. Currently
undergoing preclinical evaluation, kahalalide F (12) shows selectivity against
various solid tumor cell
lines.37
Toxicity data also show that high concentrations of
kahalalide F are toxic to CNS neurons, suggesting a potential use for the treatment
of selected tumors, such as neuroblastoma. Phase I trials are expected to be
initiated shortly.
10
H2N
H0
ON
C7)N
N
0NH
o
H
HN
NH
0
°NH
H
0
N)hhh'K\
HN-4'
0
H0
/t;s
0
NH
0
Kahalalide F (12)
Elysia rufescens
In 1993, off the northeastern coast of Australia, a new soft coral
Eleutherobia albicans was identified. The venom produced by this organism,
eleutherobin (13), has proven to be one of the most potent cytotoxins discovered
to date, displaying in vitro cancer cell inhibition with an 1050 range of 10-15 nM.38
The related sarcodyctyins (sarcodyctin A, 14) were detected earlier in the
Mediterranean Sea from a different coral species, Sarcodictyon roseum.39
)LN
OCH3
13
Eleutherobin (13)
Eleutherobia albicans
OH
Sarcodictyin A (14)
Sarcodictyon roseum
11
These diterpene glycosides are potent cancer cell inhibitors showing enhanced
potencies against selected breast and renal tumors. In mechanism of action
studies, eleutherobin was found to be a potent microtubule-stabilizing agent that
competes with Taxol® for its mictrotubule binding site.4° Bristol-Myers Squibb,
which also produces Taxol®, has licensed the compound, but natural supplies are
severely limited. Total syntheses of 13 and 14 have been achieved and thus will
provide the necessary material for further biological evaluation.41
Bioactive Metabolites from Marine Microorganisms
Despite the many successes in drug discovery from terrestrial
microorganisms, marine microbes have until recently been largely ignored as a
source of bioactive secondary metabolites. Pioneering research by Dr. William
Fenical and colleagues, however, have revealed the potential of this unique
microbial environment for production of new chemistry. For example, the
macrolactins, a novel family of cytotoxic macrolides, were discovered in 1989 from
a liquid culture of an unidentified Gram-positive bacterium collected from deep-sea
sediment. The major metabolite macrolactin A (15) displays both antineoplastic
and antiviral in vitro activities and has been the focus of several synthetic
efforts.42'43
More recent accomplishments include the structure elucidation of the
novel anti-inflammatory agent cyclomarin A (16) from a marine Streptomyces sp.
C
ON}
'iL
Macrolactin A (15)
Cyclomarin A (16)
This unique cyclic peptide is composed of an array of amino acids, four of
which were previously unobserved in nature. Cyclomarin A powerfully inhibits skin
inflammation, both when applied directly to the skin and when taken orally. This
potentially important compound has been licensed to Phytera Inc. for therapeutic
development.
Marine microorganisms are perhaps best known, however, for their
production of toxic metabolites that adversely effect human health.45 Tetrodotoxin
(17), the notorious neurotoxin once attributed to the pufferfish family
Tetraodontidae, has been shown conclusively to derive from many different marine
bacteria.46 Similarly, saxitoxin (18) and related metabolites, the renowned causes
of paralytic shellfish poisoning, originate from several dinoflagellate species and
are merely concentrated by the filter-feeding shellfish.47 Both tetrodotoxin and
saxitoxin are powerful and specific sodium channel blockers that have become
important commercially available biomedical tools for the study of ion channels.
H2N_O
OH
OH
H H
I
HO
OH
HO
N
H
Tetrodotoxin (17)
>NH
HN N1OH
OH
Saxitoxin (18)
The characteristic and by far most impressive compounds produced by
dinoflagellates are the polycyclic ethers. One of the earliest to be studied was
okadaic acid (19), a tumor-promoting agent first isolated from the marine sponge
Halichondria okadai but later demonstrated to be biosynthesized by dinoflagellate
species of the Dinophysis and Prorocentrum genera.48'49 While little was known
regarding protein phosphatases before the discovery of this significant molecule,
okadaic acid has since played a central role in our of understanding of protein
phosphorylation and signal transduction pathways of eukaryotic cells.5°
13
The brevetoxins (brevetoxin B, 20) are a family of neurotoxic shellfish
poisons produced by various red tide
dinoflagellates.51
Of special interest and
concern for the extensive damage their toxic blooms cause to marine ecosystems,
the brevetoxins have been shown to incite toxicity through selective sodium
channel activation that depolarizes excitable membranes.52 Their binding site on
sodium channels appears unlike those of other known activators, making the
brevetoxins extremely important tools in biomedical research.
Cultures of the marine dinoflagellate Gambierdiscus toxicus have also
yielded a series of novel polyether antifungals, the most potent of which, gambieric
acid A (21), is more than 2000 times more active than the clinically relevant
amphotericin B, but only moderately cytotoxic.53 While a total synthesis of
gambieric acid A has not yet been completed, the partial syntheses of several of
its complex ring systems have been achieved.
Okadaic acid (19)
HO2C
OH
HJ'
Brevetoxin B (20)
Ptychodiscus breve
Hd
j
H
H
U;
-
-I
H
OH
Gambieric acid (21)
Gambierdiscus toxicus
14
However, cyanobacteria, the most ancient of the microalgae, have been
the richest producers of novel and bioactive secondary metabolites. Several
potential anticancer agents have been identified, including the promising
antimitotic dolastatin 10 (11) presented above.
The bioactive peptide cryptophycin A (22) was originally isolated from a
cyanobacterium Nostoc sp. for its antifungal activity,55 but is now well known for its
potential application in the treatment of cancer. Interestingly, the cytotoxin
arenastatin A (23)56 reported from the Okinawan sponge Dysidea arenaria was
found to be structurally identical to cryptophycin 24. Several synthetic analogues
have been prepared, including cryptophycin 52 (24) currently in phase II trials
against a variety of solid tumors. The cryptophycins display anticancer activity by
their ability to destabilize microtubules through binding at or near the ymca-alkaloid
binding site and inducing phosphorylation of bcl-2, leading to apoptosis.
0
ço
Cryptophycin A (22)
Arenastatin A (23)
Cryptophycin 52 (24)
R1 = Cl
R1
H
R1 = Cl
R2 = CH3 R3 = H
R2 = H
R3 = H
R2 = CH3 R3 = CH3
Curacin A (25) is a novel thiazole-containing lipid isolated from the marine
cyanobacterium Lyngbya majuscula (see below). Discovered originally through its
toxicity to brine shrimp, curacin A and its structural analogues have shown in
preclinical studies a potent microtubule polymerization inhibition through
interaction at the colchicine binding site.58 Recent efforts have mainly focused on
development of synthetic curacins with greater stability and water solubility.59
15
Because of its simplicity, therapeutic versions of an active curacin agent would be
relatively inexpensive to produce compared to many of the antimitotic natural
products described above.
CH3
H
OCH3
CuracinA (25)
N
,CH3
H''/'H
Unique Chemistry of Lyngbya majuscula
Lyngbya majuscula is a filamentous, often toxic cyanobacterium found in
tropical and sub-tropical marine environments worldwide. Most infamous for its
mass outbreaks, L. majuscula can have major impacts on the health of an affected
ecosystem and humans alike. In bloom conditions, Lyngbya forms dense mats that
cover the sea floor, smothering the underlying seagrass meadows that are critical
habitats and food for many different animals. Through the accumulation of gas
bubbles these mats can rise to the surface forming large floating masses that
wash onto beaches and contaminate recreational waters, causing swelling and
irritation to swimmers unfortunate enough to make their acquaintance. To the
fortune of natural product scientists, however, the chemistry behind these toxic
conditions is both structurally intriguing and medicinally significant.
Two highly inflammatory but structurally distinct metabolites,
debromoaplysiatoxin (26)60 and lyngbyatoxin A (27),61 were isolated from a
Hawaiian strain of L. majuscula as the agents responsible for causing severe
contact dermatitis. Both classes of compounds were found to bind the phorbol
ester receptor on protein kinase C (PKC) and have played an important role in the
study of PKC signal transduction. This insight may lead to the design of
analogues useful as either tumor-suppressing agents or probes for additional
mechanistic studies of PKC.62
16
OH
OH
Debromoaplysiatoxin (26)
A major metabolic theme in
Lyngbyatoxin A (27)
L.
majuscula chemistry is the combination of
polyketide synthase and non-ribosomal peptide synthetase biosynthetic pathways
to produce a seemingly limitless number of possible secondary metabolites. One
significant structural example is antillatoxin (28), a novel lipopeptide first reported
for its potent ichthyotoxic
activity63
and later shown to depolarize membranes
through activation of voltage sensitive sodium channels.
Antillatoxin appears to
activate sodium channels through a unique binding site, adding to the array of
probes available for sodium channel investigations.
nr0LLJ
rtiL
Kalkitoxin (29)
Antillatoxin (28)
Similarly, the extract of a
L.
majuscula collection from Playa Kalki,
Curacao, displayed both ichthyotoxicity and brine shrimp toxicity. Bioassay-guided
fractionation led to the discovery of kalkitoxin (29), as the active constituent.
Subsequent screening against mouse neuro-2a neuroblastoma cells has
17
established kalkitoxin as a powerful inhibitor of sodium channels, with an EC50
eight times more potent than the model sodium channel blocker saxitoxin (18).65
Both linear and cyclic peptides are found in the chemistry of L. majuscula.
These structures most often contain both ketide portions as well as standard and
atypical amino acid residues.66 The antifungal and cytotoxic lyngbyabellins (30,
31), homologues of the molluscan metabolite dolabellin, are primary examples of
the peptides isolated from this organism.67 A C8 ketide chain with an intriguing
gem-dichloro functionality is presumed to initiate biosynthesis, with further
extensions from various amino acids including the cysteine-derived thiazole and
thiazoline residues. Unlike the curacins and dolastatin 10, the cytotoxicity of 30
and 31 derives from specific cytoskeletal disruption of microfilament networks
without effecting microtubles.
0
o
/ s
Cl
0HNo
0
cJ
N
Lyngbyabellin A (30)
0HN..o
N
Lyngbyabellin B (31)
An alternation of PKS and NRPS modules is presumed to be involved in formation
of the acyclic microcolins (32, 33), bioactive peptides reported in 1992 from a
Venezuelan L. majuscula collection.68 In vitro results suggest that the
antiproliferative and immunosuppressive properties of both compounds are
possibly associated with apoptosis-inducing events, implying an additional
potential for their development as antineoplastic agents.69
iE]
-
°OAc
0
0
0
Microcolin A (32) R = OH
Microcolin B (33) R = H
With over 150 compounds previously reported in the literature, it may seem
unlikely that new metabolites could yet be discovered from this organism. But in
fact, as the following chapters will demonstrate, Lyngbya majuscula continues to
astound natural product chemists with its production of novel and bioactive
chemistry. Thus, a thorough investigation of several L. majuscula collections was
undertaken as the focus of this Ph.D. research.
19
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3649-3651. (b) Kadota, I.; Oguro, N.; Yamamoto, Y. Tetrahedron Lett.
2001, 42, 3645-3647.
55.
(a) Smith, C. D.; Zhang, X.; Mooberry, S. L.; Patterson, G. M.; Moore, R. E.
Cancer Res. 1994, 54, 3779-3784. (b) Shih, C.; Teicher, B. A. Curr. Pharm.
Des. 2001, 7, 1259-1 276.
56.
Kobayashi, M.; Aoki, S.; Ohyabu, N.; Kurosu, M.; Wang, W.; Kitagawa, I.
Tetrahedron Lett. 1994, 35, 7969-7972.
57.
(a) Panda, D.; DeLuca, K.; Williams, D.; Jordan, M. A.; Wilson, L. Proc.
Nat!. Acad. Sd. U.S.A. 1998, 95, 931 3-9318. (b) Barbier, P.; Gregoire, C.;
Devred, F.; Sarrazin, M.; Peyrot, V. Biochemistry 2001, 40, 13510-13519.
58.
(a) Gerwick, W. H.; Proteau, P. J.; Nagle, D. G.; Hamel, E.; Blokhin, A.;
Slate, D. L. J. Org. Chem. 1994, 59, 1243-1 245. (b) Verdier-Pinard, P.;
Sitachitta, N.; Rossi, J. V.; Sackett, D. L.; Gerwick, W. H.; Hamel, E. Arch.
Biochem. Biophys. 1999, 370, 51-58.
59.
Wipf, P.; Reeves, J. T.; Balachandran, R.; Day, B. W. J. Med. Chem. 2002,
45, 1901-1917.
60.
Mynderse, J. S.; Moore, R. E.; Kashiwagi, M.; Norton, T. R. Science 1977,
196, 538-540.
61.
(a) Cardellina, J. H., II; Marner, F. J.; Moore, R. E. Science 1979, 204, 193195. (b) Fujiki, H.; Mori, M.; Nakayasu, M.; Terada, M.; Sugimura, T.;
Moore, R. E. Proc. Nat!. Acad. Sci. U.S.A. 1981, 78, 3872-3876. (c) Aimi,
N.; Odaka, H.; Sakai, S.; Fujiki, H.; Suganuma, M.; Moore, R. E.;
Patterson, G. M. L. J. Nat. Prod. 1990, 53, 1593-1596.
24
62.
(a) O'Brian, C. A.; Arcoleo, J. P.; Housey, G. M.; Weinstein, I. B. Cancer
Cells 1985, 3, 359-363. (b) Moore, R. E.; Patterson, G. M. L.; Entzeroth,
M.; Morimoto, H.; Suganuma, M.; Hakii, H.; Fujiki, H.; Sugimura, T.
Carcinogenesis 1986, 7, 641-644. (c) Molecular Probes.
http://www.probes.com/ (accessed June 2002).
63.
Orjala, J.; Nagle, D. G.; Hsu, V. L.; Gerwick, W. H. J. Am. Chem. Soc.
1995, 117, 8281-8282.
64.
(a) Berman, F. W.; Gerwick, W. H.; Murray, T. F. Toxicon 1999, 37, 16451648. (b) Li, W. I.; Berman, F. W.; Okino, T.; Yokokawa, F.; Shioiri, T.;
Gerwick, W. H.; Murray, T. F. Proc. Nat!. Acad. Sd. U.S.A. 2001, 98, 75997604.
65.
Wu, M.; Okino, T.; Nogle, L. M.; Marquez, B. L.; Williamson, R. T.;
Sitachitta, N.; Berman, F. W.; Murray, T. F.; McGough, K.; Jacobs, R.;
Colsen, K.; Asano, T; Yokokawa, F.; Shioiri, T.; Gerwick, W. H. J. Am.
Chem. Soc. 2000, 122, 12041-12042.
66.
Gerwick, W. H.; Tan, L. T.; Sitachitta, N. In The Alkaloids; Cordell, G. A.,
Ed.; Academic Press: San Diego, 2001; Vol. 57, pp 75-184.
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(a) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.; Mooberry, S. L. J.
Nat. Prod. 2000, 63, 611-615. (b) Milligan, K. E.; Marquez, B. L.;
Williamson, R. T.; Gerwick, W. H. J. Nat. Prod. 2000, 63, 1440-1443. (c)
Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J. J. Nat. Prod. 2000,
63, 1437-1439.
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Koehn, F, E.; Longley, R. E.; Reed, J. K. J. Nat. Prod. 1992, 55, 613-61 9.
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(a) Zhang, L.-H.; Longley, R. E. Life Sci. 1999, 64, 1013-1 028. (b) Zhang,
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25
CHAPTER TWO
NOVEL BIOACTIVE LIPOPEPTIDES FROM A FIJIAN MARINE
CYANOBACTERIAL MIXED ASSEMBLAGE
ABSTRACT
The organic extract from a 1997 collection of Lyngbya majuscula and
Schizothrix sp. marine cyanobacteria was found to possess activity in both brine
shrimp and neuro-2a cytotoxicity assays. Bioassay-guided fractionation of this
material led to the discovery of two novel lipopeptides, the depsipeptide somamide
A (12) and the cytotoxic dimer somocystinamide A (15). Somamide A (12)
consists of a hexanoic acid polyketide starter unit extended by seven amino acid
residues, including the unusual moieties, 3-amino-6-hydroxy-2-piperidone (Ahp)
and 2-amino-2-butenoic acid (Abu). The ester linkage in 12 arises from cyclization
of a threonine J3-hydroxyl with the carboxyl of a valine residue. Notably, this
molecule possesses strong structural homology to dolastatin 13 (13), a potent
cytostatic metabolite first reported from the marine mollusc Do/abel/a auricularia.
In stark contrast to the structure of somamide A (12), the unique dimer
somocystinamide A (15) is proposed to derive from two L-cysteine residues linked
as a disulfide. Each cysteine moiety is ketide extended with five malonyl-CoA
derived acetate units, followed by linkage of an N-methylglycine residue and then
further extension by two additional acetate units. Decarboxylation to form the
terminal olefin of 15 would complete the biosynthesis of this novel cytotoxin.
Interestingly, somocystinamide A (15) proved quite unstable under mildly acidic
conditions, rapidly and thoroughly decomposing to a characterizable derivative
(16). While somocystinamide A (15) displayspotent cytotoxicity against mouse
neuro-2a neuroblastoma cells
(IC50
1.4
gImL), 16 has no activity.
26
INTRODUCTION
Marine microalgae have provided a vast array of structurally interesting
metabolites, often possessing pharmaceutical and biomedical utility. For example,
compounds such as the curacins (1,2), microcolins (3,4) and brevetoxins (5,6) are
of considerable interest for their antiproliferative, immunosuppressive and sodium
channel-activating properties, respectively (Figure 11.1). 1-3
H N(y
Thc7Ns
OCH3
Curacin A (1) R CH3
Curacin D (2) R = H
H'7'H
'O'OO
Microcolin A (3) R = OH
Microcolin B (4) R = H
Brevetoxin B (6)
Figure 11.1. Bioactive metabolites from marine microalgae.
27
In particular, Lyngbya majuscula has been extensively studied in our
laboratory for its intriguing chemistry and has been shown to produce over 150
different compounds in diverse structural classes. A major theme in L. majuscula
chemistry is the production of metabolites deriving from a combination of
polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS)
biosynthetic pathways, often referred to as
lipopeptides.4
Examples of such mixed
biosynthetic products isolated from our laboratory include the molluscicide
barbamide (7) and the potent neurotoxins, antillatoxin (9) and kalkitoxin (8).
OCH3
iCrccl3
Barbamide (7)
Kalkitoxin (8)
0
Antillatoxin (9)
Similarly, the dolastatins are an intriguing group of polypeptides, frequently
containing ketide extended residues, which possess exceptional cytostatic and
antineoplastic
activity.8
Although originally isolated in minute quantities from the
herbivorous mollusc Dolabella auricularia, it has become evident that such
metabolites truly originate in
cyanobacteria.9
For example, dolastatin 10 (10),
currently in phase II clinical trials for the treatment of human cancers, was
originally reported from D. auricularia in 1987.10 Subsequently, the closely related
metabolite symplostatin 1(11) was discovered from the marine cyanobacterium
28
Symploca hydnoides and, in 2001, dolastatin 10 (10) was isolated from a similar
Sympioca
species.1112
OCHp
OCHD
Dolastatin 10 (10) R = H
Symplostatin 1 (11) R = CH3
The utilization of unusual ketide moieties such as the proposed 2,2-
dimethyl-propionic acid of antillatoxin (9) and the trichloroisovaleric acid of
barbamide (7), and the incorporation of non-standard and modified amino acids
like the dolaphenine (terminal thiazole-phenethylamine) residue of 7, 10 and 11,
allow for a seemingly limitless number of new structures to be produced from
these microalgal sources. Thus cyanobacteria represent a continual resource of
novel chemistry for medicinal and biochemical applications.
A phytochemical and bioassay-guided examination of the lipid extract from
a mixed assemblage of the marine cyanobacteria Lyngbya majuscula and
Schizothrix species, collected in the Fijian Islands, led to the discovery of two such
novel lipopeptides (12, 15), one possessing strong structural homology to the
molluscan cytotoxin dolastatin 13 (13), and the other, a structurally unprecedented
disulfide dimer with potent cytotoxic
properties.13
This chapter describes the
structural characterization and biological activities of these novel cyanobacterial
metabolites.
29
RESULTS AND DISCUSSION
In our continued search for pharmaceutically relevant agents from marine
cyanobacteria, brine shrimp and mouse neuroblastoma cell toxicity assays were
utilized to evaluate our cyanobacterial extract library.1415 A crude organic extract
from a Somo Somo, Fiji collection of L. majuscula and Schizothrix sp. proved quite
active in both assays, and thus was fractionated by silica gel vacuum liquid
chromatography (VLC) to produce a series of chemically distinct fractions. Polar
fractions eluting between 90% ethyl acetate in hexanes and 5% methanol in ethyl
acetate displayed significant cytotoxicity and thus were subjected to further
fractionation by C18 solid phase extraction (SPE) and reversed-phase HPLC to
afford two novel metabolites, somamide A (12) and somocystinamide A (15)
(Figure 11.2).
Lyngbya majuscula/Schizothrix sp.
Somo Somo, Fiji
(VSO 8/Feb 97/02)
1.0 g organic extract
VLC, Normal phase Si
Hexanes:EtOAc:MeOH
I
I
3:1
100%
9:1
EtOAc/Hex EtOAc/Hex
EtOAc
19:1
3:1
EtOAc/MeOH EtOAc/MeOH
Mega-Bond
C18 Sep-Pak
I
3:2
I
7:3
4:1
I
9:1
MeOH/H20 MeOH/H20 MeOH/H2O MeOH/H20
RP HPLC
3:1 MeOH/H20
somamide A (12)
3.1 mg
100% MeOH
RP HPLC
19:1 MeOH/H20
somocystinamide A (15)
20.8 mg
Figure 11.2. Isolation and purification of compounds 12 and 15.
'Is
Somamide A (12) was isolated as a clear oil with a molecular composition
of C48H67N7012S as determined by HRFABMS (m/z 948.4587 for [M + H - H2O]).
The lR spectrum of 12 displayed strong absorption bands at 1730 and 1641 cm1,
indicating the presence of ester and amide functionalities, and 1H and 13C NMR
spectra were indicative of a depsipeptide (Figure 11.3, Table 11.1).
Somamide A (12)
MeO
0 HN
Ne
Nc
O
N
H
OHN
H
HO
Dolastatin 13 (13)
OH
/
0
HN
'N
O
N
OHN
L7J°
HO
Symplostatin 2 (14)
Os
OH
31
Table 11.1. NMR
unit
position
Val
1
2
3
4
5
spectral data for somamide A (12) in DMSO-d6.
13C
172.5
55.5
30.5
16.9
18.9
NH-i
N-MeTyr
6
7
8
9
10/14
11/13
Phe
Ahp
Abu
12
15
16
17
18
19
20/24
21/23
22
25
26
27
28
29
NH-2
OH
30
31
Thr
MetSO
32
33
NH-3
34
35
36
37
NH-4
38
39
40
41
Hex
42
NH-5
43
44
45
46
47
48
169.2
60.4
32.5
127.0
130.1
114.9
155.8
30.0
170.0
49.8
34.9
136.3
129.0
127.4
125.8
168.9
47.8
21.5
28.9
73.2
1H (J in Hz)
HSQCTOCSYa
HMBCb
4.74, d (11.3)
3, NH-i
6
2.09,m
2,4,5
1,4,5
0.75, d (6.9)
0.88, dd (6.9, 2.3)
7.47, brs
3, 5
3, 4
2, 3, 5
2, 3, 4
4.91,d(ii.7)
8
7
6,8
3.08, 2.70
6.99, d (8.4)
6.76, d (8.3)
11/13
10/14
8, 11/13, 12
9, 12
2.76, s
7, 9, 10/14
7
4.74, d (11.3)
2.78, 1.81
18
17
16, 18, 19,25,29
6.84, d (7.3)
21/23
20/24, 22
21/23
18, 21/23, 22
19, 20/24
20/24
27, 28, NH-2
26,28, NH-2
27, 29
28, OH
25
7.14,m
7.18, m
3.79, m
2.41,1.57
1.70, 1.57
5.07, brs
7.19
6.08, brs
17, 19, 20/24
162.1
129.9
131.7
12.8
30,31,33
6.53, q (7.1)
1.50, d (7.1)
9.21, brs
33
32
4.53, m
5.52, brs
1.25, d (6.6)
7.89
36, NH-4
36
35, 36
51.7,50.0c
4.62,m
40,41,NH-5
38
24.4, 24.1c
49.3, 48.9c
37.7
1.95, 1.90, m
2.79, 2.70, m
41
39,41
39 40
39
173.4
55.5
71.3
17.5
30, 31, 32
35,37
172.1
2.53,2.51,cs
41
8.14, brs
174.4
34.6
24.5
30.5
21.5
13.5
2.14, m
1.50
1.28
1.28
0.86, t (7.0)
45, 46/47, 48
44, 46/47, 48
45, 48
48
46/47
43, 45, 46
43, 44, 46, 47
47
Proton showing TOCSY correlation to indicated proton. " Proton showing HMBC
c
correlation to indicated carbon. Indicates the presence of multiple conformers.
a
32
9
170
160
7
8
150
140
130
6
120
110
4
5
100
90
80
70
3
60
2
50
40
1
30
20
ppm
ppm
Figure 11.3 'H and 13C NMR spectra of somamide A (12).
Analysis of 2D NMR spectra including HSQC, HSQC-TOCSY, ROESY and
HMBC (Figures 11.4-11.7) allowed for the construction of eight partial structures,
accounting for all of the atoms in 12. Four standard amino acid residues were
deduced as phenylalanine (Phe), N-methyl-tyrosine (N-MeTyr), threonine (Thr)
and valine (Val). The presence of the more unusual moieties, 2-amino-2-butenoic
acid (Abu) and 3-amino-6-hydroxy-2-piperidone (Ahp), were also deduced by 2D
NMR and suggested the close relationship of 12 to the previously isolated
cyanobacterial metabolite symplostatin 2 (14) as well as the molluscan
depsipeptide dolastatin 13
(13).1316
The two remaining partial structures were
deduced as hexanoic acid (Hex) and a methionine sulfoxide (MetSO), observed
as a 1:1 mixture of R and S sulfoxide diastereomers (Table 11.1). It is possible that
this diastereomeric mixture formed during the isolation process from a naturally
occurring methionine residue.
33
ppm
20
30
40
50
60
70
80
90
100
110
120
130
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
Figure 11.4. HSQC spectrum of somamide A (12).
2.5
2.0
1.5
1.0
ppm
34
ppm
20
30
40
50
60
70
80
90
100
110
120
130
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
Figure 11.5. HSQC-TOCSY spectrum of somamide A (12).
2.0
1.5
1.0
ppm
35
ppm
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
Figure 11.6. ROESY spectrum of somamide A (12).
3.5
3.0
2.5
2.0
1.5
1.0
ppm
60
yb
ppm
99ctflcco
37
Correlations observed in the HMBC spectrum of 12 allowed for the
formation of the partial sequences Val-N-MeTyr-Phe-Ahp and Thr-MetSO--Hex
(Figure 11.8). In addition, a ROESY correlation observed between the NH-3 of Abu
(6 9.21) and H35 (64.53) linked this moiety to the Thr residue (Figure 11.8).
0
9.21
4.53
0ROESY correlation
(\ HMBC correlation
Abu-Thr
/ N-.--/SO
Ahp-Phe-N-Melyr-Val
OH
Thr-MetSO-Hex
Figure 11.8. Partial sequencing of 12 by HMBC and ROESY correlations.
However, FABMS fragmentation was instrumental in completion of the
planar structure of somamide A (Figures 11.9-11.10). A peak at m/z 503,
corresponding to the fragment N-MeTyr-Phe-Ahp-Abu, and a peak at m/z 295 for
the fragment Thr-Abu-Ahp, were both observed and thus placed the Abu unit
between Thr and Ahp. Fragment ions at m/z 420 and 348, corresponding to N-
MeTyr-Phe-Ahp and Thr-MetSO-Hex, respectively, were also detected, and
further supported the HMBC sequence assignments (Figure 11.8). The final degree
of unsaturation of 12 required a connection between the Val carbonyl (Cl) and the
Thr hydroxyl (C36-OH), thereby completing the planar structure of somamide A.
38
Figure 11.9. Low resolution positive FABMS of somamide A (12).
m/z 503
nilz 348
nz 420 4
m/z 246
Iy
HO
m/z295
Figure 11.10. Amino acid sequencing of 12 by mass spectral fragmentation
analysis.
39
The absolute stereochemistries of the residues in somamide A (12) were
determined by Marfey's analysis, and indicated that the Val, N-MeTyr, Phe, Thr,
and MetSO were all of L
configuration.17
The absolute configuration of C26 (Ahp)
could not be determined by Marfey's analysis despite several attempts to isolate
the corresponding glutamate residue following oxidation and acid hydrolysis of 12.
However, proton and carbon chemical shifts for the Ahp unit of 12 were nearly
identical to those observed in symplostatin 2 (14), suggesting it was of the same
stereochemistry. In addition, ROESY correlations observed throughout the Ahp
ring and between H17 (Phe) and H29 (Ahp) supported a 26S, 29R configuration,
analogous to the stereochemical assignments in symplostatin 2 (14).16
The NH of the Abu residue did not show a ROESY correlation to either the
C32 methine or the C33 methyl protons, preventing determination of the double
bond geometry by homonuclear dipolar coupling. However, this was circumvented
by application of a 1 D HSQMBC experiment from which a coupling constant of 3.5
Hz between the methine proton H32 (66.53) and C30 (6 162.1) was measured,
thus establishing the geometry as
Z.18
OH
Hi37
i')
ON-ö
0
H
341
HN
0
Somamide A (12)
Somamide A (12) demonstrates the extensive biosynthetic capabilities of
marine cyanobacteria to produce secondary metabolites of novel structure, in
particular, products of NRPS/PKS
origins.4
While 12 proved inactive in the
40
cytotoxicity assays, the related metabolite dolastatin 13 (13) strongly inhibited
growth of P388 lymphocytic leukemia cells, exhibiting an EC50 of 0.013
jtg/mL.13
This metabolite as well as other members of the dolastatin family have previously
been reported from marine invertebrates, including the sea hare D. auricularia,
well known for its ability to sequester compounds acquired from its cyanobacterial
diet.9
Thus, isolation of this depsipeptide provides further evidence that such
metabolites are truly of cyanobacterial origin.
41
The cytotoxic activity associated with this extract, however, was attributed
to the less polar metabolite, somocystinamide A (15), isolated as a white
amorphous solid in good yield (20.8 mg, 2.1% of the crude extract). While
dissolving readily in CH2Cl2 and CHCI3, compound 15 was not appreciably soluble
in other solvents, and thus, all NMR experiments were performed in CDCI3.
1H
and 13C NMR spectra initially indicated that 15 was a small molecule having
approximately 21 carbon atoms and existing in two conformations, as shown by an
approximate 7:3 ratio of several 1H and 13C signals (Figure 11.11).
Figure liii. 1H and 13C NMR spectra of somocystinamide A (15).
42
HN
i.
HN1-
19
0
Somocystinamide A (15)
HN1
11
HN
HNj2-
14
Decomposition Product (16)
0
A 1H-1H COSY spectrum of 15 (Figure 11.12) was obtained to begin
developing spin system. However, 2D heteronuclear data acquisition was delayed
and the sample remained in CDCI3 for several days. Unexpectedly, marked
differences were displayed in the 1H NMR spectrum run at this later time, including
the appearance of an aliphatic aldehyde signal (8 9.78, t, J= 1.2 Hz) and several
alterations in the high-field region of the spectrum (Figure 11.13). 2D NMR data
including HSQC, HSQC-COSY and HMBC were acquired but proved difficult to
interpret due to an absence of key connectivities. Therefore, the sample was
again prepared for further NMR experiments.
43
TI
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
Figure 11.12. COSY spectrum of somocystinamide A (15).
2.0
1.5
1.0
ppm
44
9
200
190
8
180
170 160 150
7
140
6
130
120
5
110 100
3
4
90
80
70
60
50
2
ppm
40
ppm
Figure 11.13. 1H and 13C NMR spectra of intermediate product mixture.
Interestingly, the sample was no longer soluble in CDCI3 or CD3OD but
dissolved only in an approximate 1:1 mixture of these two solvents. A 1H NMR
spectrum acquired under these new conditions again produced surprising results
(Figure 11.14). Not only had the new aldehyde and high-field signals disappeared,
but several signals present in the initial 1H NMR spectrum of 15 were additionally
absent (Figure 11.15). 1H and 13C NMR data suggested this compound (16) was a
structurally simplified derivative of the original natural product, and therefore, 2D
NMR and mass spectral data were obtained (Table 11.2, Figures 11.16-11.19).
45
7.5
170
7.0
160
6.5
150
140
6.0
130
5.5
120
5.0
110
4.5
100
4.0
90
3.5
80
70
3.0
60
2.5
50
Figure 11.14. 1H and 13C NMR spectra of degradation product (16).
2.0
40
1.5 ppm
30
ppm
46
Somocystinamide A (15)1
Intermediate Mixture
Degradation Product (16)
9
8
7
6
5
4
ppm
2
3
Figure 11.15. 1H NMR spectral comparison of the somocystinamide decomposition
process.
HRFABMS of 16 produced an [M + H] molecular ion at m/z 627.3976 for
the formula C32H58N404S2, indicating that 16 was actually a symmetrical dimer. 1 D
NMR spectra showed that 16 possessed a
trans
double bond and two amide
carbonyl carbons. Thus, as a dimer, all six degrees of unsaturation inherent to the
molecular formula were assigned, and 16 was shown to be acyclic.
HSQC-COSY and HMBC were used to connect the low-field methine signal
CH-2/2' to an acetylated amine moiety (C-i 5/1 5', CH3-i 6/1 6'), the
trans
double
bond (CH-3/3', CH-4/4') and a mid-field methylene signal (CH2-i/1'). This
methylene showed no further correlations, and its proton and carbon shifts were
47
consistent with the presence of a sulfur substituent. 2D NMR data were then
utilized to extend outward from the double bond to a methylene (CH2-5/5') followed
by a (CH2)5 envelope and a high-field methylene. This latter methylene (CH2-
11/11') was connected to yet another mid-field CH2 unit (CH2-1 2/1 2') that, by
HMBC, terminated with the carbonyl of an N-methylated amide (Table 11.2).
Dimerization of this linear chain through a disulfide functionality accounted for all
atoms in 16, thus completing its planar structure.
Table 11.2. NMR spectral data for compound 16 in 1:1 CDCI3/CD3OD.
position
13C
1H (J in Hz)
HSQC-COSY°
HMBC b
1/1'
43.8
2.88, m
4.62
50.5, 127.6
2/2'
50.5
4.62, ddd (6.6, 6.6, 6.6)
2.88, 5.45
43.8, 127.6, 133.0, 170.9
3/3'
127.6
5.45, dd (15.4, 6.6)
4.62, 5.65
31.9, 43.8, 50.5
4/4'
133.0
5.65, dt(15.4, 6.6)
2.04, 5.45
31.9, 50.5
5/5'
31.9
2.04, m
1.35-1.28, 5.65
127.6, 133.0
6/6'-9/9'
28.7
1.35-1 .28c
10/10'
28.9
1.351.28c
1.60
11/11'
25.6
1.60, m
1.33, 2.20
28.7, 35.9, 175.3
12/12
35.9
2.21-2.17, m
1.60
25.6, 28.9, 175.3
13/13'
175.3
14/14'
25.4
15/15'
170.9
16/16'
22.0
2.74, s
35.9 (weak), 175.3
1.99, s
50.5 (weak), 170.9
Proton showing COSY correlation to indicated proton.
C
to indicated carbon. Obscured
b
Proton showing HMBC correlation
4B
30
ppm
gufe
t6 4SQG 9peCtV
o degradatton
49
ppm
30
40
50
60
70
80
90
100
110
120
130
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
Figure 11.17. HSQC-TOCSY spectrum of degradation product (16).
1.5
ppm
50
70
80
ppm
BG 9pec
9ro0
51
50d.t..pt.fs4Ak0915O1.m.2
PS. Nan,.
P0.751.
14.1.,
Nag.
S
LIsa Nogi., 1.504 113432-C
1.3-aba
Seas 3-7. 0,0.14. 100% InLnl.47001. FAa. P05.
50
50
70
60
50
3131
40
267.1
50
1
430
246.0
I
256.1
175.0
000
100
520.2
60
I0
527.2
70
50
50
40
50
..
1- .l.
.1
--
445
540
i-..
560
s..1-i1.J-.rrt
040
. . I
fl
ISO
Figure 11.19. Low resolution positive FABMS of degradation product (16).
To our fortune, additional somocystinamide A (15) was detected in side
fractions associated with its original chromatographic isolation. Realizing the
apparent instability of the compound, mass spectra and NMR data in acid
neutralized CDCI3 were quickly and carefully collected, and the structure of this
unique metabolite was finally resolved (Figures 11.20-11.22).
A HR FABMS [M + H] molecular ion at m/z 759.4295 identified the
molecular composition of 15 as C42H70N404S2, indicating that 15 was also a
symmetric, acyclic dimer. I D NMR spectra showed that each symmetric unit of 15
possessed a terminal olefin, two trans-disubstituted double bonds and two amide
carbonyls, accounting for all ten degrees of unsaturation. 2D NMR data confirmed
52
that the intact structure of 16 was conserved within 15 and assembled the
remaining
C10H10
component missing from 16 as two five-carbon 1 ,4-diene
moieties. These units were connected to the terminal amide nitrogens by the key
HMBC correlation from H-14/14' ( 6.67) to C-I 3/13' ( 171.7, see Table 11.3), thus
completing the planar structure of somocystinamide A (15). The mixture of
conformers of 15 is presumed to arise from cis/trans isomerization of these tertiary
amides.
Table 11.3. NMR spectral data for somocystinamide A (15) in CDCI3.
position
13C
1/1'
44.7
8
b
1H (J in Hz)
HSQC-COSY
3.08, dd (13.4, 6.2)
4.67
50.8, 127.6
HMBC
2.83c
2/2'
50.8
4.67, m
2.83, 3.08, 5.43, 6.34
44.7, 127.6, 133.7, 169.7
3/3'
127.6
5.43, dd (15.5, 6.5)
5.66
32.2, 44.7, 50.8
4/4'
133.7
5.66, dt (15.5, 6.6)
2.02, 5.43
29.3, 32.2, 50.8
5/5'
32.2
2.02, q (6.7)
1.33, 5.66
29.3, 127.6, 133.7
6/6'
29.3
1.33c
2.02
7/7'-9/9'
29.2
1 33C
10/10'
28.9
1.33"
1.64
11/11'
25.0
1.64, m
1.33,2.43
28.9, 171.7
12/12'
33.8
2.43, m
1.64
25.0, 28.9, 171.7
6.67, d (13.9)
4.99
29.7, 34.7, 109.0, 171.7
2.83, 6.67
34.7, 129.2, 137.0
13/13'
14/14'
1717/1714d
129.2
736d d (14.6)
109.0
4.99, m
1084d
497d
16/16'
34.7
2.83"
4.99
109.0, 115.4, 129.2, 137.0
17/17'
137.0/137.4"
5.84, m
5.06
34.7
115.4/115.0"
5.06, m
5.84
137.0
15/15'
18/18'
19/19'
2971321d
NH
20/20'
169.7
21/21'
23.4
a
3.07, s13.O8", S
129.2, 171.7
6.34
50.8, 169.7
2.01, s
169.7
Proton showing COSY correlation to indicated proton.
C
to indicated carbon. Obscured dMinor conformer.
U
Proton showing HMBC correlation
The absolute stereochemistry of 15 was determined by ozonolysis, acid
hydrolysis and oxidation to release the corresponding cysteic acid residues. An
HPLC comparison and coinjection of this product with standard D- and L-cysteic
53
acid revealed only L-cysteic acid; hence somocystinamide A (15) is of 2R, 2'R
absolute configuration.
It appears that trace quantities of HCI and H20 present in the original NMR
solvent initiated the cleavage of 15 leading to the formation of 16 and the release
of the C14-C18 and C14'-C18' units as aldehyde 17. Although this fragment was
not isolated and was likely evaporated during the second preparation of the NMR
sample, the presence of 17 is strongly supported by the 2D NMR data obtained on
the crude decomposition mixture.
Somocystinamide A (15) exhibits potent cytotoxicity to mouse neuro-2a
neuroblastoma cells with an
IC50
of 1.4 p.gImL. While derivative 16 displayed no
cytotoxicity, its insoluble nature may contribute to this apparent inactivity.
Somocystinamide A (15) represents a new and unique cyanobacterial metabolite
class of likely mixed PKS/NRPS biosynthetic origin. We envisage L-cysteine is
ketide extended with five malonyl CoA-derived acetate units, followed by linkage of
an N-methyl glycine moiety and then further extension by two additional acetates.
Decarboxylation to produce the terminal olefin and dimerization completes the
proposed biosynthesis of somocystinamide A (15).
54
pm
30
40
50
60
70
80
90
100
110
120
130
140
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.'.)
Figure 1120. HSQC spectrum of somocystinamide A (15).
.0
i.
i.0
ppm
55
ppm
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
Figure 11.21. HMBC spectrum of somocystinamide A (15).
2.0
1.5
1.0
ppm
56
F. P4,n.
FsT0i.
NSss
1..Nog
LN041200
n3-.*.
S.n 3.7. &b.nb.. 100% kt.0.N000. FA6. P06.
00
00
70
SO
50
40
50.0
07.1
--
376.3
107.2
20
1300
107.1
10
200.2
2403
2063
4123
I
7500
50
50
40
30
20
7U0
00
0
707.6
.,I.,.,i
.
500
560
550
700
750
050
V.6
Figure 11.22. Low resolution positve FABMS of somocystinamide A (15).
57
EXPERIMENTAL
General Experimental Procedures. IR spectra were recorded on a
Nicolet 510 Fourier transform IR spectrophotometer and optical rotations were
measured on a Perkin-Elmer 141 polarimeter. All NMR spectral data were
recorded on Bruker DRX600 and AC300 spectrometers, with the solvents used as
internal standards [CDCI3 (6c 77.23 oH 7.27), CD3OD (0c 49.15
d6
0H
3.31), or DMSO-
(Oc 39.51 0H 2.50)]. Chemical shifts are reported in ppm and coupling constants
(J) are reported in Hz. Mass spectra were recorded on a Kratos MS5OTC mass
spectrometer.
HPLC isolation of somamide A (12) and somocystinamide A (15)
was performed using a Waters Millipore Lambda-Max model 480 LC
spectrophotometer with Waters 515 HPLC pumps.
Cyanobacterial Collection. The mixed assemblage of Lyngbya
majuscula and Schizothrix sp.
marine cyanobacteria (voucher specimen available
as collection number VSO-8/Feb/97-02) was collected by M. Graber and G.
Hooper near the island of Somo Somo, Fiji from a depth of 3-6 meters, on
February 8, 1997. The material was stored in 2-propanol at reduced temperature
until extraction.
Extraction and Isolation. Approximately 43 grams dry weight of the
cyanobacterial material was repetitively extracted with 2:1 CH2Cl2/MeOH to yield
1.3 g of crude extract. Approximately 1.0 g of the extract was fractionated by VLC
over silica gel. Fractions eluting between 90% EtOAc in hexanes and 5% MeOH
in EtOAc were recombined and further purified by C15 SPE cartridge. The fraction
eluting from the
C18
cartridge with 7:3 MeOH/H20, followed by reversed-phase
HPLC using a Phenomenex Sphereclone 5tm ODS column (250
x
10 mm) with
3:1 MeOH/H20 yielded 3.1 mg of somamide A (12). The fraction eluting with 9:1
MeOH/H20 from the
C18
cartridge was also purified on the same HPLC column
using 19:1 MeOH/H20, affording 20.8 mg of somocystinamide A (15).
58
Somamide A (12): glassy oil;
[cx]22D
..3o (c 0.08, MeOH); IR (neat) 3370,
3270, 2930, 2852, 1730, 1641, 1535, 1516, 1202, 1025 cm1; 1H and 13C NMR
data, see Table 11.1; FABMS (positive ion, 3-nitrobenzyl alcohol) mlz 988 (85), 948
(100), 884 (6), 703 (6), 503 (7), 420 (24), 348 (9), 295 (8), 246 (36), 226 (38), 150
(63), 99 (18), 56 (96); HRFABMS m/z [M + H - H2OJ 948.4587 (calculated for
C48H66N7011S, 948.4541).
Somocystinamide A (15): white amorphous solid;
[aJ220
+14° (c 0.75,
CHCI3); IR (neat) 3299, 2922, 2852, 1642, 1533, 1446 cm1; 1H and 13C NMR data,
see Table 11.3; FABMS (positive ion, 3-nitrobenzyl alcohol) m/z 759 (56), 412 (6),
379 (23), 347 (15), 333 (13), 197 (19), 98 (100); HRFABMS m/z [M + HJ 759.4925
(calculated for
C42H71 N404S2,
759.4917).
Decomposition Product 16: white amorphous solid;
EcLJ22D
-17° (c 0.15,
1:1 CHCI3/MeOH); IR (neat) 3317, 2918, 2848, 1642, 1542 cm; 1H and 13C NMR
data (referenced to CD3OD), see Table 11.2; FABMS (positive ion, 3-nitrobenzyl
alcohol) m/z 627 (68), 346 (14), 313 (36), 281 (22), 267 (29), 225 (20); HRFABMS
m/z [M + H] 627.3976 (calculated for CH59N4O4S2, 627.3979).
Brine Shrimp Toxicity
Bioassay.14
Evaluation of the crude extract,
chromatography fractions, and pure compounds for brine shrimp
(Artemia sauna)
toxicity was performed as previously described.
Cytotoxicity Assay.15 Neuro-2a mouse neuroblastoma cells (ATCC CCL131) were grown in RPMI 1640 medium supplemented with 10% heat-inactivated
fetal bovine serum, 1 mM sodium pyruvate, 50 1g/mL streptomycin, and 50
units/mL penicillin in an atmosphere of 5% CO2 at 37 °C. Growth medium (200 tL)
containing the cell suspension (1
x
i0 cells/mL) was added to 96-well culture
plates. After 24 hours, 30 j.tL of sample (dissolved in EtOH and serially diluted with
medium to make the final concentration of EtOH less than 1 %) was added to the
cells. Cultures were incubated for 24 h, and cytotoxicity determined by the MU
59
(3-[4,5-dimethylthiazol-2-yll-2,5-diphenyltetrazolium bromide) assay with
colorimetric measurement at 570 nm.
Absolute Configuration of Amino Acids in Somamide A (12).17
Somamide A (0.5 mg) was hydrolyzed with 6N HCI at 110°C for 16 h. The
hydrolysate was evaporated to dryness and dissolved in H20 (50 tL). A 0.1% 1fluoro-2,4-dinitrophenyl-5-L-alaninamide (FDAA, Marfey's reagent) solution in
acetone (50 tL) and 20 ML of 1 N NaHCO3 were added, and the mixture was
heated at 40°C for 1 hour. The solution was cooled to room temperature,
neutralized with 10 ML of 2N HCI and evaporated to dryness. The residue was
resuspended in 100 ML of 1:1 DMSO/H20, and the solution was analyzed by
reversed-phase HPLC using a Waters Nova-Pak
C18
column (3.9
x
150 mm) with
UV detection at 340 nm, using a linear gradient of 10% CH3CN in H20 containing
0.05% TFA to 50% CH3CN.
The retention times
(tR,
mm) of the derivatized amino acids in the
hydrolysate of somamide A (12) matched those of L-MetSO (18.6), L-Thr (18.9), LVal (33.0) and L-Phe (38.5) but not D-MetSO (19.3), D-Thr (23.6), L-allo-Thr (19.4),
D-allo-Thr (21.0), D-Val (37.9), or D-Phe (42.7).
Absolute Configuration of Somocystinamide A (15). A stream of ozone
was bubbled through 15 (0.3 mg) dissolved in CH2Cl2 (2 mL) at -78 °C until the
solution turned a pale blue color (Ca. 2 mm). The solvent was removed under a
stream of N2, and 6N HCI (1 mL) was added. This solution was microwaved in an
Ace high-pressure tube (1 mm) and again dried under N2. Formic acid (500 ML)
and 30% H202 (10 ML) were added, and the solution was stirred at room
temperature (30 mm) and then dried under N2.
The material was resuspended in H20 (50 tL) and analyzed by reversedphase chiral HPLC using a Phenomenex, Chirex phase 3126 column (4.6
x
50
mm) in 2 mM CuSO4/CH3CN (95:5) with UV detection at 254 nm. The retention
times of the authentic amino acids were 3.5 mm and 4.1 mm for L- and D-cysteic
acid, respectively, Injection of the reaction product from 15 under these HPLC
conditions produced only a peak at 3.5 mm, and coinjection of this material with
the standards confirmed the presence of L-cysteic acid.
61
REFERENCES
(a) Gerwick, W. H.; Proteau, P. J.; Nagle, D. G.; Hamel, E.; Blokhin, A.;
Slate, D. L. J. Org. Chem. 1994, 59, 1243-1 245. (b) Yoo, H.-D.; Gerwick,
W. H. J. Nat. Prod. 1995, 58, 1961-1 965. (c) Marquez, B.; Verdier-Pinard,
P.; Hamel, E.; Gerwick, W. H. Phytochemistiy 1998, 49, 2387-2389.
2.
(a) Zhang, L.-H.; Longley, R. E. Life Sc!. 1999, 64, 1013-1 028. (b) Zhang,
L.-H.; Longley, R. E.; Koehn, F. E. Life Sd. 1997, 60, 751-762.
3.
(a) Berman, F. W.; Murray, T. F. J. Neurochem. 2000, 74, 1443-1451. (b)
Purkerson, S. L.; Baden, D. G.; Fieber, L. A. Neurotoxicology 1999, 20,
909-920.
4.
Gerwick, W. H.; Tan, L. 1.; Sitachitta, N. In The Alkaloids; Cordell, G. A.,
Ed.; Academic Press: San Diego, 2001; Vol. 57, pp 75-184.
5.
Orjala, J.; Gerwick, W. H. J. Nat. Prod. 1996, 59, 427-430.
6.
Orjala, J.; Nagle, D. G.; Hsu, V. L.; Gerwick, W. H. J. Am. Chem. Soc.
1995, 117, 8281-8282.
7.
Wu, M.; Okino, T.; Nogle, L. M.; Marquez, B. L.; Williamson, R. T.;
Sitachitta, N.; Berman, F. W.; Murray, T. F.; McGough, K.; Jacobs, R.;
Colsen, K.; Asano, T; Yokokawa, F.; Shioiri, T.; Gerwick, W. H. J. Am.
Chem. Soc. 2000, 122, 12041-12042.
8.
Pettit, G. R. Prog. Chem. Org. Nat. Prod. 1997, 70, 1-79.
9.
Harrigan, G. G.; Luesch, H.; Moore, R. E.; Paul, V. J. Spec. PubI. - R. Soc.
Chem. 2000, 257, 126-1 39.
10.
(a) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Tuinman, A. A.; Boettner, F. E.;
Kizu, H.; Schmidt, J. M.; Baczynskyj, L.; Tomer, K. B.; Bontems, R. J. J.
Am. Chem. Soc. 1987, 109, 6883-6885. (b) Bagniewski, P. G.; Reid, J. M.;
Pitot, H. C.; Sloan, J. A.; Ames, M. M. Proc. Am. Assoc. Cancer Res. 1997,
38, 221-222.
62
11.
Harrigan, G. G.; Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Nagle, D. G.;
Paul, V. J.; Mooberry, S. L.; Corbett, T. H.; Valeriote, F. A. J. Nat. Prod.
1998, 61, 1075-1077.
12.
Luesch, H.; Moore, R. E.; Paul, V. J.; Mooberry, S. L.; Corbett, T. H. J. Nat.
Prod. 2001, 64, 907-910.
13.
Pettit, G. R.; Kamano, Y.; Herald, C. L.; Dufresne, C; Cerny, R. L.; Herald,
D. L.; Schmidt, J. M.; Kizu, H. J. Am. Chem. Soc. 1989, 111, 5015-501 7.
14.
Meyer, B. N.; Ferrigni, N. R.; Putnam, J. E.; Jacobsen, L. B.; Nichols, D. E.;
McLaughlin, J. L. Pianta Med. 1982, 45, 31-34.
15.
(a) Tan, L. T.; Okino, T.; Gerwick, W. H. J. Nat. Prod. 2000, 63, 952-955.
(b) Manger, R. L.; Leja, L. S.; Lee, S. Y.; Hungerlord, J. M.; Hokama, Y.;
Dickey, R.W.; Granade, H. R.; Lewis, R.; Yasumoto, T.; Wekell, M. M. J.
AOAC Intern. 1995, 78, 521-527.
16.
Harrigan, G. G.; Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Nagle, D. G.;
Paul, V. J. J. Nat. Prod. 1999, 62, 655-658.
17.
Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596.
18.
Williamson, R. T.; Marquez, B. L.; Gerwick, W. H.; Kover, K. E. Magn.
Reson. Chem. 2000, 38, 265-273.
63
CHAPTER THREE
NEW AND DIVERSE SECONDARY METABOLITES FROM A PUERTO RICAN
COLLECTION OF LYNGBYA MAJUSCULA
ABSTRACT
A mammalian sodium channel modulation bioassay was used to guide the
fractionation of a crude organic extract from a Puerto Rican collection of Lyngbya
majuscula. Remarkably, both the potent sodium channel blocker kalkitoxin (3) and
the sodium channel activator antillatoxin (4) were discovered from this extract as
well as the new secondary metabolite antillatoxin B (15), an unusual N-methyl-
homophenylalanine analogue of the neurotoxin 4. The structure of antillatoxin B
(15) was deduced from 2D NMR and data comparisons with 4. Similar to
antillatoxin (4), 15 exhibits significant sodium-channel activating (EC50 = 1.77 tM)
and ichthyotoxic
(LC50 =
1 tM) properties. During this extensive fractionation
process several additional nontoxic metabolites were identified, including a new
malyngamide (16), a quinoline alkaloid (17) and a tryptophan derivative (18). The
structures of 16, 17 and 18 were deduced by NMR and mass spectral data
interpretation, and suggest the existence of a convergent biosynthetic pathway for
these new and structurally dissimilar metabolites.
64
INTRODUCTION
Marine microalgae are known for their production of noxious compounds
responsible for human intoxication and extensive fish
mortality.1
White
dinoflagellates are the notorious cause of neurotoxic and paralytic shellfish
poisonings, marine cyanobacteria like Lyngbya majuscula also produce a variety
of toxic secondary metabolites, including such structurally diverse substances as
the lipopeptide apratoxin (1) and the macrolide debromoaplysiatoxin (2) 23 In
addition to their ecological and economic significance, such molecules also
possess considerable potential as biochemical and pharmaceutical tools.
OCH3
a
OH
0
ss'sO
0
N} N-2O'<
Apratoxin (1)
The potent ichthyotoxic
OH
OH
Debromoaplysiatoxin (2)
L.
majuscula metabolite, antillatoxin (4), was first
reported from our research laboratory in 1995 and subsequently shown to induce
NMDA receptor-mediated
neurotoxicity.45
On the basis of this finding and the
prospect of discovering new neurotoxins of novel structure and pharmacology, we
evaluated our library of cyanobacterial extracts for sodium channel modulators
using a mouse neuroblastoma cell
assay.6
Approximately 150 cyanobacterial
extracts were initially screened, identifying nine that displayed either significant
sodium channel activating or sodium channel blocking properties. Assay-guided
65
fractionation of one chemically prolific
L.
majuscula extract from a collection
acquired in Puerto Rico (PRLP-16/Sept/97-03) led to the isolation of several active
constituents, including the sodium channel blocker kalkitoxin (3), the sodium
channel activator antillatoxin (4) and the cytotoxic hermitamides A (5) and B
(6).478
Moreover, a novel bioactive structural homologue of 4, antillatoxin B (15), was also
discovered in minute quantity from this extract.
0
r!4
N
0
L
Antillatoxin (4)
Kalkitoxin (3)
0
OCH3
H
Hermitamide A (5)
OCH3
NH
0
Hermitamide B (6)
H
\,2
During this extensive fractionation process, several other previously
reported compounds were also detected from this one extract, including the
quinoline alkaloid 7 and various malyngamide-type structures (8-14, Figure
lll.1).9.b0
While the malyngamides are an especially abundant and interesting
class of L. majuscula compounds which combine a lipid portion (typically, 7(S)methoxytetradec-4(E)-enoic acid =lyngbic acid (8)) with a variety of amine-derived
moieties, quinoline alkaloids have rarely been observed from this marine
cyanobacterium.9
Along with the isolation of antillatoxin B (15), three additional secondary
metabolites, malyngamide T (16), the quinoline alkaloid 17 and the tryptophan
derivative 18, were also discovered. Herein, the chromatographic purification and
structure elucidation of these four new compounds (15-18) are presented, and,
based on shared structural characteristics, a plausible biogenic connection
between several dissimilar metabolites from this chemically diverse
L. majuscula
collection is proposed.
H
MeO0
O
Quinoline alkaloid (7)
0
OCH3
OH
7(S)-methoxytetradec-4(E)-enoic acid (8)
0
OCH3
R
H
110%J
I
Malyngamide C (9) R = OH
Malyngamide C acetate (10) R = OAc
OCH3
0
HftI
Cl
Malyngamide F (11) R = OH
Malyngamide F acetate (12) R = OAc
Malyngamide K (13) R = H
R
0
OCH3
Malyngamide J (14)
H
0"
I
OCH3
Figure 111.1. Previously reported non-toxic metabolites from collection PRLP1 6ISept/97-03.
67
RESULTS AND DISCUSSION
Antillatoxin B (15) was isolated as a colorless oil using vacuum liquid
chromatography and reversed-phase HPLC. Initial inspection of the 1H NMR data
of 15 (Figure 111.2) indicated the close similarity of this metabolite to antillatoxin
(4)4
A.
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0 p
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0 p
Figure 111.2. 1H NMR spectral comparison of (A) antillatoxin (4) and (B) antillatoxin
B (15).
By high resolution FABMS, compound 15 analyzed for C33H48N305 and
hence, was larger than antillatoxin by a C5H2 unit, indicating the presence of
additional unsaturation. Moreover, several signals associated with the N-methyl
valine moiety in 4 were absent and were replaced by a mono-substituted aromatic
ring and two mid-field methylenes, coupled to a down-field methine by TOCSY
correlations (Figures 111.3 and 111.4).
Two-dimensional NMR spectral data, especially HMBC (Figure 111.5), were
used to delineate this new residue as an unusual N-methylated homophenylalanine unit (Table 111.1). While homophenylalanine has been detected
previously in a few fresh-water cyanobacterial metabolites such as [Dha7]-
microcystin-HphR and nodulapeptins A and B, it was previously unreported from
marine cyanobacteria.'12 2D NMR spectral analysis and data comparisons
confirmed that the remainder of the planar structure of antillatoxin B (15) was
identical to that of antillatoxin (4).
22
H
I
Q
20
Antillatoxin B (15)
The absolute stereochemistry of antillatoxin (4) has recently been firmly
established by total
synthesis.13
The close comparability of NMR shifts and
coupling constants at equivalent centers (e.g., 3JH21H23 = 11.0 Hz as seen for 4),
along with its similar biological properties (see below), suggested that antillatoxin B
(15) was of the same enantiomeric series. Additionally, the absolute
stereochemistry of the alanine residue in 15 was determined by Marfey's analysis
to be
S,14
and the presence of a strong nOe correlation between H-4 and H-i 5
supported an S stereochemistry for the N-methyl homophenylalanine residue as
well.
Both natural products 4 and 15 are potent activators of the voltagesensitive sodium channel in mouse neuro-2a neuroblastoma cells, with EC50
values of 0.18 tM and 1.77 MM, respectively. In addition, antillatoxin B (15) is
strongly ichthyotoxic to goldfish, with an LC50 of 1.0 MM (antillatoxin LC50 = 0.1
tM).15
It is interesting to note that in both measures of antillatoxin B's biological
activity, a ten-fold decrease in potency is observed compared to antillatoxin. It
seems likely that substitution of the larger N-methyl homophenylalanine residue for
the N-methyl valine accounts for this decrease.
Antillatoxin B (15) represents an unusual addition to the growing number of
bioactive peptide-derived metabolites from L. majuscula. The presence of
homophenylalanine is particularly unique in that only a few cyanobacterial isolates
have been shown to possess this structural feature. It is also worth noting that
dolastatin 16, a cyclic peptide originally reported from the marine mollusc,
Do/abe/Ia auricu/aria, contains a similar aromatic residue (3-methyl
homophenylalanine =
dolaphenvaline).16
It has been demonstrated that several of
the dolastatins and related compounds are of cyanobacterial origin, and are likely
sequestered by these herbivorous animals from their cyanobacterial diet (see
chapters two and four).17
70
Table 111.1. NMR spectral data for antillatoxin B (15) in ODd3.
1H (J in Hz)
TOCSY
HMBCa
4.67, dd (18.4, 9.7)
NH-i
NH-i
1/3
3.57, dd (18.4, 1.5)
7.98, d (9.5)
2
1/3
4.77, t (7.3)
5, 6
3, 5, 13, 14
2.39, 2.05
4, 6
3, 7
2.68,2.54
4,5
7
7.19, d (7.3)
9/11
6, 10
7.31,t(7.3)
8/12,10
7
126.1
7.22, d (7.3)
9/li
28.5
172.9
42.9
18.3
2.88, s
position
167.9
41.4
2
NH-i
3
4
5
6
7
8/12
9/11
10
13
14
15
16
167.8
59.3
29.9
31.6
140.2
128.0
128.5
NH-2
17
18
170.9
46.4
5.06, m
1/3
4, 14
NH-2, 16
NH-2, 15
16, 17
1.29,.d (6.7)
6.46, d (9.1)
15, 16
17
2.98, d (12.9)
14, 15
17, 19, 20, 21
2.80, d (12.9)
19
20
21
22
23
24
25
26
27
28
29
30
31/32/33
a
144.6
113.7
38.9
18.8
5.06, 5.02
18
18, 19,21
2.17, m
22, 23
18, 19, 20, 22
0.87, d (7.0)
21,23
21,22
19,21,23
1,19,21,24,25,26
83.3
128.9
12.2
5.17,d(il.0)
26,29
23,24,26
137.1
5.94, s
25, 28, 29
23, 25, 28, 29
130.2
17.5
141.3
32.5
30.8
1.80, brs
26, 29
26,27, 29
5.29, S
25, 26, 28
26, 28, 31-33
l.56
1.13, s
Proton showing HMBC correlation to indicated carbon. ° Obscured.
29,30
1\
4O
QG sP°
0
72
ppm
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
Figure 111.4. TOCSY spectrum of antillatoxin B (15).
3.0
2.5
2.0
1.5
1.0
ppm
73
ppm
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
Figure 111.5. HMBC spectrum of antillatoxin B (15).
3.0
2.5
2.0
1.5
1.0
ppm
74
Along with identification of the agents responsible for the observed sodium
channel modulation, 1H and TLC analyses indicated the presence of additional
secondary metabolites of equally intriguing chemical structure. To fully
characterize the extraordinary chemistry of this marine cyanobacterial collection, a
detailed examination of the crude organic extract was undertaken. Using vacuum
liquid chromatography and reversed-phase HPLC, several metabolites of known
composition as well as three new chemical entities (16, 17, 18) were identified.
15'
0
OCH3
14
0
0
1
N
Malyngamide I (16)
OCH3
10
MeO0
3'
Cl
OMe1'
H
I
11
Quinoline derivative (17)
OH°
H
Tryptophan derivative (18)
Compound 16 produced an [M + HJ peak at m/z 468.2518 by HRFABMS
for a molecular formula of C25HNO5Cl (7° unsaturation). 1 D and 2D NMR data
(Figures 111.6-111.8) suggested a strong resemblance between 16 and the.
malyngamide family of metabolites (Table 111.2), including the presence of the 7(S)-
methoxytetradec-4(E)-enoate moiety (lyngbic acid, 8), a common feature of this
structural class.10'18
75
'1
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0 ppm
Figure 111.6. 1H NMR spectrum of malyngamide T (16).
Table 111.2. NMR spectral data for malyngamide T (16) in CDCI3.
position
1H (J in Hz)
13C
COSY
HMBCa
1
3.94, d (6.1)
42.9
NH
2, 3, 1'
133.4
2
3
6.27,s
119.9
1,2,4,5
4
3.38, s
33.2
1, 2, 3, 5, 6
160.8
5
5.92, d (1.9)
6
100.8
8
4,5, 7,8
6
6, 7, 9
171.3
7
8
5.42, d (1.9)
87.8
164.4
9
10
3.80, s
NH
5.89, brt (6.0)
7
55.8
1
1, 1'
1', 3', 4
172.8
1'
2'
2.27, m
36.2
3'
3'
2.34, m
28.4
2', 4'/5'
5.48, m
130.6
4'
1', 2', 4, 5'
2', 3', 6'
5'
5.48, m
127.8
6'
2.18, m
36.1
4'IS', 7'
4', 5', 7', 8'
7'
3.16, m
80.7
6', 8'
5', 8', 9', 15'
8'
1.43
33.2
7', 9
6', 7', 9'
9'
1.33
25.4
8'
10'
10'
1.28
29.4
11'
1.28
29.9
12'
1.29
22.7
13'
1.27
31.8
14'
0.89,t(7.0)
14.3
15'
3.31,s
56.3
8
3', 6'
13', 14'
12'
13'
Proton showing long-range correlation to indicated carbon.
12', 13'
7
76
ppm
0
30
4
40
4
50
4
60
70
80
$
90
100
110
120
I
130
140
150
0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
Figure 111.7. HSQC spectrum of malyngamide T (16).
2.5
2.0
1.5
1.0
ppm
77
ppm
D
20
30
$
$
:
1
I
40
50
I
I
60
70
ID
80
90
100
110
120
$1
9
130
4
140
150
I
I
160
I
170
'l'rI
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
.b
3.0
Figure 111.8. HMBC spectrum of malyngamide T (16).
2.5
..I0000I0000I..
2.0
1.5
1.0
ppm
78
HMBC correlations were essential in constructing the remaining portion of
16 as homonuclear couplings were virtually absent (Figure 111.8, Table 111.2). In the
COSY spectrum, one cross-peak was observed between an amide proton (oH
5.89) and a low-field methylene signal (CH2-1, 0c 42.9,
0H
3.94). HMBC data
showed this methylene was separated from a second low-field methylene (CH2-4,
Oc 33.2, 8H 3.38) by a vinyl chloride functionality, also a common feature of the
malyngamides.
Both UV spectral data and HMBC connectivities were used to assemble
the a-pyrone ring of 16, notably H8 to C9, C7 and C6, and H6 to C8, C7 and C5.
This substructure has been observed in several metabolites from red algae and
marine bacterial species, but only the 'y-pyrone metabolite kalkipyrone has
previously been isolated from this marine
cyanobacterium.192°
The placement of
the 0-methyl substituent on the a-pyrone ring was determined by an HMBC
correlation observed between the 0-methyl protons (H3-1O, 0 3.80) and C7, as well
as the homonuclear meta coupling (1.9 Hz) measured between H8 and H6.
The subunits were linked by HMBC correlations seen from both the amide
proton
(OH
5.89) and the H2-1 methylene protons to CI'. The E geometry of the
vinyl chloride in 16 was deduced by measurement of a 3.9 Hz coupling between
H3 and Cl, and a 7.1 Hz coupling between H3 and C4, by HSQMBC, completing
the structure of malyngamide T (16).21
Compound 17 showed a HRCIMS [M] ion at m/z 369.1339, for a molecular
formula of C18H24N05C1, and therefore, possessed seven degrees of unsaturation.
1H and 13C NMR spectra were well dispersed in C6D6 and indicated the presence
of four carbon-carbon double bonds. Thus, three rings were required to account
for the remaining degrees of unsaturation (Figure 111.9).
79
7.0
6.0
6.5
5.5
5.0
4.5
4.0
3.5
2.5
3.0
2.0
ppm
Figure 111.9. 1H NMR spectrum of quinoline 17.
2D NMR experiments, including HSQC (Figure 111.10), COSY and
HSQMBC (Figure 111.11), were mainly used for the structure elucidation of 17. 1H
NMR coupling constants and COSY spectral data were used to construct the 2, 4dimethoxyxylose moiety (Figure 111.12, Table 111.3), a residue previously observed
in the L. majuscula metabolites malyngamide J (14) and the quinoline alkaloid 7,
both of which were also isolated during the fractionation of this extract (Figure
111.1). The remaining signals in 17 were assembled to form a quinoline-like
structure similar to compound 7. However, signals forming the methyl pyridine ring
in 7 were replaced by two coupled methylene signals (CH2-1,
CH2-2, E, 26.4,
10, ö 111.3,
metabolites.
6H
H
c
41.6,
6H
2.75 and
2.61) and an exocyclic olefin possessing a vinyl chloride (CH-
6.34), more reminiscent of the malyngamide class of secondary
ppm
20
30
40
50
60
70
80
90
100
110
120
130
7.0
6.5
6.0
5.5
5.0
4.5
4.0
Figure 111.10. HSQC spectrum of quinoline 17.
3.5
3.0
2.5
2.0
ppm
81
ppm
44
20
30
40
50
60
...
-70
0 S
9
S.
-
80
90
100
.
110
45
4
120
S
4
130
140
150
7.0
6.5
6.0
5.5
5.0
4.5
4.0
Figure 111.11. HSQMBC spectrum of quinoline 17.
3.5
3.0
2.5
2.0
ppm
82
Table 111.3. NMR spectral data for quinoline alkaloid 17 in C6D6.
position
1H (J
2
in Hz)
COSY
HsQMBca
2.75,brt(5.9)
41.6
2
2.61, brt(5.9)
26.4
1
2,3,9
1,3,10
7
3,6,7,9
5
5,6,9, 11
3
135.1
4
119.3
5
111.8
7.14
149.6
6
7
121.5
6.90, d (1.5)
124.0
8
139.4
9
2,3,4
7,8,9
10
6.34,s
111.3
11
1.66,brs
17.4
1'
4.78, d (7.4)
103.9
2'
6, 2', 5'
2'
3.32, dd (8.8, 7.4)
83.8
1', 3'
1', 3', 2'-OMe
3'
3.66, dd (8.8, 8.8)
76.4
2', 4'
2', 4', 5'
4'
3.21, ddd (9.6,8.8,5.1)
79.6
3', 5a', 5b'
3', 5', 4'-OMe
5a'
2.98, dd (11.8, 9.6)
63.6
4', 5b'
1', 3', 4'
5b'
3.81, dd (11.8, 5.1)
4', 5a'
1', 3', 4'
2'-OMe
4'-OMe
3.60, s
60.7
2'
3.16, s
58.6
4'
8
Proton showing long-range correlation to indicated carbon.
The sugar and quinoline units of 17 were connected through an HSQMBC
correlation observed from Hi' to C6 (Figure 111.12).
I-'
9.6 Hz
6.8 Hz
zI
-'
7.4Hz
-
42Hz
)
/'' 1H -H coupling constant
H.JleH
/''\ HSQMBC correlation
8.8Hz
8.8Hz
Figure 111.12. Key coupling constants and HSQMBC correlations for 17.
83
A 4.2 Hz heteronuclear coupling constant between H10 and C4 and a 6.8 Hz
coupling constant between H1O and C2 were also measured from the HSQMBC
experiment, establishing an E geometry for the vinyl chloride functionality on 17
and completed the planar structure and relative stereochemistry of this unusual
quinoline derivative.
Compound 18 was assigned a molecular composition of C11H13N04 by
HRFABMS (m/z [M + HJ 224.0920). The 1H (Figure 111.13) and 13C NMR spectra
revealed the existence of aldehyde and ester functionalities as well as two olefins.
This accounted for four of the six degrees of unsaturation in 18 and indicated the
presence of two rings. 2D NMR data (Table 111.4) were used to sequence a
hydroxy-substituted methine signal (CH-4, c 67.8, oH 4.92) adjacent to a second,
acetoxy-substituted methine (CH-5, 8c 75.9, 0H 5.06), which in turn was adjacent to
two consecutive methylene signals (CH2-6, Oc 26.4, 8H 2.20,1.94 and CH2-7, Oc
20.5, 8H 2.77, 2.64).
9
Figure 111.13.
8
7
6
5
4
1H NMR spectrum of tryptophan derivative 18.
3
ppm
[;ril
Table 111.4. NMR spectral data for compound 18 in CDCI3.
position
1H (J in Hz)
1
NH, 8.79, brs
2
7.39, d (2.3)
13C
HsQc-1-ocsr
132.5
HMBCb
1
3, 3a, 7a, 8
125.7
3
118.8
3a
4
4.92, d (6.7)
67.8
5
3a, 5, 7a
5
5.06, ddd (10.9, 6.7, 3.3)
75.9
4, 6
3a, 4, 9
6
2.20, 1.94
26.4
5, 7
4, 5,7, 7a
7
2.77, ddd (15.9, 10.2, 5.5)
20.5
6
3a, 5, 6, 7a
2.64, brdt (15.9, 4.6)
132.1
7a
8
9
10
188.5
9.65, s
3, 3a
172.6
9
21.2
2.11,s
4, 5
5.36, brs
OH
0
a
Proton showing long-range correlation to indicated proton. Proton showing long-range
correlation to indicated carbon.
Due to its small molecular size and the presence of several quaternary
carbons, four possible structure types were initially conceived for 18.
OH°
OH°
OH
H
Ac
AcO
NH
I 8a
H
AcO
OH
I 8b
H
18c
18d
However, an HMBC cross-peak observed between H2 (6H 7.39) and C8 (c
188.5) excluded structures of type I 8a. Additionally, because the proton spectrum
of 18 showed H2 as a finely split doublet coupled to an NH proton (HI, 68.79) in
the HSQC-TOCSY experiment, structure 18b was also rejected. While structure
types 18c and 18d differed only in the location of substituents about the six-
membered ring, HMBC correlations observed from the C6 methylene protons to
C1a
5toG
ôeflt'
iBó
conect p\3
The relative stereochemistry of H4 and H5 was deduced through proton
coupling constant analysis. H5 appears as a ddd with 3J coupling constants of
10.9 Hz and 3.3 Hz to the C6 methylene protons and 6.7 Hz to H4, establishing an
axial orientation for H5 and indicating a trans relationship with H4. In addition, no
dipolar coupling was observed between H4 and H5 from 1-D nOe experiments, as
anticipated for the
trans
geometry. Due to the instability of compound 18, attempts
to determine the absolute stereochemistry at C4 by Mosher ester analysis were
unsuccessful
22
Isolation of these new
L.
majuscula metabolites, in addition to the twelve
known compounds detected from this single collection, demonstrates the capability
of this organism to produce a remarkable variety of fundamentally different
chemotypes. While this species was not viable in culture, it is interesting to
speculate on the biogenesis of such an intriguing spectrum of metabolites.
Lyngbic acid (8) appears to serve as substrate for extension by a variety of amino
acids, producing a suite of related
L.
majuscula natural products (Figure 111.15).
For example, hermitamide B (6) can be formed directly from condensation of 8
with tryptamine. Similarly, we envisage that 8 is condensed with either J3-alanine
or glycine, extended with additional malonyl-00A derived acetate units, and further
elaborated with chlorination, methylation, and glycosylation events, to produce
malyngamides J (14) and T (16), respectively. Supporting data for the utilization of
13-alanine as a constituent in
L.
majuscula secondary metabolite production has
been obtained from recent biosynthetic studies conducted in our laboratory (see
chapter six).
Moreover, while the Lyngbya-derived malyngamide and quinoline alkaloids
may previously have appeared as disparate structural families (quinolines are
known to derive from anthranilate biosynthetic origins in plants),23 the discovery of
17 with its distinctive vinyl chloride functionality suggests that a potential biogenic
relationship could exist between these classes (Figure 111.15) and an alternative
route to quinoline metabolite formation may exist in
L.
majuscula. Should
compounds of this nature be produced in a cultured L. majuscula, exploration of
their molecular assembly (through stable-isotope labeling experiments, for
example) will determine the validity of this proposed biosynthetic convergence.
87
NH
0
OCH3
NH2
COH
N
H
0
H
OCH3
N/
OH
8
17
H2NAOH
0
0
H2N..tL
OCH3
o\J30
14
OH\
H30
H3CO0
OCH3
16
OCH3
Figure 11115. Proposed biogenic relationships between secondary metabolites
from Lyngbya majuscula. Lyngbic acid (8) serves as the substrate for
condensation with various amino acid residues, which upon further ketide
extensions and chemical elaborations, leads to the numerous malyngamide
structures. Additionally, acetate-extended 13-alanine may cyclize via discrete
pathways, producing either quinoline alkaloid 17 or the head group of
malyngamide J (14).
EXPERIMENTAL
General Experimental Procedures. Optical rotations were measured on
a Perkin-Elmer 141 polarimeter. IR and UV spectra were recorded on Nicolet 510
and Beckman DU64OB spectrophotometers, respectively. NMR spectra were
recorded on a Bruker DRX600 and AM400 spectrometers with the solvent (CDCI3
or C6D6) used as an internal standard. Chemical shifts are reported in ppm and
coupling constants (J) are reported in Hz. Mass spectra were recorded on a
Kratos MS5OTC mass spectrometer. HPLC isolations were performed using a
Waters Millipore Lambda-Max model 480 LC spectrophotometer with Waters
model 515 HPLC pumps.
Cyanobacterial Collection. The marine cyanobacterium Lyngbya
majuscula (voucher specimen available as collection number PRLP-1 6/Sept/97-
03) was obtained from shallow waters (0.5 m) near Collado Reef, Puerto Rico, on
September 16, 1997. The material was stored in 2-propanol at -20°C until
extraction.
Extraction and Isolation. Approximately 170 g (dry wt) of the
cyanobacterium was extracted repeatedly with 2:1 CH2Cl2/MeOH to produce 6.8 g
of crude organic extract. A portion of the extract (5.9 g) was subjected to Si VLC
to produce 14 fractions. The fraction eluting with 45-50% EtOAc in hexanes was
further purified, first by a
C18
solid phase extraction (SPE) cartridge (3:2
MeOH/H20), followed by reversed-phase HPLC (3:1 MeOH/H20, Phenomenex
Spherisorb 10
t
ODS) to yield 1.3 mg of quinoline alkaloid 17. The fraction eluting
with 55-65% EtOAc in hexanes was purified using a
C18
SPE cartridge (7:3
MeOH/H2O) and RP HPLC (7:3 MeOH/H2O, Phenomenex Spherisorb 10
jt
ODS)
to produce 0.4 mg of antillatoxin B (15). Likewise, the fraction eluting with 70-75%
EtOAc was also subjected to
C18
SPE (3:2 MeOH/H20) and RP HPLC (3:1
MeOH/H20, Phenomenex Sphereclone 5
.t
ODS), affording 13.2 mg of tryptophan
derivative 18. Finally, 80-90% EtOAc in hexanes VLC fraction was purified by
C18
SPE (7:3 MeOH/H20) and RP HPLC (4:1 MeOH/H20, Phenomenex Spherisorb 10
t
ODS) to give 2.3 mg of malyngamide T (16).
Biological Assays. The ichthyotoxicity of pure 4 and 15 to the common
goldfish (Carassius auratus) was determined as previously described.15 Activation
of the voltage sensitive sodium channel in mouse neuro-2a neuroblastoma cells
was also determined as previously described.6
Antillatoxin B (15): colorless oil;
Xmax
[a}23D
-110° (c 0.21, MeOH); UV (MeOH)
209 nm (log c 4.70), 240 nm (log c 4.06); lR (neat) 3445, 3294, 3080, 2959,
1734, 1645, 1543, 1455, 1259 cm; 1H and 13C NMR data, see Table 111.1;
LRFABMS (positive ion, 3-nitrobenzylalcohol) m/z 588 (18), 566 (34), 491 (41),
316 (15), 307 (21), 251 (53), 217 (15), 148 (100); HRFABMS m/z [M + H]
566.3596 (calculated for C33H48N305, 566.3594).
Malyngamide T (16): pale yellow oil;
(MeOH)
max
218 nm (log s 4.45), 284 nm (log
[a]22D
-14° (c 0.20, CHCI3); UV
4.l4); IR (neat) 3309, 2927, 2855,
1725, 1650, 1567, 1457, 1415, 1247, 1095, 1037 cm1; 1H and 13C NMR data, see
Table 111.2; LRFABMS (positive ion, 3-nitrobenzylalcohol) m/z 470 (36), 468 (100),
438 (32), 436 (91), 367(11), 325 (21), 230 (24), 213 (26), 143 (23); HRFABMS
m/z [M + H] 468.2518 (calculated for C25H39N05C1, 468.2517).
Quinoline alkaloid (17): white amorphous solid;
CHCI3); UV (MeOH)
Xmax
[a]220
-21°
(C
0.12,
212 nm (log s 4.58), 241 nm (log c4.29), 269 nm (log c
3.90); IR (neat) 3407, 2922, 2853, 1632, 1091, 1074 cm1; 1H and 13C NMR data,
see Table 111.3; LRCIMS m/z 371 (32), 369 (92), 263 (69), 209 (100); HRCIMS m/z
[M] 369.1339 (calculated for C18H24N05C1, 369.1343).
Tryptophan derivative (18): white amorphous solid;
CHCI3); UV (MeOH)
max
[a]22D
+105° (c 0.50,
214 nm (log c 4.39), 254 nm (log c 4.04), 287 nm (log
c 3.67); lR (neat) 3274, 2935, 2890, 1731, 1637, 1245, 1042 cm1; 1H and 13C NMR
data, see Table 111.4; LRFABMS (positive ion, 3-nitrobenzylalcohol) m/z 224 (83),
206 (100), 163 (64), 146 (32); HRFABMS m/z [M + H] 224.0920 (calculated for
C11H14N04, 224.0923).
Absolute Stereochemistry of the Alanine Residue in 15 by Marfey's
Derivatization.14
Approximately 0.2 mg of antillatoxin B (15) was hydrolyzed with
6N HCI (Ace high pressure tube, microwave, 1.5 mm), and the hydrolysate was
evaporated to dryness and resuspended in H20 (50 .tL). A 0.1% 1-fluoro-2,4dinitrophenyl-5-L-alaninamide solution in acetone (Marfey's reagent, 20 jiL) and
1N
NaHCO3 (10 j.tL) were added, and the mixture was heated at 40°C for 1 h. The
solution was cooled to room temperature, neutralized with 2N HCI (5 iL) and
evaporated to dryness. The residue was resuspended in H20 50 (tL) and
analyzed by reversed-phase HPLC [Waters Nova-Pak C18, 3.9
x
150 mm, UV
detection at 340 nm] using a linear gradient (10% CH3CN in H20 containing 0.05%
TFA to 50% CH3CN). The retention time
(tR,
mm) of the derivatized alanine
residue in the hydrolysate of antillatoxin B (15) matched that of L-Ala (25.8) but not
that of D-Ala (30.2).
REFERENCES
1.
(a) Shimizu, Y. Chem. Rev. 1993, 93, 1685-1698. (b) Yasumoto, T.;
Murata, M. Chem. Rev. 1993, 93, 1897-1909.
2.
Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.; Corbett, T. H. J. Am.
Chem. Soc. 2001, 123, 5418-5423.
3.
(a) Mynderse, J. S.; Moore, R. E.; Kashiwagi, M.; Norton, T. R. Science
1977, 196, 538-540. (b) Molecular Probes. http://www.probes.com/
(accessed June 2002).
4.
Orjala, J.; Nagle, D. G.; Hsu, V. L.; Gerwick, W. H. J. Am. Chem. Soc.
1995, 117, 8281-8282.
5.
Berman, F. W.; Gerwick, W. H.; Murray, T. F. Toxicon 1999, 37, 16451648.
6.
Manger, R. L.; Leja, L. S.; Lee, S. Y.; Hungerford, J. M.; Hokama, Y.;
Dickey, R. W.; Granade, H. R.; Lewis, R.; Yasumoto, T.; Wekell, M. M. J.
AOAC Intern. 1995, 78, 521-527.
7.
Wu, M.; Okino, T.; Nogle, L. M.; Marquez, B. L.; Williamson, R. T.;
Sitachitta, N.; Berman, F. W.; Murray, T. F.; McGough, K.; Jacobs, R.;
Colsen, K.; Asano, 1; Yokokawa, F.; Shioiri, 1.; Gerwick, W. H. J. Am.
Chem. Soc. 2000, 122, 12041-12042.
8.
Tan, L. T.; Okino, T.; Gerwick, W. H. J. Nat. Prod. 2000, 63, 952-955.
9.
Orjala, J.; Gerwick, W. H. Phytochemistry 1997, 45, 1087-1090.
10.
(a) Cardellina, J. H., II; Dalietos, D.; Marner, F.-J.; Mynderse, J. S.; Moore,
R. E. Phytochemisttyl978, 17, 2091-2095. (b)Ainslie, R. D.; Barchi, J. J.,
Jr.; Kuniyoshi, M.; Moore, R. E.; Mynderse, J. S. J. Org. Chem. 1985, 50,
2859-2862. (c) Gerwick, W.H.; Reyes, S.; Alvarado, B. Phytochemistry
1987, 26, 1701-1704. (d) Orjala, J.; Nagle, D.; Gerwick, W. H. J. Nat. Prod.
1995, 58, 764-768. (e) Wu, M.; Milligan, K. E.; Gerwick, W. H. Tetrahedron
1997, 53, 15983-15990. (f) Milligan, K. E.; Marquez, B.; Williamson, R. T.;
Davies-Coleman, M.; Gerwick, W. H. J. Nat. Prod. 2000, 63, 965-968.
11.
Namikoshi, M.; Sivonen, K.; Evans, W. R.; Carmichael, W. W.; Rouhiainen,
L.; Luukkainen, R.; Rinehart, K. L. Chem. Res. Toxicol. 1992, 5, 661-666.
12.
Fujii, K.; Sivonen, K.; Adachi, K.; Noguchi, K.; Sano, H.; Hirayama, K.;
Suzuki, M.; Harada, K. Tetrahedron Lett. 1997, 38, 5525-5528.
92
13.
(a) Yokokawa, F.; Fujiwara, H.; Shioiri, T. Tetrahedron 2000, 56, 17591775. (b) Yokokawa, F.; Fujiwara, H.; Shioiri, T. Tetrahedron Lett. 1999,
40, 1915-1916. (C) Yokokawa, F.; Shioiri, T. J. Org. Chem. 1998, 63, 86388639.
14.
Marfey, P. Car!sberg Res. Commun. 1984, 49, 591-596.
15.
(a) Bakus, G. J.; Green, G. Science 1974, 185, 951-953. (b) Orjala, J.;
Nagle, D.; Gerwick, W. H. J. Nat. Prod. 1995, 58, 764-768.
16.
Pettit, G. R.; Xu, J.; Hogan, F.; Williams, M. D.; Doubek, D. L.; Schmidt, J.
M.; Cerny, R. L.; Boyd, M. R. J. Nat. Prod. 1997, 60, 752-754.
17.
Harrigan, G. G.; Leusch, H.; Moore, R. E.; Paul, V. J. Spec. Pub!. - R. Soc.
Chem. 2000, 257, 126-139.
18.
While the geometry of the olefin in the lipid portion of 2 was obscured in
CDCI3, a 15.1 Hz coupling was deduced from a proton spectrum run in
C6D6, and thus the E geometry at this position, observed in all
malyngamides, was confirmed. The absolute stereochemistry at C-16 was
indicated as S through isolation of the free methoxy acid (lyngbic acid) from
the crude extract. The optical rotation of this isolate ([a]24D -14.1°, c 0.22,
CHCI3) was comparable to the reported literature value (see reference lOa)
for 7(S)methoxytetradec-4(E)-enoic acid ([a]26D -11.1°, c 3.9, CHCI3).
19.
(a) Shin, J.; Paul, V. J.; Fenical, W. Tetrahedron Lett. 1986, 27, 5189-5192.
(b) Toske, S. G.; Jensen, P. R.; Kauffman, C. A.; Fenical, W. Nat. Prod.
Lett. 1995, 6, 303-308. (c) Linde!, T.; Jensen, P. R.; Fenical, W.
Tetrahedron Lett. 1996, 37, 1327-1330. (d) Sitachitta, N.; Gadepalli, M.;
Davidson, B. S. Tetrahedron 1996, 52, 8073-8080.
20.
Graber, M. A.; Gerwick, W. H. J. Nat. Prod.
21.
Williamson, R. T.; Marquez, B. L.; Gerwick, W. H.; Kover, K. E. Magn.
Reson. Chem. 2000, 38, 265-273.
22.
(a) Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34, 25432549. (b) Ninomiya, M.; Hirohara, H.; Onishi, J.; Kusumi, T. J. Org. Chem.
1999, 64, 5436-5440.
23.
Herbert, R. B. In The Biosynthesis of Secondary
and Hall: New York, NY, 1989, pp162-164.
1998, 61,
677-680.
Metabolites;
Chapman
93
CHAPTER FOUR
THE ANTANAPEPTINS, NEW CYCLIC DEPSIPEPTIDES FROM A
MADAGASCAN LYNGBYA MAJUSCULA
ABSTRACT
Phytochemical examination of a Lyngbya majuscula collection from Antany
Mora, Madagascar, led to the discovery of a new series of cyclic depsipeptides,
the antanapeptins A-D (9-12). Their structures were deduced through extensive
analysis of 1 D and 2D NMR spectroscopic data and supported by high resolution
positive FAB mass spectrometry. Antanapeptins A (9) and D (12) contain the
unique 3-hydroxy acid unit 3-hydroxy-2-methyloct-7-ynoic acid (Hmoya), while
antanapeptins B (10) and C (11) possess the reduced double bond and single
bond equivalents of Hmoya, respectively. In addition to the discovery of the
antanapeptin series, several previously reported metabolites were also identified
from this microalgal collection, including the cyanobacterial cytotoxin hermitamide
A and the promising antineoplastic agent dolastatin 16 (17), first isolated in small
yield from the marine mollusc Dolabella auricularia. Dolastatin 16 (17) also
possesses unusual structural features such as 3-methyl-homophenylalanine and
the p-amino acid 3-amino-2,4-dimethylpentanoate. Isolation of this marine
invertebrate metabolite from a cyanobacterial source, as well as the close
structural relationship of the antanapeptins to the molluscari kulolides (18-20),
kulomo'opunalides (21, 22), and onchidin B (23), gives further credence that
cyanobacteria are, in fact, the true producers of these compounds.
INTRODUCTION
Several current literature reviews have discussed the close relationship of
metabolites isolated from marine invertebrates to those reported from marine
cyanobacterial sources. Most notably, the dolastatins, a highly bioactive family of
peptides from the marine mollusc
have long been speculated
Do/abe/Ia auricularia,
to derive from the cyanobacterial diet of this generalist
herbivore.1'2
In recent years
this hypothesis has been substantiated by the discovery of several identical
metabolites (1-3) from cyanobacterial origins (Figure
ONH
H(NS{'
0
0
N
NNU'
0
0
0
/
Dolastatin 12 (2)
Dolastatin 3 (1)
I
IV.1).3
o
0
OMeO
OMeO
N
S
Dolastatin 10 (3)
Figure IV.1. Dolastatins isolated from both marine invertebrate and
cyanobacterial sources.
95
The marine cyanobacterium Lyngbya
ma] uscula,
in particular, is an
established source of such unique and bioactive peptides (Figure lV.2). Examples
include the antimicrobial and actin polymerization-inhibiting lyngbyabellins A (4)
and B (5) the cytotoxic lyngbyastatin 2 (6) and the pro-inflammatory lyngbyatoxins
A (7) and B
(8).46
Cl
C
ii '0
ir\-
0HNo
0
T\fs0HNo
0
0
0
I
s
H\Lyngbyabellin A (4)
I
H
N
Lyngbyabellin B (5)
OMe
N
0
010 01 0Y
0\
0
OH
9
OMe
Lyngbyastatin 2 (6)
OH
OH
Lyngbyatoxin A (7)
Figure IV.2. Bioactive peptides from Lyngbya
OH
Lyngbyatoxin B (8)
majuscula.
As part of our ongoing search for structurally and pharmacofogically
interesting substances from this cyanobacterium, a detailed exploration of a
Madagascan
L.
ma] uscula collection was undertaken. During this study, several
metabolites of known identity were isolated, including the cytotoxin hermitamide A7
and, rather significantly, dolastatin 16 (17), a potential antineoplastic metabolite
first reported in 1997 from D.
auricularia.8
In addition, four new compounds,
antanapeptins A-D (9-12), were discovered in the same organic extract. This
chapter describes the chromatographic isolation and structure elucidation of this
new series of L. majuscula peptides.
[11
RESULTS AND DISCUSSION
The collection of L. majuscula acquired from Antany Mora, Madagascar in
April 2000, was extracted with 2:1 CH2Cl2/MeOH and fractionated by silica vacuum
liquid chromatography (Figure IV.3).
Lyngbya majuscula
Antany Mora, Madagascar
(MAM 25/Apr 00/02)
12.8 g organic extract
VLC, Normal phase Si
Hexanes:EtOAc:MeOH
I
I
I
100%EtOAc
1:1
17:3
7:3
EtOAc/Hexanes EtOAc/Hexanes EtOAc/Hexanes
Mega-Bond
Mega-Bond
C18 Sep-Pak
Mega-Bond C18 Sep-Pak
7:3 MeOH/H20
RP HPLC 19:1 CH3CN/H20
I
hermitamide A
16.5 mg
7:3
I
4:1
C18 Sep-Pak
7:3 MeOH/H20
RP HPLC 3:1 cH3cN,H2o
dolastatin 16 (17)
11.0 mg
MeOH/H20 MeOH/H20
RP HPLC
9:1 CH3CN/H20
antanapeptin A (9)8.5mg
antanapeptin D (12)1.0mg
RP HPLC
19:1 CH3cN/H2o
antanapeptin B (10)1.2mg
antanapeptin C (11)1.0mg
Figure IV.3. Isolation scheme for the antanapeptins.
TLC and 1H NMR analyses of the crude polar fractions indicated the
presence of several structurally intriguing metabolites of peptide origin. The
fraction eluting from the VLC with 85% EtOAc in hexanes was subjected to C18
solid-phase extraction and reversed-phase HPLC to afford four new depsipeptides,
antanapeptins A-D (9-12).
13
13
/ 18
27
12
HN
10
Oo4
15
HN
00
JQ33O
0
15
R4
35
40"
"'38
38
Antariapeptin D (12)
Antanapeptin A (9) R =
Antanapeptin B (10) R =
Antanapeptin C (11) R
Antanapeptin A (9) was isolated as a pale yellow oil with a molecular
composition of C1 H60N408, as determined by HRFABMS (observed [M+H] at m/z
737.4473). Analysis of 10 and 2D NMR spectra in CDCI3 allowed construction of
six partial structures (Table lV.1, Figures lV.4-IV.7). Four standard amino acid
residues were deduced as N-methyl isoleucine (N-Melle), N-methyl phenylalanine
(N-MePhe), proline (Pro), and valine (Val), while a fifth, non-amino acid moiety
was identified as 2-hydroxyisovaleric acid (Hiv).
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
Figure IV.4. 1H NMR spectrum of antanapeptin A (9).
2.0
1.5
1.0
ppm
ppm
20
30
40
50
60
70
80
90
100
110
120
130
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
Figure IV.5. HSQC spectrum of antanapeptin A (9).
2.0
1.5
1.0
0.5
ppm
100
ppm
20
30
40
50
60
70
80
90
100
110
120
130
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
Figure IV.6. HSQC-TOCSY spectrum of antanapeptin A (9).
1.5
1.0
0.5
ppm
101
pm
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
Figure IV.7. HMBC spectrum of antanapeptin A (9).
2.0
1.5
1.0
0.5
ppm
102
Table IV.1. NMR spectral data for antanapeptin A (9) in CDCI3.
unit
Hmoya
Val
N-MePhe
position
13C
1H (J in Hz)
HSQCTOCSYa
HMBCb
1
76.7
4.87, d (10.4)
2, 3, 4
2, 3, 7, 8, 9
2
27.7
1.99, 1.60
3
25.2
1.63, 1.45
4
18.0
2.20,m
1,3,4
1,2,4
1,3
1,3,4,7
1,2,4,5
2,5,6
5
83.5
6
68.9
1.92, bit (2.6)
4
5
7
42.5
3.36, dd (7.3, 1.5)
8
1, 2, 8, 9
8
14.9
1.23,d(7.3)
7
1,7,9
9
171.3
NH
9, 10, 14
6.01, d (8.9)
10
52.4
4.40, dd (8.9, 7.3)
NH, 11, 12, 13
9, 11, 14
11
31.2
1.46,m
12
17.7
0.62,d(6.4)
10,12,13
NH, 10,11,13
10,12,13,14
10,11,13
13
18.8
0.28, d (6.4)
NH, 10, 11, 12
10, 11, 12
14
173.4
15
30.5
2.95, S
16
62.4
5.15, dd (10.5, 3.8)
17
14, 15, 17, 18,24
17
35.3
3.50, dd (14.3, 3.7)
16
16, 18, 19/20,24
10, 14, 16
3.00, dd (10.6, 3.7)
Hv
Pro
18
137.0
19/20
129.3
7.25
17, 21/22, 23
21/22
128.9
7.30
18, 19/20
23
24
25
26
27
28
29
30
127.2
7.24
19/20
170.4
77.4
5.03, d (9.2)
26, 27, 28
24, 26, 29
29.6
2.19, m
25, 27, 28
25, 27,29
17.9
0.96
25, 26, 28
25, 26,28
18.6
0.95
25, 26, 27
25, 26, 27
3.89, q (7.3)
31, 32, 33
29, 31, 32, 33
165.6
47.3
3.65, q (7.3)
N-MefIe
31
25.0
2.13, 1.97
30, 32, 33
30, 32,33
32
33
34
35
36
37
38
39
40
29.2
2.22, 1.83
30,31,33
30,31,33,34
56.9
4.99, dd (7.7, 3.9)
30, 31,32
29, 30,31,32,34,
41
170.9
172.6
33, 34, 36
28.8
3.02, s
64.2
4.17, d (10.4)
37, 39
34, 35, 38, 39, 37,
34.5
2.08, m
36, 39
36, 38, 40
15.5
0.97
36, 37
36, 40
25.6
1.49, 0.96
36, 37
36, 37,38, 40
11.2
0.96
36, 37, 39
a Proton showing TOCSY correlation to indicated proton.
correlation to indicated carbon.
Proton showing HMBC
i[i]
The sixth and final residue required the composition C9H1202 to complete
the molecular formula of 9. The 13C NMR spectrum showed two distinctive carbon
signals at 6 83.5 and 6 68.9, consistent with a terminal acetylenic functionality. As
previously observed, the carbon at 6 68.9 exhibited no HSQC correlations but
showed a
JCH
coupling of 249 Hz to a methine proton at 6 1.92 in the HMBC
HMBC correlation to the quaternary
spectrum.9
This proton also exhibited a
carbon at
83.5, confirming the presence of an acetylene.
2JCH
The remaining signals were then assembled by 2D NMR, identifying this
unit as 3-hydroxy-2-methyloctynoic acid (Hmoya), a substructure observed in the
molluscan metabolites onchidin B (23) and the kulomo'opunalides (21,
22).b0
However, Ca alkynoate moieties have also been reported recently from several
cyanobacterial metabolites, including the yanucamides (13, 14), georgamide (15)
and dragonamide (16) (Figure
lV.8).9'11'12
o
H
0
0
NH
0
N' iii
/
H
JOHO
Yanucamide A (13) R = H
Yanucamide B (14) R =
Georgamide (15)
CH3
Nk0
°
NJ
I
N
o____I
NH2
0
Dragonamide (16)
Figure IV.8. Cyanobacterial metabolites containing C8 alkynoate moieties.
104
HMBC connectivities were used to unambiguously sequence the residues
of antanapeptin A (9) (Figure IV.9), with a final ester linkage between the Hmoya
hydroxyl and the carbonyl carbon of N-Melle, completing its planar structure (Table
IV.1).
The absolute stereochemistries of 9, determined by Marfey's analysis,
included L-N-Me-lle, L-N-Me-Phe, L-Pro and L-Val.13 An L-configuration was also
confirmed for the Hiv residue by a GC-MS comparison of the corresponding methyl
ester derivative with methylated Hiv standards. However, a lack of commercially
available standards precluded the absolute configuration determination of the
Hmoya residue.
N-MePhe
Val
'
HMBC correlation
1734
N
0
6
25
0
Hiv
30.5,
5.03 1
0165.6 /
4870
T7l30
76.7\
0
0
N-Melle
fl172
302
28.8
3.8a65
2.22.1.83
29.2
Pro
Figure lV.9. Partial structure sequencing of 9 by HMBC correlations.
105
Antanapeptins B (10) and C (11) were isolated by reversed-phase HPLC
from the crude fraction containing antanapeptin A (9). Their high structural
homology to 9 was demonstrated by nearly identical 1H and 13C NMR chemical
shifts (Figure IV.1O, Table IV.2). However, both metabolites displayed obvious
differences in the Hmoya unit. While lacking the characteristic acetylene carbon
signals of 9, antanapeptin B (10) contained new methine (6 138.5,65.75) and
methylene signals (6 115.1,64.97,4.94) indicative of a mono-substituted olefin.
Similarly, the acetylenic carbons were absent from the '3C spectrum of
antanapeptin C (11) and were replaced by two additional high-field carbons at 6
22.7 and 6 14.1. These observations suggested that 10 and 11 were likely the
reduced double bond and single bond equivalents of 9, respectively.
Subsequently, 2D NMR and mass spectral data (Figure lV.11) confirmed these
assignments.
Hydrolysis and stereoanalysis of metabolites 10 and 11 were not
undertaken due to their limited quantities and the desire to preserve as much
sample as possible for biological testing. However, based on the comparable
spectroscopic and physical properties of 10 and 11 to those of 9, the three
metabolites are likely of the same enantiomeric series.
A.
B.
C.
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
ppm
Figure IV.1O. 1H NMR spectral comparison of the antanapeptins. (A) antanapeptin
A (9), (B) antanapeptin B (10), (C) antanapeptin C (11), (D) and antanapeptin D
(12).
107
Table IV.2. 1H and 13C data for antanapeptins B (10), C (11) and D (12) in CDCI3.
Antanapeptin B (9)
Antanapeptin D (11)
13C
1H
1
77.2
4.86
77.5
4.86
76.8
4.87
2
28.1
1.89, 1.35
29.0
1.89, 1.36
27.7
1.97, 1.60
3
25.0
1.65
26.3
1.39, 1.21
25.2
1.62, 1.44
4
33.3
2.35,2.04
31.7
1.26
18.1
2.21
5
138.3
5.75
22.7
1.28
83.4
497,4948
13C
6
115.1
14.1
0.87
68.8
1.92
7
42.6
3.34
42.7
3.33
42.5
3.36
8
14.7
1.21
14.8
1.21
14.9
1.23
9
171.4
NH
8
Antanapeptin C (10)
position
171.6
171.3
6.02
6.02
6.02
10
52.4
4.41
52.5
4.40
52.5
4.39
11
31.1
1.46
31.3
1.45
31.4
1.45
12
17.6
0.62
17.7
0.62
17.6
0.62
13
18.8
0.28
19.1
0.28
18.9
0.26
14
173.4
173.4
173.6
15
30.5
2.94
30.6
2.95
30.6
2.95
16
62.4
5.15
62.6
5.15
62.6
5.14
17
35.3
3.51, 3.00
35.5
3.50, 3.00
35.4
3.50, 3.00
18
19/20
136.9
129.3
7.25
129.4
7.25
129.4
7.25
21/22
128.9
7.30
129.2
7.30
129.1
7.30
23
24
25
26
27
28
29
30
127.3
7.24
127.3
7.24
127.3
7.24
137.1
137.1
170.4
170.6
170.4
77.6
5.03
77.6
5.03
77.6
5.03
29.6
2.19
29.9
2.19
29.6
2.19
18.0
0.97
18.2
0.97
18.2
0.98
18.6
0.94
18.7
0.94
18.9
0.95
165.6
165.8
165.6
47.3
3.89, 3.65
47.4
3.89, 3.65
47.4
3.90, 3.65
31
25.5
2.13, 1.98
25.3
2.13, 1.97
25.2
2.12, 1.98
32
33
34
35
36
37
38
39
40
29.2
2.23, 1.84
29.4
2.23, 1.83
29.4
2.25, 1.84
56.9
4.99
57.1
4.99
57.0
5.00
41
171.0
172.4
172.8
172.6
28.9
3.01
29.1
3.01
28.9
64.2
4.15
64.4
4.16
65.3
3.02
4.078
34.6
2.08
34.8
2.07
28.3
2.33
15.7
0.96
15.7
0.97
19.7
0.95
25.7
1.50,0.96
25.9
1.50,0.96
19.7
0.99
11.3
0.95
11.4
0.95
170.7
171.0
Proton(s) with coupling constants differing from 9: Antanapeptin B H-6, ö 4.94, d (10.5
Hz); Antanapeptin C H3-6, 50.87, t (7.2 Hz); Antanapeptin D H-36 54.07, d (10.5 Hz).
108
FOotO.
CN.S50dO.200OS100G.4dN00N07..M2
tJ0N0cM. U4C4C
Nol..
03-100
F. N..,,.
Antanapeptin A (9)
00
75..
00
7.
SO
30
1401
30
302.2
itv.r
I205.2
to
331.3
ni.
073.4
o
10500.1103
C.1MO3000á200t
FM N.e,.
(N0I. LOG.
FM TO.
MOM
.
03-10.
Antanapeptin B (10)
N
50
70
13&I
to
337.2
1t17.vl&t::1!It.,;;
ITO
35.
300
400
200
45.
700
,it .
000
OM
3000
Figure IV.1 1. Low resolution positive FABMS of antanapeptins A-D.
110*
13O
109
Figure IV.11 (Continued)
110
Antanapeptin D (12) also showed a close structural relationship to 9 (Table
IV.2). However, HRFABMS data showed an [M+HJ ion at mlz 723.4336,
indicating 12 differed from 9 by loss of a CH2 unit. 2D NMR was again used to
assemble the planar structure of 12, confirming that antanapeptin 0 (12) was
identical to 9, except for substitution of an N-methyl valine (N-MeVal) residue for
the N-Melle in 9. The absolute stereochemistries of 12 were determined in the
same manner as 9 and found to be L-N-MeVal, L-N-Me-Phe, L-Pro, L-Val and LHiv.
The antanapeptins were screened for biological activity using brine shrimp
toxicity (10
j.tglmL),14
bioassays (100
sodium channel modulation (10
tg/disk).16
j.iglmL)15
and antimicrobial
This series, however, proved inactive in all of these
assays. Similarly, the related metabolite georgamide (15),12 the major secondary
metabolite from an Australian cyanobacterial collection, was not reported to
possess biological properties. Interestingly, two other related metabolites,
yanucamides A (13) and B (14) from a Fijian collection of L. majuscula, do display
relatively potent brine shrimp toxicity
(LD50
5 tgImL).9 Thus, a specific biological
function for this structural class of compounds remains uncertain.
Dolastatin 16 (17) was isolated as a white amorphous solid (11.0 mg,
0.09% extract) following reversed-phase chromatographic purification of the VLC
fraction eluting with 100% EtOAc. The identity of this compound was established
by comparison with the reported spectroscopic and physical data, including I D
and 20 NMR, HRFABMS, and optical
rotation.8
c'0i
o"oo
Dolastatin 16 (17)
111
The recent isolation of dolastatins 3(1), 10(3), 12(2), and now 16(17),
as well as several dolastatin analogs from various microalgal collections,
demonstrates that this bioactive family of metabolites in fact originates in
cyanobacteria.1'3
Additionally, the high homology of the antanapeptins (9-12) to
the kulolides (18-20) and kulomo'opunalides (21, 22) supports a cyanobacterial
origin for these metabolites as well (Figure
IV.12).1°
R
:g
R-"7C
'D
0
K\Lro 0
N)(
s's
H
b
Kulolide-1 (18) R =
Kulolide-2 (19) R =
Kulomo'opunalide-1 (21) R = CH3
Kulolide-3 (20) R =
Kulomo'opunalide-2 (22) R = H
0
N'0)Oy
"sss
0
0
Onchidin B (23)
Figure IV.12. Marine invertebrate metabolites with structural homology to the
antanapeptins.
112
EXPERIMENTAL
General Experimental Procedures. Optical rotations were measured on
a Perkin-Elmer 243 polarimeter. IR and UV spectra were recorded on Nicolet 510
and Beckman DU64OB spectrophotometers, respectively. All NMR spectra were
recorded on Bruker DRX600 and AM400 spectrometers with the solvent CDCI3
used as an internal standard (6c 77.23
oH
7.27). Chemical shifts are reported in
ppm and coupling constants (J) are reported in Hz. Mass spectra were recorded
on a Kratos MS5OTC mass spectrometer. HPLC isolations were performed using
a Waters Millipore Lambda-Max model 480 LC spectrophotometer with Waters
515 HPLC pumps.
Cyanobacterial Collection. The marine cyanobacterium Lyngbya
majuscula (voucher specimen available as collection number MAM-25/Apr/00-01)
was collected by E. H. Andrianasolo, W. H. Gerwick, B. L. Marquez and K. E.
Milligan from shallow waters (1-3 m) in Antany Mora, Madagascar, on April 25,
2000. The material was stored in 2-propanol at -20 °C until extraction.
Extraction and Isolation. Approximately 602 g dry weight of the
cyanobacterium was extracted repeatedly with 2:1 CH2Cl2/MeOH to produce 13.5
g of crude organic extract. A portion of the extract (12.8 g) was then fractionated
by silica gel vacuum liquid chromatography. The fraction eluting with 85% EtOAc
in hexanes was further purified with a
C18
solid phase extraction (SPE) cartridge
(7:3 MeOH/H2O) and reversed-phase HPLC (9:1 CH3CN/H20, Phenomenex
Sphereclone 5
j.t
ODS) to yield 8.5 mg of antanapeptin A (9) and 1.0 mg of
antanapeptin D (12). A second fraction eluting from the
C18
cartridge (4:1
MeOH/H20) was also purified by reversed-phase HPLC (19:1 CH3CN/H20, YMCPack 5
.t
ODS-AQ) to yield 1.2 mg of antanapeptin B (10) and 1.0 mg of
antanapeptin C (11). The fraction eluting with 100% EtOAc from the Si VLC was
purified with a
C18
cartridge (7:3 MeOH/H20) and reversed-phase HPLC (3:1
CH3CN/H20, Shiseido
C18
Capcell Pak 5 i)to yield 11.0mg of dolastatin 16(17).
113
Antanapeptin A (9): pale yellow oil;
[ct]210
500 (c. 0.13, MeOH); UV
(MeOH) ?max 212 nm (log 64.5); IR (neat) 2966, 2936, 2876, 1734, 1650, 1447,
1243, 1182 cm; 1H and 13C NMR data, see Table IV.1; HRFABMS m/z[M + H]
737.4473 (calculated for C41H61N405, 737.4489).
Antanapeptin B (10): pale yellow oil;
[ct]210
-42° (c. 0.09, MeOH); UV
(MeOH) ?max 212 nm (log 64.6); IR (neat) 2964, 2931, 2874, 1734, 1651, 1447,
1241, 1183 cm1; 1H and 13C NMR data, see Table IV.2; HRFABMS m/z [M +
739.4636 (calculated for C41H63N408, 739.4646).
Antanapeptin C (11): pale yellow oil;
[a]210
-42° (c. 0.09, MeOH); UV
(MeOH) ?max 212 nm (log 64.6); IR (neat) 2963, 2932, 2873, 1734, 1651, 1450,
1242, 1183 cm1; 1H and 13C NMR data, see Table IV.2; HRFABMS m/z [M + H]
741.4815 (calculated for C41H65N408, 741.4802).
Antanapeptin D
(12):
pale yellow oil;
[a]210
-30° (c. 0.05, MeOH); UV
(MeOH)Xmax 212 nm (log c4.4); IR (neat) 2965, 2936, 2874, 1734, 1650, 1448,
1242, 1182 cm; 1H and 13C NMR data, see Table IV.2; HRFABMS m/z [M + H]
723.4336 (calculated for C40H59N408, 723.4333).
Biological Assays. The brine shrimp (Artemia sauna) toxicity assay was
performed as previously described with all samples at 10 tg/mL'4 Modulation of
the voltage-sensitive sodium channel in mouse neuro-2a neuroblastoma cells was
also examined as previously
described.15
Antimicrobial activity was evaluated
using standard paper disk/agar plate methodology against Candida albicans
(ATCC 14053), Pseudomonas aeruginosa (ATCC 10145) and Escherichia co/i
(ATCC 11775). The antanapeptins displayed no antimicrobial activity to any of the
test organisms at concentrations of 100
tg/disk'6
Marfey Analysis of Antanapeptins A (9) and D
mg of antanapeptin A (9) and 0.3 mg of antanapeptin D
(12).13
(12)
Approximately 0.8
were separately
114
hydrolyzed with 6N HCI (Ace high pressure tube, microwave, 1.0 mm). The
hydrolysates were evaporated to dryness and resuspended in H20 (100 pL). A
0.1% 1 -fluoro-2,4-dinitrophenyl-5-L-alaninamide solution in acetone (L-Marfey's
reagent, 20 tL) and 1 N NaHCO3 (10 tL) were added to a portion of each
hydrolysate, and the mixtures were heated at 40°C for I h. The solutions were
cooled to room temperature, neutralized with 2N HCI (5 jtL) and evaporated to
dryness. The residues were resuspended in H20 (50 jiL) and analyzed by
reversed-phase HPLC [LiChrospher 100
C18,
5 t, 4
x
125 mm, UV detection at
340 nm] using a linear gradient of 9:1 50 mM triethylamine phosphate (TEAP)
buffer (pH 3.1)/CH3CN to 1:1 TEAP/CH3CN over 60 mm.
The retention times (tR, mm) of the derivatized residues in the hydrolysate
of 9 matched L-VaI (28.5; 0-Val, 35.7), L-Pro (22.3; 0-Pro, 25.8), and N-Me-L-Phe
(38.4; N-Me-o-Phe, 39.3). Given that only N-Me-L-lle and N-Me-L-a//o-lle were
commercially available, the D-Marfey's reagent was synthesized and used to make
the N-Me-D-lle and N-Me-D-a//o-lle derivatives. The retention time of the
hydrolysate matched that of N-Me-L-lle (41.0; N-Me-L-a//o-lle, 41.5; N-Me-D-lle,
44.9; N-Me-o-aIio-lle, 45.5). Similarly, the retention times of the derivatized
residues in the hydrolysate of 12 matched L-Val, L-Pro, N-Me-L-Phe, and N-Me-LVal (36.4; N-Me-D-Val, 40.2)
Chiral GCMS Analyses of the Hiv Moieties in 9 and 12. Portions of
each hydrolysate of 9 and 12 were separately diluted in 50 tL MeOH and treated
with diazomethane for 10 mm. Excess CH2N2 and solvent were removed with a
stream of N2 and the residues were resuspended in CH2Cl2. Capillary GCMS
analyses were conducted using a Chiralsil-Val column (Alltech, 25 m
x
0.25 mm)
using the following conditions: column temperature held at 40 °C for 10 mm
after
injection of the sample, then increased from 40 °C to 100 °C at a rate of 3 °C/min,
then from 100°C to 150°C at a rate of 15 °C/min. The retention time of the
methylated Hiv residue in both 9 and 12 matched that of the methylated L-Hiv
standard (7.7 mm) but not the methylated D-Hiv standard (8.5 mm).
115
REFERENCES
Gerwick, W. H.; Tan, L. T.; Sitachitta, N. In The Alkaloids; Cordell, G. A.,
Ed.; Academic Press: San Diego, 2001; Vol. 57, pp 75-1 84.
2.
Harrigan, G. G.; Luesch, H.; Moore, R. E.; Paul, V.J. Spec. Pub!. - R. Soc.
Chem. 2000, 257, 126-1 39.
3.
(a) Mitchell, S. S.; Faulkner, D. J.; Rubins, K.; Bushman, F. D. J. Nat. Prod.
2000, 63, 279-282. (b) Harrigan, G. G.; Yoshida, W. Y.; Moore, R. E.;
Nagle, D. G.; Park, P. U.; Biggs, J.; Paul, V. J.; Mooberry, S. L.; Corbett, 1.
H.; Valeriote, F. A. J. Nat. Prod. 1998, 61, 1221-1225. (c) Luesch, H.;
Moore, R. E.; Paul, V. J.; Mooberry, S. L.; Corbett, T. H. J. Nat. Prod. 2001,
64, 907-91 0.
4.
(a) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.; Mooberry, S. L. J.
Nat. Prod. 2000, 63, 611-615. (b) Milligan, K. E.; Marquez, B. L.;
Williamson, R. T.; Gerwick, W. H. J. Nat. Prod. 2000, 63, 1440-1443. (c)
Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J. J. Nat. Prod. 2000,
63, 1437-1439.
5.
Luesch, H.; Yoshida, W. Y.;Moore, R. E.; Paul, V. J. J. Nat. Prod. 1999,
62, 1702-1 706.
6.
(a) Cardellina, J. H. 2nd; Marner, F. J.; Moore, R. E. Science 1979, 204,
193-195. (b) Fujiki, H.; Mori, M.; Nakayasu, M.; Terada, M.; Sugimura, T.;
Moore, R. E. Proc. Nat!. Acad. Sd. U.S.A. 1981, 78, 3872-3876. (c)Aimi,
N.; Odaka, H.; Sakai, S.; Fujiki, H.; Suganuma, M.; Moore, R. E.;
Patterson, G. M. L. J. Nat. Prod. 1990, 53, 1593-1596.
7.
Tan, L. T.; Okino, T.; Gerwick, W. H. J. Nat. Prod. 2000, 63, 952-955.
8.
Pettit, G. R.; Xu, J. P.; Hogan, F.; Williams, M. 0.; Doubek, D. L.; Schmidt,
J. M.; Cerny, R. L.; Boyd, M. R. J. Nat. Prod. 1997, 60, 752-754.
9.
Sitachitta, N.; Williamson, R. T.; Gerwick, W. H. J. Nat. Prod. 2000, 63,
197-200.
116
10.
(a) Rogelio, F.; Rodriguez, J.; Quinoa, E.; Riguera, R.; Munoz, L.;
Fernandez-Suarez, M.; Debitus, C. J. Am. Chem. Soc. 1996, 118, 1163511643. (b) Nakao, Y.; Yoshida, W. Y.; Szabo, C. M.; Baker, B. J.; Scheuer,
P. J. J. Org. Chem. 1998, 63, 3272-3280.
11.
Jimenez, J. I.; Scheuer, P. J. J. Nat. Prod. 2001, 64, 200-203.
12.
Wan, F.; Erickson, K. L. J. Nat. Prod. 2001, 64, 143-146.
13.
Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596.
14.
Meyer, B. N.; Ferrigni, N. R.; Putnam, J. E.; Jacobsen, L. B.; Nichols, D. E.;
McLaughlin, J. L. Planta Med. 1982, 45, 31-34.
15.
(a) Tan, L. T.; Okino, T.; Gerwick, W. H. J. Nat. Prod. 2000, 63, 952-955.
(b) Manger, R. L.; Leja, L. S.; Lee, S. Y.; Hungerford, J. M.; Hokama, Y.;
Dickey, R.W.; Granade, H. R.; Lewis, R.; Yasumoto, T.; Wekell, M. M. J.
AOAC Intern. 1995, 78, 521-527.
16.
Gerwick, W. H.; Mrozek, C.; Moghaddam, M. F.; Agarwal, S. K.
Experientia, 1989, 45, 115-121.
117
CHAPTER FIVE
WEWAKAZOLE, A NOVEL CYCLIC DODECAPEPTIDE FROM A PAPUA NEW
GUINEA COLLECTION OF LYNGBYA MAJUSCULA
ABSTRACT
A phytochemical study of a Lyngbya majuscula strain collected in Wewak
Bay, Papua New Guinea, resulted in the discovery of the novel peptide
wewakazole (5). Typical of
L.
majuscula peptides, 5 is a cyclic structure
comprised of several aliphatic residues including alanine, valine, isoleucine and
two phenylalanines. However, wewakazole also possesses unique peptide
features not generally observed from this cyanobacterium. Relative to typical
L.
majuscula secondary metabolites, 5 is a large compound, consisting of 59 carbon
atoms and twelve amino acid residues. Even more extraordinary, wewakazole
contains no ketide-derived portions but is solely of peptide biosynthetic origin.
Additionally, while heterocyclized threonines and serines are infrequently found,
wewakazole contains both oxazole and methyloxazole moieties. Structure
elucidation, particularly the sequencing of amino acid residues, was difficult for this
large and extensively signal-overlapped molecule. Thus, multiple one- and twodimensional NMR experiments and MS/MS data were required to assemble its
planar structure. Specifically, the application of a ID HMBC was utilized to orient
a certain three amino acid fragment that otherwise could not be confirmed by
standard spectroscopic methods. Through a combination of HPLC and GCMS
techniques, stereochemical analysis of wewakazole established that all residues
were of L-configuratiOfl.
118
INTRODUCTION
Lyngbya majuscula is best recognized for its production of lipopeptides like
lyngbyastatin 2 (1), a cytotoxic metabolite reported in 1999, from Apra Harbor,
Guam.1'2
H3CO
OH
p
OCH3
Lyngbyastatin 2 (1)
In fact, compounds of purely amino acid derivation are comparatively rare.
A small number of examples have recently been cited in the literature, including
dolastatin 3 (2), homodolastatin 3 (3) and kororamide (4), isolated from a L.
majuscula collected in a shallow water lagoon near Big Goby marine lake,
Palau.3
Kororamide is particularly noteworthy, not only because it derives solely
from peptide origins, but also for its amino acid composition. Although aliphatic
residues like leucine and isoleucine are common constituents of L. majuscula
peptides, polar residues like the serine and asparagine found in 4 are rarely
observed in marine cyanobacterial compounds.1
119
)C
N
00
HN
0
\
0NH
0NH
OLCONH2
SN)N
0
N
HNYH
CONH2
Dolastatin 3 (2) R = H
Homodolastatin 3 (3) R = CH3
0
OH
Kororamide (4)
A phytochemical study of a Lyngbya majuscula collected in 1998 from
Wewak Bay, Papua New Guinea, resulted in the discovery of a novel cyclic
peptide wewakazole (5). Though possessing some metabolic commonalities,
wewakazole also integrates several structural aspects that are unique to L.
majuscula peptide chemistry. This chapter describes the isolation, structure
elucidation and stereochemical determination of this novel secondary metabolite.
120
RESULTS AND DISCUSSION
A Lyngbya majuscula strain was collected from a shoreline growth of coral
(<1.5 m depth) in Wewak Bay, Papua New Guinea on September 5, 1998. The
extract from this cyanobacterium was chromatographed over silica gel and
resulting fractions were analyzed by TLC. Although highly pigmented, polar
fractions eluting from the VLC showed the existence of UV-active metabolites and
thus were further fractionated by LH-20 size exclusion chromatography. Proton
NMR spectra of the subsequent fractions indicated the presence of a peptidic
metabolite. Additional purification by
C18
solid-phase extraction and reversed-
phase HPLC led to the isolation of wewakazole (5) as a clear glassy oil.
23
\/
HO
36
ON
15
9NN
31
Y
41N
H
0
N
N
H HN'(J\
17-L
54
Wewakazole (5)
The 1H and 13C NMR spectra of wewakazole indicated that 5 was a large
molecule of peptide origin (Figure V.1). Twelve carbon resonances observed
between 158 ppm and 174 ppm, and a molecular composition of C59H73N12O12
determined by HRFABMS, suggested that wewakazole possessed twelve amino
acid residues.
121
9
170
160
7
8
150
140
130
120
5
6
110
100
90
4
80
70
3
60
50
ppm
2
40
30
20 ppm
Figure V.1. 1H and 13C NMR spectra of wewakazole (5).
Although a high degree of spectral overlap existed in both the proton and
carbon dimensions, twelve individual units were assembled from the HSQC,
TOCSY and HMBC data (Figures V.2-V.4). Among the deduced partial structures
were the standard amino acids glycine (Gly), alanine, (Ala), valine (Val), isoleucine
(lie), two phenylalanines (Phe) and three prolines (Pro) (Table V.1).
Interestingly, wewakazole also displayed the two methine singlets CH-18 (6c
140.8,
H
8.04) and CH-41 (6c 142.7,
H
7.78) indicative of two oxazole (Oxz)
moieties, and the methyl singlet CH3-54 (6c 12.2,
methyl oxazole residue (MeOxz).
H
2.61), characteristic of a
122
ppm
20
30
40
50
60
70
80
90
100
110
120
130
140
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
Figure V.2. HSQC spectrum of wewakazole (5).
3.0
2.5
2.0
1.5
1.0
ppm
123
ppm
1.0
tQ+
1.5
2.0
2.5
3.0
S
3.5
S
4.0
5
0
4.5
3
.
-
S
5.0
a
S
5.5
p
6.0
6.5
44.4
7.0
0
00
7.5
ft
8.0
8.5
9.0
p
*
9.5
10.0
9
8
7
6
5
4
Figure V.3. TOCSY spectrum of wewakazole (5).
3
2
1
ppm
124
ppm
I
P
20
a
30
*
a
40
ii
50
-
60
70
S
80
90
100
110
120
,
130
ci
a
* 1,
140
150
le
I*
a
I
S
9
8
160
p.
0*
170
a
7
6
5
Figure V.4. HMBC spectrum of wewakazole (5).
4
3
2
1
ppm
125
Table V.1. NMR spectral data for wewakazole (5) in CDCI3.
position
Clv
1
2
Proi
3
5
6
7
8
9
10
11
Ala
Oxzl
He
Pro3
Phe 1
12
13
14
15
8
HMBCOb
4.31, dd (17.4. 8.2)
3.52, dd (17.4. 4.3)
7.00. m
NH-i
1. 3,4
2
3
3.74. dd (8.0. 5.1)
1.57. 1.02
1.82. 0.96
3.40
5, 6
4.6. 7
3, 5,6. 8
3.6. 7
4, 5. 7
5. 6
5, 7
6
59.1
4.62.m
10.11.12
8,10
27.6
26.2
47.7
171.0
46.5
18.5
2.39. 1.86
2.09
3.85. 3.40
9, 11. 12
12
9. 10. 11
11. 13
4.79. m
1.28, d (6.9)
7.89. brs
15. NH-2
14. NH-2
14. 15
13. 15, 16. 17
13. 14
13. 14, 16. 17
8.04.s
53.1
21
37.7
25.8
11.9
16.5
5.05. dd (9.2. 6.1)
2.19
22
23
24
NH-3
25
26
27
28
29
30
33
34/38
35/37
36
NH-4
39
40
42
43
44
45
46/50
47/49
48
NH-5
Val
TOCSY
159.8
136.2
140.8
165.0
41
MeOxz
'H (J in Hz)
16
17
18
19
20
32
Phe2
172.9
61.4
28.6
24.8
47.5
171.2
NH-2
31
Oxz2
169.4
43.3
NH-i
4
Pro2
1JC
51
52
53
54
55
56
57
58
59
NH-6
9.10.12
17.19
136.9
130.5
129.2
128.0
160.7
135.8
142.7
163.6
48.8
40.8
135.7
129.7
129.0
127.7
161.3
129.0
154.0
12.2
19. 21. 22. 24.25
1.43
21. 22. 24. NH-3
20. 22. 23. 24
20. 21, 23. 24
0.87
0.93
9.34. d (9.4)
21.22
20. 21.22
21,223.24
21.22
21.22
20
20. 25
3.34
2.08. 0.92
1.62. 1.51
3.45
27.28
26.28.29
26.27.29
25. 27. 28. 29. 30
25. 26. 28. 29
26
4.68, m
3.79, dd (11.5, 3.7)
3.02. dd (11.5, 11.2)
32. NH-4
7.52. d (6.6)
7.30
7.29
7.86. d (7.8)
35/37
34/38
172.1
61.5
31.5
22.6
47.0
170.8
55.9
37.5
10.12
7.78.
20
27. 28
31
31
30. 39
30. 31. 33, 34/38
32. 35/37. 36
33, 34/38
35/37
30. 31. 32. 39
40.42
S
5.62. brq (6.9)
3.17, dd (13.5. 6.9). 3.29
44. NH-5
43. NH-5
42. 44. 45
42.43. 45. 46/50
6.90, d (7.2)
7.14. t (7.2)
7.23. d (7.2)
7.35
47/49. 48
46/50. 48
47/49
43. 44
44. 47/49, 48
45. 46/50
47/49
42. 43. 51
51. 52. 53
2.61. s
162.1
53.3
30.8
19.7
20.9
4.83. m
2.33
0.87
0.93
7.38
Proton showing correlation to indicated carbon.
57, 58. 59. NH-6
56. 58. 59. NH-6
56. 57. NH-6
56. 57. NH-6
56. 57. 58. 59
1. 55. 57
58
56. 57, 59
56. 57. 58
1. 56. 57
Optimized for 4 and 8 Hz coupling.
126
The heterocyclization of cysteine residues to form thiazole and thiazoline
rings, like those found in dolastatin 3 (2) and kororamide (4), occurs frequently in L
majuscula peptides. In fact, free reduced cysteine is practically never detected
(e.g. somocystinamide A, see chapter two). Conversely, the serine and threonine
counterparts, oxazoles and methyloxazoles, are rare to marine cyanobacteria,
more often observed in metabolites from marine macroorganisms (Figure V.5).
/N
, III,
0
HN
Cycloxazoline/Westiellamide (7)
Ceratospongamide (6)
OH C...
OCH3O
OCH3OO
1
OH
Halichondramide (8)
HO
N
N0
OCH
Figure V.5. Oxazole and methyloxazole containing metabolites from marine
macroorganisms.
For example, the phospholipase A2 inhibitor, ceratospongamide (6), was
isolated from a symbiotic red alga (Ceratodictyon spongiosum)Isponge
(Sigmadocia symbiotica) association, while the antifungal and cytotoxic
127
halichondramides (halichondramide, 8), derive from various sponge and
One particularly noteworthy metabolite, however, is the cell
cycle inhibitor cycloxazoline (7)6 First isolated from the marine tunicate
nudibranch
species.5
Lissoclinum bistratum, an identical structure, westiellamide (7), was later reported
from the terrestrial cyanobacterium Westiellopsis prolifica.7
HMBC correlations and chemical shift comparisons with literature values,
however, confirmed the presence of both the methyloxazole and two oxazole units
in the structure of wewakazole, and thus accounted for all remaining atoms in its
molecular formula. While a few other oxazole-containing metabolites have been
reported from terrestrial
(muscoride
A,1°
(raocyclamides,8
dendroamides9) and freshwater
microcyclamide11) cyanobacteria, the occurrence of oxazole and
methyloxazole residues in 5 is exceptional for marine cyanobacterial chemistry. In
addition, the presence of six, five-membered heterocyclic rings (3 prolines, 2
oxazoles, and one methyloxazole) in one cyclic peptide is unprecedented in
marine chemistry.
An 8 Hz optimized HMBC experiment was used to initiate amino acid
sequencing. Despite several proton and carbon resonances in 5 having close or
overlapping chemical shifts, four fragments accounting for all twelve amino acid
units could be constructed from this HMBC data (Figure V.6). Though each was
linked to one other residue by HMBC, neither the methyloxazole nor the oxazole
moieties showed further connectivities necessary for subsequent amino acid
sequencing, and thus, additional 2D NMR experiments were conducted.
While no new sequencing information was obtained from an HMBC
optimized for 12 Hz coupling, 4 Hz optimization showed both the 3J HMBC
correlation from NH-2 to C17 and 4J correlation from H14 to C17, linking the
alanine residue of fragment B to the oxazole of fragment C. In addition, this
experimental data more clearly distinguished between the Ala carbonyl resonance
(öc 171.0) and that of the Pro-i carbonyl (öc 171.2), allowing for connection of Pro-
I and Pro-2 and thus fragments A and B. Support for the sequencing of this
portion of wewakazole was also obtained from MS/MS analysis (Figure V.7).
128
1.57,
102
698
0
8N47
3jH
N 4.83
3.85,
/
340
62.1
1.28
738
171.2
fragment A
fragment B
O'
160.7"
136')\ 8.04
682 o
7.86
C 0
NJ(''
3.17
N 1708
"T
Ii
08,
0
5j\/5.62 N\)-
N 5.05
7.35//0
0
2.6J
9
0.92
fragment C
fragment D
Figure V.6. Partial amino acid sequencing by an 8 Hz optimized HMBC.
rm'z1114
m/z765
nVz5l4
0
'N
0
f.
Lm,z1017
m/z571
nilz 668
Figure V.7. MS/MS fragmentation of the [M-fH] parent ion m/z 1142.
129
Though no HMBC connectivities or dipolar couplings were observed for
either the oxazole or methyloxazole of fragment D, only two partial structures
remained, and thus, there appeared only one obvious direction for connecting this
cyclic peptide (Figure V.8).
,'-_
Phe-I
Phe-2
-
\
NH
MeOxz
o J,o
lie
çLN
0'
Oxz-I
N
4
--
NH
00
N
Gly
0N0
H
N
Ala
Pro-2
Pro-I
Figure V.8. Remaining structure fragments of 5, with arrows indicating the
expected ring closing linkages.
However, upon further consideration, it was realized that the orientation of
the three amino acid unit could be reversed if the proposed 3J correlation from the
C44 methylene protons to C42 ( 163.6) was actually a 4J correlation. In this
opposite orientation, all previously observed HMBC connectivities were still
plausible and therefore, alternative methods were explored to resolve this
sequencing issue.
Structure analyses of peptides containing oxazole and methyloxazole
residues have often reported this lack of HMBC correlations to oxazole moieties.
In fact, most connections through oxazoles are assumptions based on calculated
130
double bond equivalents from the molecular formula (dolastatin I, 9)12 However, a
new decoupled HMBC was recently reported to show such long-range couplings,
as demonstrated in the antibiotic promothiocin B (1O).13 Though this experiment
was applied to wewakazole, it unfortunately was not successful.
,/\ long-range correlation observed
by decoupled HMBC
No HMBC
observed
ON
H
N\)
NH
HN
HLJ:
ykri
XN
NN
N HN
oi:,co
S
NH
/
NO
H2N
0
S
Promothiocin B (10)
Dolastatin I (9)
Further literature research revealed another newly described experiment
with potential to resolve this orientation dilemma. Therefore, the 1D HMBC14 was
also employed in the structure elucidation of wewakazole (Figure V.9).
Precise irradiation of C39 ( 160.7) was difficult to achieve, and thus, the
carbon resonance at
161.3 (C51) was also irradiated. Nevertheless, only one
molecular arrangement was feasible for the observed correlations. Specifically,
couplings were seen to the protons at
and 6 2.61 (H3-54).
7.86 (NH-4), 6 7.78 (H41), 6 7.35 (NH-5),
In the originally proposed orientation A, these signals
represent two-, three- or four-bond correlations (Figure V.IOA). However, in the
converse orientation (Figure V.1 OB), enhancement of the proton at 6 7.78 would
require an unlikely six-bond coupling from the carbon resonance at 6 161.3. This
final piece of experimental evidence unequivocally resolved the amino acid
sequence of wewakazole and completed its planar structure.
131
7.5
7.0
6.5
6.0
5.0
5.5
4.5
4.0
Figure V.9. ID HMBC of 5. Irradiation of both C39 and C51 displayed couplings
to NH-4 (6 7.86), H41 (6 7.78), NH-5 (6 7.35) and H3-54 (6 2.61).
i1
A
N
o
7.86
7.78
0
10
2.61
B
cN7 OO\o
"
0
7.86
/
Q\73.78Q
2.61
Figure V.10. Possible orientations for the Meoxz-Phe-OxZ fragment in 5 based
on either a (A) 3J coupling or (B) a 4J coupling from 6H 3.29/3.17 to 6c 163.6.
132
The absolute stereochemistry of 5 was determined through a combination
of HPLC analysis using Marfey methodology and chiral GCMS analysis using N-
pentafluoropropyl isopropyl ester (PFPA) derivatives. By Marfey analysis, the
retention times of the derivatized residues in the hydrolysate of 5 matched L-Ala, LVal and L-Phe. However, the retention times for the L-lle and L-aIIo-lle standards
were identical, as were the retention times for the D-lle and D-aIIo-lle standards.
Thus, we could only conclude that the isoleucine residue of 5 was either of L- or L-
allo configuration. Additionally, though Marfey analysis of the hydrolysate
indicated the presence of at least one L-Pro residue, the underivatized Marfey
reagent eluted simultaneously with the 0-Pro standard, precluding the
stereochemical analysis of the three proline residues of 5 by this method. Thus
chiral GCMS was employed to resolve these final stereochemical issues. The
PFPA derivatized residues in the wewakazole hydrolysate confirmed an L-
configuration for the Ala, Val and Phe residues. Additionally, the lie residue of 5
was determined to be of an L-configuration and all three proline residues were
clearly shown to also possess L-stereochemistry.
° ON
o
NJyo
NNH HN(<3
o
N
N
I
0
0
Wewakazole (5)
133
Isolation and structure elucidation of wewakazole demonstrates the
enormous potential of cyanobacteria, and especially Lyngbya majuscula, to
produce novel chemistry yet to be discovered. While L. majuscula is known for its
synthesis of metabolites deriving from mixed PKS/NRPS origins, wewakazole
represents a completely new class of compound for this species, deriving solely
from amino acids and comprised of residues atypical for this marine
cyanobacterium. The presence of six heterocyclic rings in wewakazole is also
without precedent in marine-derived cyclic peptides.
134
EXPERIMENTAL
General Experimental Procedures. Optical rotations were measured on
a Perkin-Elmer 243 polarimeter. lR and UV spectra were recorded on Nicolet 510
and Beckman DU64OB spectrophotometers, respectively. All NMR spectra were
recorded on Bruker DRX600 and AM400 spectrometers with the solvent CDCI3
used as an internal standard (c 77.23
6H
7.27). Chemical shifts are reported in
ppm and coupling constants (J) are reported in Hz. FABMS and MS/MS data were
recorded on Kratos MS5OTC and Perkin-Elmer Sciex API3 mass spectrometers,
respectively. HPLC isolations were performed using a Waters Millipore LambdaMax model 480 LC spectrophotometer with Waters 515 HPLC pumps.
Cyanobacterial Collection. The marine cyanobacterium Lyngbya
majuscula Gomont (Oscillatoriaceae) (voucher specimen available as collection
number PNSB-5/Sept/98-02) was collected by B. Marquez, W. Gerwick and S.
Ketchum in Wewak Bay, Papua New Guinea, on September 5, 1998. The material
was stored in 2-propanol at -20 °C until extraction.
Extraction and Isolation. Approximately 280 g dry weight of the
cyanobacterium was extracted repeatedly with 2:1 CH2Cl2/MeOH to produce 7.5 g
of crude organic extract. A portion of the extract (5.9 g) was then fractionated by
silica gel vacuum liquid chromatography. The fraction eluting between 5% and
15% MeOH in EtOAc was chromatographed over LH-20 (4
100% MeOH. Additional purification by a
C18
x
36 cm), eluting with
solid phase extraction (SPE)
cartridge (7:3 MeOH/H20) and reversed-phase HPLC on a phenylhexyl column
(4:1 MeOH/H20, Phenomenex Luna 5
Wewakazole (5): glassy oil;
t)
yielded 6.9 mg of wewakazole (5).
[a]210
-47° (c. 0.41, MeOH); UV (MeOH) Xrnax
213 nm (log c 4.55), 221 nm (log c 4.39); IR (neat) 3371, 3275, 2961, 2932, 2878,
1635, 1514, 1452 cm'; 1H and 13C NMR data, see Table V.1; HRFABMS m/z [M +
H] 1141.5463 (calculated for C59H73N12012, 1141.5471); MS/MS fragmentation of
135
parent ion m/z 1142: 1114 (100), 1017 (28), 765 (96), 694 (40), 668 (77), 597 (50),
571 (43), 514 (27), 497 (39), 349 (44), 321 (54), 295 (57), 172 (69).
ID HMBC of Wewakazole (5)14 A total of 32K scans were acquired with
selective irradiation at C39 (6 160.7) on a 13.8 mM solution of wewakazole (6.3mg
dissolved in 0.4 mL of CDCI3). The delay for the evolution of long-range
heteronuclear coupling was optimized for 4 Hz (125 msec). The spectrum was
processed with 1 Hz line broadening.
Stereochemical Analysis of Wewakazole (5). A stream of ozone was
bubbled through approximately 1.0 mg of 5 dissolved in 2 mL of CH2Cl2 at -78 °C
until the solution turned pale blue (Ca. 1 mm). The solvent was removed under a
stream of N2, and the sample was hydrolyzed with 6N HCI at 110 °C for 14 h. A
portion of the hydrolysate was evaporated to dryness and resuspended in H20
(100 tL). A 0.1% 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide solution in acetone
(L-Marfey's reagent, 20 tL) and iN NaHCO3(10 tL) were added to the
hydrolysate, and the mixture was heated at 40 °C for 1 h. The solution was cooled
to room temperature, neutralized with 2N HCI (5 j.L) and dried. The residue was
resuspended in 1:1 DMSO/H20 (50 jtL) and analyzed by reversed-phase HPLC
[LiChrospher 100
C18,
5 t, 4
x
125 mm, UV detection at 340 nm] using a linear
gradient of 9:1 50mM triethylamine phosphate (TEAP) buffer (pH 3.1)/CH3CN to
1:1 TEAP/CH3CN over 60 mm.
The retention times
(tR,
mm) of the derivatized residues in the hydrolysate
of 5 matched L-Ala (19.7; D-Ala, 21.2), L-Val (28.4; 0-Val, 33.9), and L-Phe (36.7;
D-Phe, 40.1). However, the retention times for the L-Ile and L-a!Io-lle standards
(34.3) were identical, as were the retention times for the D-Ile and o-aIIo-lle
standards (35.8). Thus, we could only conclude that the isoleucine residue of 5
was either of L- or L-aIIo configuration. Additionally, though Marfey analysis of the
hydrolysate indicated the presence of at least one L-Pro residue (22.2), the
underivatized Marfey reagent eluted simultaneously with the D-Pro standard
136
(25.4), precluding the stereochemical analysis of the three proline residues of 5 by
this method.
The second portion of the hydrolysate was also dried under N2 and 100 jL
of a 1:5 acetyl chloride/2-propanol mixture was added. The solution was heated to
105 °C for 45 mm, dried under N2 and resuspended in 200 tL of
CH2Cl2.
Approximately 100 jtL of pentafluoropropanoic acid (PFPA) were added and the
solution was heated to 105 °C for 15 mm. After drying under N2, the derivatized
hydrolysate was resuspended in CH2Cl2 and analyzed by capillary GC-MS (Alltech
Chiralsil-Val 25 m
x
0.25 mm column, oven temperature held at 70 °C for 0.5 mm
after injection of the sample, then increased from 70°C to 180°C at a rate of 4
°C/min). The
tR
(mm) of derivatized residues in the wewakazole hydrolysate
confirmed an L-configuratiOn for the L-A!a (5.1; 0-Ala, 4.5), L-Val (6.5; D-Val, 6.1),
and L-Phe (19.5; D-Phe, 19.2) residues of 5. Additionally, the lIe residue was also
determined to be of an L-configuration
(tR
8.3, L-aJIo-He, 8.0). Chiral GC-MS
analysis, using a temperature ramp of 0.5 °Clmin, clearly demonstrated that all
three proline residues of wewakazole were also of L-stereochemistry (L-Pro, 25.7
and 0-Pro, 25.3 mm).
137
REFERENCES
1.
Gerwick, W. H.; Tan, L. T.; Sitachitta, N. Jn The Alkaloids; Cordell, G. A.,
Ed.; Academic Press: San Diego, 2001; Vol. 57, pp 75-184.
2.
Leusch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J. J. Nat. Prod. 1999,
62, 1702-1 706.
3.
Mitchell, S. S.; Faulkner, D. J.; Rubins, K.; Bushman, F. D. J. Nat. Prod.
2000, 63, 279-282.
4.
Tan, L. T.; Williamson, R. T.; Gerwick, W. H.; Watts, K. S.; McGough, K.;
Jacobs, R. J. Org. Chem. 2000, 65, 419-425.
5.
(a) Kernan, M. R.; Faulkner, D. J. Tetrahedron Left. 1987, 28, 2809-2812.
(b) Kernan, M. R.; Molinski, T. F.; Faulkner, D. J. J. Org. Chem. 1988, 53,
5014-5020. (c) Kobayashi, J.; Murata, 0.; Shigemori, H.; Sasaki, T. J. Nat.
Prod. 1993, 56, 787-791. (d) Kobayashi, J.; Tsuda, M.; Fuse, H.; Sasaki,
T.; Mikami, Y. J. Nat. Prod. 1997, 60, 150-1 54.
6.
Hambley, T. W.; Hawkins, C. J.; Lavin, M. F.; Van den Brenk, A.; Watters,
D. J. Tetrahedron 1992, 48, 341-348.
7.
Prinsep, M. R.; Moore, R. E.; Levine, I. A.; Patterson, G. M. L. J. Nat. Prod.
1992, 55, 140-142.
8.
Admi, V.; Afek, U.; Carmeli, S. J. Nat. Prod. 1996, 59, 396-399.
9.
Ogino, J.; Moore, R. E.; Patterson, G. M.; Smith, C. D. J. Nat. Prod. 1996,
59, 581-586.
10.
Nagatsu, A.; Kajitani, H.; Sakakibara, J. Tetrahedron Left. 1995, 36, 40974100.
11.
Ishida, K.; Nakagawa, H.; Murakami, M. J. Nat. Prod. 2000, 63, 1315-1317.
12.
Sone, H.; Kigoshi, H.; Yamada, K. Tetrahedron 1997, 53, 8149-81 54.
13.
Furihata, K.; Seto, H. Tetrahedron Left. 1995, 36, 281 7-2820.
14.
Meissner, A.; Sorensen, 0. W. Magn. Reson. Chem. 2001, 39, 49-52.
138
CHAPTER SIX
BIOSYNTHETIC STUDIES OF HECTOCHLORIN AND THE JAMAICAMIDES
ABSTRACT
Stable isotope incorporation experiments were used to investigate the
biosynthesis of hectochlorin (8) and jamaicamide A (12), two halogenated
secondary metabolites produced by a cultured strain of Lyngbya majuscula
originally collected in 1996 from Hector Bay, Jamaica. While both compounds
derive from mixed polyketide synthase (PKS) and non-ribosomal peptide
synthetase (NRPS) biosynthetic pathways, each is uniquely constructed from a
variety of simple metabolic precursors, creating two chemically distinct structure
classes. The C1-C14 lipid portion of jamaicamide A (12), including the brominated
acetylene moiety, is assembled from intact acetate units. Additionally, C20-C21 of
the pyrrolidone ring and C17-C18 are derived from intact acetate units while the
vinyl chloride C27 derives specifically from C2 of acetate. Both the pendant
methyl C26 and the 0-methyl C25 arise from S-adenosyl methionine (SAM) while
the remainder of the methyl pyrrolidone ring originates from S-alanine. Most
noteworthy, however, was the finding that f3-alanine is the direct precursor to the
NH through C17 portion of 12, demonstrated by a [13C3,15N]13-alanine feeding
experiment. Secondary metabolite production studies using serially diluted
bromide media have indicated that halogen ion concentration is not limiting in
jamaicamide A (12) biosynthesis. Similar to 12, the C1-C8 lipid portion of
hectochlorin (8) and C26-C27 originate from intact acetate units while the pendant
methyl C-9 derives from SAM. Feeding experiments utilizing 13C-labeled pyruvate
and 13C-labeled alanine to probe biosynthesis of the putative dihydroxyisovalerate
moieties, however, have produced inconclusive results.
139
INTRODUCTION
Contrary to terrestrial secondary metabolism, the halide rich marine
environment has facilitated the incorporation of halogen atoms into organic
structures of diverse chemotypes, many of which display valuable biological
properties (Figure Vl.1).1'2
NH2
Hoxnçj
H3N
5'-Deoxy-5-lodotubercidin (2)
Hypnea valetiae
adenosine kinase inhibitor
Halomon (1)
Portieria hornemannhi
antitumor agent
HNO
Eudistomin C (3)
Eudistoma olivaceum
antiviral agent
o
H
H
N
(jBr
Jaspamide (4)
Jaspis sp.
actin polymerization inhibitor
ci;
El
Methyl Chlorosarcophytoate (5)
Sarcophyton glaucum
cytotoxin
Figure VII. Bioactive halogenated metabolites of marine origin.
'Cl
140
While the potential medicinal development of these novel compounds (for
example, through combinatorial biosynthesis) necessitates a basic understanding
of their molecular and genetic assembly, the producing organisms are by and large
unculturable, and thus, biosynthetic origins of such intriguing molecules remain
unknown. Our laboratory, however, has been fortunate in successfully cultivating
and maintaining several marine cyanobacterial strains, thus allowing unique
secondary metabolic pathways to be probed through stable-isotope feeding
studies and genetic
manipulations.3'4
Two molecules that have been extensively examined from our culture
collections are the antimitotic curacin A (7) and the molluscicide barbamide (6),
isolated from the same strain of a Curaçao
Lyngbya majuscula.3'4
Figure VI.2
summarizes the basic biosynthetic units that comprise these bioactive agents.
Acetate was shown to form both the lipid chain and the cyclopropyl ring of curacin
A (7) and to provide the sole ketide unit of 6. Interestingly, S-adenosyl methionine
(SAM) labeled not only the N-methyl and 0-methyl carbon resonances of 6 and 7
as expected, but was additionally responsible for C-methyl synthesis on the lipid
chain of 7. This apparent cyanobacterial biosynthetic trend is also supported in
the current study (see results and discussion).5
0
/\
SAM
Phenylalanine
CH
Acetate
Leucine
V"0H
Glycine
/
Cysteine
OCH3CCI3
oxr-
H3
Glycine
\\
SAM
Cysteine
Barbamide (6)
Curacin A (7)
Figure Vl.2. Summary of the biosynthetic units forming barbamide (6) and curacin
A(7).
141
While 13C-labeled phenylalanine and leucine were successfully
incorporated into the carbon skeleton of barbamide, 13C-labeled cysteine was not a
viable precursor of the thiazole and thiazoline rings in 6 and 7, respectively.
Because cysteine and its direct metabolic precursor serine both proved highly toxic
to L. majuscula, 13C-labeled glycine was used instead as an indirect method for
establishing the biosynthetic origins of the thiazole and thiazoline
rings.6
A L. majuscula strain acquired from Hector Bay, Jamaica was found to
produce in culture both a new actin-polymerization inhibitor hectochlorin (8) and a
novel series of compounds, the jamaicamides (12-14), with jamaicamides A and C
(12) displaying sodium channel blocking activity against a mammalian
neuroblastoma cell
line.8
Planar structures of hectochlorin and the jamaicamides
were elucidated by NMR methods and mass spectral data. The three-dimensional
structure of hectochlorin was deduced by X-ray crystallography and the Sstereochemistry of C23 in jamaicamide A was established by Marfey analysis.
While hectochlorin is analogous to the lipopeptides dolabellin
(9)9
and the
lyngbyabellins (1 0,11)10 isolated from Dolabella auricularia and L. majuscula,
respectively, the jamaicamides are a chemically distinct group of molecules
bearing a slight structural resemblance to the malyngamide family of L. majuscula
metabolites (see chapter three).11
Isolation of hectochlorin and the jamaicamides from a cultured organism
provided the unique opportunity to explore their biosynthesis, particularly the
origins of structurally intriguing elements such as the bromoacetylene and methyl
pyrrolidone of jamaicamide A (12) and the putative dihydroxyisovalerates of
hectochlorin (8). This chapter describes the stable-isotope feeding experiments
and altered growth media studies used to probe our biosynthetic hypotheses of
these two significant molecules. Although both compounds appear to originate
from mixed biosynthetic origins, possessing ketide and amino acid-derived
portions alike, their subunit composition and molecular assembly differ significantly
between the structure classes. As such, their biosyntheses are subsequently
described as independent projects.
142
ct
CI
,,
os
Nç..O
0es
NOH
00H
'"0
0
00
-\
HC<
S
DolabeIlin (9)
Hectochlorin (8)
cI
CI
"0
0HNo
=o
0<NJJ
j s
N
H'\
H0
Lyngbyabellin A (10)
N
HF
s-
Lyngbyabellin B (11)
HfNA(JTA
CI
0
0
0
Jamaicamide A (12)
HN)(C#
HN)(C
0
Jamaicamide B (13)
0'__
N
0
0
Jamaicamide C (14)
Br
143
RESULTS AND DISCUSSION
Biosynthetic Hypotheses of Jamaicamide A (12)
Initial inspection of the jamaicamide A structure suggested molecular
assembly begins with a polyketide chain (Cl-C 14) deriving from seven intact
malonyl-CoA-derived acetate units (Figure Vl.3). Based on results obtained from
curacin A biosynthetic studies (see
above),3'5
the branching methyl carbon C26
was postulated to derive from S-adenosyl methionine (SAM) rather than from a
C1O-C9-C26 propionate unit.
0
Acetate = u
V'OH
SAM
\25
H3C,0
Alanine\ 24
CH3
22
20
-alanine
26
SAM
0
Jamaicamide A (12)
Figure VL3. Proposed biosynthetic composition of jamaicamide A (12).
However, in contrast to the origin of C26, C27 was hypothesized to
originate from C2 of acetate. Similar proposals have also been suggested in the
biosynthesis of both
oncorhyncolide12
and
virginiamycin.13
This HMG CoA
synthase-like mechanism is thought to involve addition of a malonyl CoA-derived
acetate to a C6 keto-carbon. The -hydroxyacyl acid intermediate would then be
decarboxylated and halogenated to form the vinyl chloride.
144
OCH3
O
N
0
0
HO
o
Dolastatin 15 (13)
N
/
0
Puketeimide A (14)
OCH3
-
OH
0
OAc
0
Majuscutamide D (15)
Figure Vl.4. Pyrrolidone-containing metabolites from marine sources.
The methyl pyrrolidone moiety of 12 was conceived to derive from a
Claisen-like condensation and cyclization of an acetate unit (C20-C21) with Salanine (C22-C24 and the tertiary nitrogen). Various pyrrolidone-containing
metabolites have been isolated from marine organisms (Figure
similar biosynthetic schemes have been proposed in the
Vl.4),14'6
literature.17
and
However,
only the lactam antibiotic andrimid (16), produced by fermentation of
Pseudomonas fluorescens obtained from an unidentified Alaskan tunicate, has
been probed experimentally. Feeding studies of 16 utilizing [1,2-13C2]acetate and
[13C2,15N]glycine support this hypothesis for pyrrolidone
formation.18
[13C1 5Nglycine
NH
H
N
H
Andrimid (16)
010
[1 ,21 3C2jacetate
145
A major point of interest in jamaicamide biosynthesis is the origin of the
NH-C17 portion of 12. Structurally, this unit appears to arise from 13-alanine, and
other biosynthetic schemes based on the utilization of this amino acid in
cyanobacteria have been proposed (see chapter three). However, no previous
studies have demonstrated the incorporation of 3-alanine in marine cyanobacterial
secondary metabolism. To fully explore the biosynthetic origin of the carbon
atoms within 12, various isotopically labeled precursors were supplied to the
producing organism.
[13CJ-Acetate Feeding Experiments of Jamaicamide A
Two initial isotope feeding experiments, [1-13C]acetate and [2-13C]acetate,
were separately conducted as described in the experimental section of this
chapter. The 13C NMR spectrum of jamaicamide A isolated from the [1-13C]acetate
feeding (compared to natural abundance jamaicamide A, Figure Vl.5A) showed
that carbons 2,4,6,8, 10, 12, 14, 19, and 20 all arise from Cl of acetate (Figure
VI.5B, Table Vl.1). Similarly, analysis of the 13C NMR spectrum from the [2-
13Cjacetate experiment established that carbons 1,3,5,7,9, 11, 13, 18,21 and
importantly, C27, are all derived from C2 of acetate (Figure Vl.5C, Table VI.1).
Next, a doubly labeled [1 ,2-13C2]acetate feeding experiment was performed
to observe integration of intact acetate units into the jamaicamide backbone
(Figure Vl.6). Although a high degree of background incorporation from acetate
degradation was observed, analysis of the coupling constants determined that
intact acetates were incorporated into carbon pairs C1-C2 (Jcc = 170.7 Hz), C3-C4
(Jcc = 33.9 Hz), C5-C6 (Jcc = 43.3 Hz), C7-C8 (Jcc = 33.3 Hz), C9-C10 (Jcc = 40.3
Hz), C11-C12 (Jcc = 43.3 Hz), C13-C14 (Jcc
49.7 Hz), C18-C19 (Jcc = 71.9 Hz)
and C20-C21 (Jcc = 63.1 Hz) as predicted in our original hypothesis. Results from
the above three experiments were confirmed by comparison with the labeling
patterns observed in jamaicamide B and clearly demonstrate that, starting from
Cl, seven intact acetate units are linearly assembled to form the lipid portion of
jamaicamide A. In addition, C27 derives from C2 of acetate while carbons C18C19 and C20-C21 are also incorporated as intact acetate units.
146
A.
13 9 716/2 26
10
22
1714
6
I
2019
1121
27
II
I
18
I
HH
2
I
i
2325 1,15&/'/4 '3
I24
I
II
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
ppm
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
ppm
C.
21
95
18
13
11
27
j
I
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
ppm
Figure Vl.5. 13C NMR spectra of (A) natural abundance jamaicamide A, (B)
jamaicamide A isolated from cultures provided with [1-13C]acetate and (C)
jamaicamide A isolated from cultures provided with [2-13C]acetate. Bolded/
italicized numbers indicate significantly enriched carbon resonances (see Table
Vl.1).
147
10
i21
22
63.1
J76a1719Li
170
175
165
160
150
155
140
145
135
ppm
130
27
25
110
105
100
95
90
85
80
75
70
ppm
60
65
24
15
512
13
H
8
716
39 38 37 36 35 34 33 32 31
3
26
30 29 28 27 26 25 24 23 22 21
i
20 19
18
ppm
Figure V16. 13C NMR spectrum of jamaicamide A (12) isolated from cultures
provided with [1 ,2-13C2]acetate. Coupling constants for the intact acetate units
incorporated in 12 are given in Hz.
148
S-[Methyl-13C]Methionine Feeding Experiment of Jamaicamide A
Similar to results obtained from studies with curacin A (7) and barbamide
(6), we predicted that both the 0-methyl carbon C25 and the branching methyl
group C26 originate from methionine via S-adenosyl methionine (SAM). Analysis
of the 13C NMR spectrum of jamaicamide A isolated from a S-[methyl-
13C]methionine feeding experiment revealed that both C25 and C26 arise from the
Cl pool via SAM, displaying incorporation enhancements of 2.6 fold and 2.5 fold,
respectively (Figure Vl.7, Table Vl.1).
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
ppm
Figure Vl.7. 13C NMR spectrum of jamaicamide A isolated from cultures provided
with S-[methyl-13C]methionine. Bolded/italicized numbers indicate significantly
enriched carbon resonances (see Table Vl.1).
149
Table Vl.1. Relative enhancements of carbon resonances from jamaicamide A
biosynthetic feeding experiments. Methods for quantitation are detailed in the
experimental section of this chapter.
position
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Chemical
[1-13C ]
[2-13C]
[CH3-'3C1
[1-13CJ
[3-13C]
[1-13C]
shift (ppm)a
acetate
acetate
methionine
alanine
alanine
pyruvate
38.1
1.0
1.7
0.9
1.5
1.0
1.6
0.9
1.9
1.0
1.6
1.1
1.2
0.9
1.0
1.3
1.1
0.6
1.5
0.9
0.9
2.0
0.9
1.4
1.0
1.0
1.2
1.0
1.0
0.9
1.0
1.0
1.0
1.0
0.9
1.0
0.8
0.9
0.9
1.0
0.9
0.9
0.9
0.9
0.9
1.0
0.9
79.8
19.5
25.8
29.2
141.7
32.5
34.7
36.2
136.5
127.5
28.5
36.6
172.4
2.0
0.9
2.0
2.0
0.9
1.8
1.0
1.1
2.0
2.1
38.1
1.1
1.0
1.3
32.3
175.3
94.9
165.9
169.9
125.7
1.1
153.1
1.1
58.0
17.8
56.0
20.7
11.7
1.0
1.3
1.3
0.9
1.0
1.0
1.0
1.0
1.0
0.9
1.0
0.9
0.9
1.0
1.0
1.1
1.1
1.1
1.0
1.0
0.9
1.0
1.1
1.2
1.1
1.1
1.1
2.3
0.9
0.9
2.4
1.0
1.0
1.3
1.0
1.1
1.0
1.0
1.8
1.7
1.1
1.1
1.1
1.0
1.2
1.2
1.0
1.7
1.4
1.0
1.5
0.9
1.3
1.0
1.4
1.0
1.5
1.0
1.5
0.9
1.0
1.1
1.1
0.9
0.9
1.0
1.5
1.1
0.8
0.7
1.5
1.1
1.0
1.1
1.0
0.9
3.2
2.6
2.5
1.1
1.1
1.0
1.0
1.1
1.0
1.5
0.9
0.9
1.0
0.9
1.0
1.0
0.9
1.0
1.1
2.0
0.9
0.9
1.1
1.0
1.0
Referenced to CDCI3 centerline, 77.0 ppm. Bolded/italicized numbers indicate 'C
enrichment.
8
S-[13CJAIanine Feeding Experiments of Jamaicamide A
To examine the C22-C24 origin of the jamaicamide pyrrolidone ring, both
S-[1-13C]alanine and S-[3-13C]alanine were separately provided to the jamaicamide
producing L. majuscula. Analysis of the 13C NMR spectra of isolated jamaicamide
A revealed a 2.0-fold enhancement of C22 from the S-[1-13C]alanine feeding
(Figure Vl.8A) and a 3.2-fold enhancement of C24 from the S-[3-13C]alanine
feeding (Figure Vl.8B), supporting our hypothesis that S-alanine is the precursor of
C22-24 (and the tertiary nitrogen) in the jamaicamides.
150
A.
22
170
160
150
140
130
120
110
90
100
80
70
60
50
40
30
ppm
24
B.
1121
170
160
150
140
130
27
120
110
18
100
90
80
70
60
50
40
30
ppm
Figure Vl.8. 13C NMR spectra of (A) jamaicamide A isolated from cultures
provided with S- [1 -13C]alanine and (B) jamaicamide A isolated from cultures
provided with S-[3-13C]alanine. Bolded/italicized numbers indicate significantly
enriched carbon resonances (see Table VI.1).
The 13C NMR spectrum of jamaicamide A isolated from the S-[3-13C]alanine
experiment displayed additional enrichment at carbon resonances 1, 3, 5, 7, 9, 11,
13, 18, 21 and 27 (Table Vl.1). Pyridoxal phosphate-dependent transamination of
alanine results in formation of pyruvate which subsequently undergoes
decarboxylation to produce acetate. As shown in Figure Vl.9, a 13C-labeled
carbon at position 3 of alanine is converted into position 2 of acetate, thus
providing a source of [2-13C]acetate for incorporation into all acetate-derived
subunits of jamaicamide A.
151
glutamate
a-ketoglutarate H2N_t_OH
alanine
pyruvate
acetate
hydroxyethyl-TPP
pyruvate
TPP
HoLJL
2-acetolactate
valine
2,3-dihydroxyisovalerate
Figure Vl.9. Metabolic pathways of alanine and pyruvate. The black circle and
black diamond indicate the location of a 13C carbon labeled in the 1-position or 3position of alanine/pyruvate, respectively.
Based on this biosynthetic relationship, a feeding experiment utilizing
[1-13C]pyruvate was anticipated to label the Cl position of all acetate units in 12.
Interestingly though, no enrichment of any resonances in jamaicamide A was
observed from this feeding (Table Vl.1). Thus the biosynthetic conversion of
exogenously supplied pyruvate to acetate appears to dominate over the anabolic
formation of alanine.
[13C3,
15N]/3-A!anine Feeding Experiment of Jamaicamide A
To directly evaluate whether -alanine is incorporated into the NH-C17
portion of 12, [13C3,15N]-alanine was fed to the jamaicamide producer. Analysis of
the 13C NMR spectrum of jamaicamide A (Figure Vl.1O) showed intact
incorporation of the nitrogen and all three isotopically-labeled carbons, C15-17,
with coupling constants of Jc15N = 10.5 Hz, Jc15c16 = 34.5 Hz and Jc16-c17 = 47.9
Hz. These results unequivocally confirm that p-alanine is the precursor for this
region of jamaicamide A.
152
47.9 Hz
34.5 Hz
47.9 Hz
10.5 Hz
r1i
ppm
176
Cl 7
ppm
38.5
C15
ppm
33
C16
Figure VilO. Expansions of the 13C NMR spectrum of jamaicamide A isolated
from cultures provided with [13C3,15N]3-alanine. Coupling constants are shown to
indicate intact incorporation of the labeled precursor.
Bromide Ion Concentration Studies
As the L. majuscula cultured in standard artificial seawater produces both
the brominated jamaicamide A (12) and its debrominated equivalent, jamaicamide
B (13), in nearly the same quantity, bromide ion could be the limiting factor in
jamaicamide A production. To explore the role of bromide concentration on
metabolite synthesis, cultures were grown in media containing varying amounts of
bromide ion (see experimental). Interestingly, media supplemented with 10-fold or
100-fold excess sodium bromide had no effect on jamaicamide composition
compared to controls. An artificial seawater control completely depleted of
bromide ion was also prepared. As anticipated, the quantity of brominated
metabolite proportionally declined while that of the debromo- compound increased
(Figure VI.1 1). These results demonstrate that bromide ion does not limit
production of jamaicamide A at naturally occurring halogen concentrations in
seawater.
153
A. 100-fold bromide ion
hectochiorin
jamaicamide B
jamaicamide A
Minutes
Figure VI.1 1. Qualitative HPLC comparison of jamaicamide production in (A)
100-fold bromide ion media, (B) normal bromide ion control and (C) bromide ion
depleted media.
Biosynthetic Hypotheses of Hectochlorin (8)
Structural examination of hectochiorin also suggested that molecular
assembly initiates from an acetate-derived Cl -C8 polyketide chain (C26-C27 was
also postulated to derive from intact acetate) with the pendant methyl carbon C9
arising from SAM. The remainder of the molecule was hypothesized to derive
from amino acid origins; specifically, cysteine was proposed to form the two
thiazole moieties, C10-C12 and C18-C20, as observed in barbamide and curacin
A biosynthesis, while dihydroxyisovalerate, a precursor in valine biosynthesis, was
proposed to form both the C13-C17 and C21-C25 units of hectochlorin (Figure
Vl.12).
154
Acetate
Cysteine
CI\q
/10
(
H3C-1
V'OH
27
I
0
0
O%O
SI
16
C
0
Dihydroxyisovalerate
24..
HO
S
25
17\
Dihydroxy-
isovalerate
20
Cysteine
Hectochlorin (8)
Figure Vl.12. Proposed biosynthetic composition of hectochlorin (8).
13C Acetate Feeding Experiments of Hectochiorin
[1-13C]acetate, [2-13C]acetate (Figure Vl.13) and the doubly labeled [1,2-
13C2]acetate (Figure Vl.14) were separately supplied to the cultures of L.
ma] uscula, and hectochiorin was isolated from each feeding experiment by VLC
and HPLC (see experimental). Analysis of the [1 ,2-13C2]acetate coupling
constants (Figure VLI4) determined that intact acetate units were incorporated
into carbon pairs C1-C2 (Jcc
57.9 Hz), C3-C4 (Jcc = 38.6 Hz), C5-C6 (Jcc = 36.8
Hz), C7-C8 (Jcc = 40.2 Hz) and C26-C27 (Jcc = 60.2 Hz). As predicted, the 13C
NMR spectrum of 8 isolated from the [1-13Cjacetate feeding showed enhancement
of carbons resonances 1, 3, 5, 7, and 26 while the [2-13C]acetate experiment
showed enhancement of carbon resonances 2, 4, 6, 8 and 27, compared to natural
abundance hectochlorin (Figure Vl.13, Table Vl.2).
155
160
170
t V
'-
1___i.jt
150
140
130
120
110
V t
-
100
90
VVVt
U_J .__
L-.
80
70
60
40
50
ppm
30
5
B.
26
LL
170
160
ii
.
150
140
130
120
110
100
90
80
70
60
40
50
ppm
30
27
8
C.
6
2
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
ppm
Figure Vl.13. '3C NMR spectra of (A) natural abundance hectochlorin, (B)
hectochlorin isolated from cultures provided with [1 -13C]acetate and (C)
hectochiorin isolated from cultures provided with [21 3C]acetate. Bolded/italicized
numbers indicate significantly enriched carbon resonances (see Table Vl.2).
26
570i31j1JJ
170
160
165
155
145
150
135
140
22
23
3
I
ppm
130
7
14
40.2
91
90
38.6
15
89
88
87
86
85
84
83
82
81
80
79
78
77
76
75
74
73
ppm
72
5, 27
24
8
6
9
2
36.8
50
40.2
36.8
I
45
40
35
30
25
20
ppm
Figure Vl.14. 13C NMR spectrum of hectochlorin (8) isolated from cultures
provided with [1 ,2-13C2]acetate. Coupling constants for the intact acetate units
incorporated in 8 are given in Hz.
157
Table Vl.2. Relative enhancements of carbon resonances from hectochiorin
biosynthetic feeding experiments. Methods for quantitation are detailed in the
experimental section of this chapter.
chemical
position
1
2
3
4
shift (ppm)a
173.0
42.6
75.1
7
8
9
30.9
20.8
49.3
90.4
37.2
15.0
10
161.1
11
147.0
128.5
166.2
74.7
81.9
24.4
21.9
160.4
147.4
127.7
165.2
77.9
71.6
26.7
25.8
168.7
20.8
5
6
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
[2-13cJ
[cH3-'3c]
[1-13c]
[3-13c]
[1-'3c1
acetate
acetate
methionine
alanine
alanine
pyruvate
1.9
1.0
1.7
1.0
0.8
2.6
0.9
1.2
1.0
1.0
0.9
2.0
1.0
1.0
1.2
1.9
0.7
1.0
1.0
0.7
1.1
0.9
1.0
1.1
2.8
1.0
1.2
2.2
1.4
1.1
1.1
1.2
1.4
1.0
3.2
1.3
1.3
1.0
0.9
1.0
1.1
1.1
0.9
0.7
0.9
0.9
3.5
0.9
0.8
0.6
0.6
0.8
0.7
0.8
0.7
0.6
1.9
1.2
2.0
1.2
0.9
0.9
1.4
1.0
0.9
1.2
1.2
0.9
1.3
0.9
1.6
1.0
1.3
1.2
1.2
1.6
0.7
0.8
0.6
1.0
0.7
1.4
1.3
0.7
1.4
0.9
0.7
1.2
1.0
2.1
1.0
1.0
[1-13C I
1.4
2.2
1.0
1.5
0.9
0.9
1.5
1.4
1.0
1.5
0.8
1.0
0.8
0.7
1.2
1.0
0.8
1.1
1.3
0.9
0.8
0.8
0.8
1.5
1.7
1.3
1.8
2.8
1.2
0.9
1.1
1.2
1.6
4.1
1.3
0.7
0.7
1.0
0.9
1.1
0.7
1.0
1.2
1.8
1.1
0.9
1.9
1.0
1.0
1.3
1.1
0.9
1.2
0.8
1.2
0.9
1.1
0.9
1.2
1.5
2.6
°
Referenced to CDCI3 centerline, 77.0 ppm. Bolded/italicized numbers indicate 'C
enrichment.
S-[Methyl-13C]Methionine Feeding Experiment of Hectochiorin
Analysis of the 13C NMR spectrum of 8 isolated from the S-[methyl13C]methionine feeding experiment revealed that only C9 arises from SAM (Figure
Vl.15, Table Vl.2).
158
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
ppm
Figure V1.15. 13C NMR spectrum of hectochiorin isolated from cultures provided
with S-[methyl-13C]methionine. Bolded/italicized numbers indicate significantly
enriched carbon resonances (see Table VI.2).
13C Labeled Pyruvate and Alanine Feeding Experiments of Hectochiorin
Although the C1O-C12 and C18-C20 units of hectochlorin were proposed to
derive from dihydroxyisovalerate (DHiv), a precursor in valine biosynthesis,
verification of this hypothesis proved exceptionally difficult. Isotope-labeled DHiv
substrates are not commercially available, and isotope-labeled valine could not be
used, as the anabolic and catabolic pathways are not reversible (Figure Vl.9). In
addition, this significantly polar precursor may not be favorably transported from
the media into cyanobacterial cells if labeled compound was synthetically
accessible. Therefore, an exploration of alternative routes was undertaken.
As shown in Figure VI.9, DHiv ultimately derives from the condensation of
two pyruvate units followed by reduction and isomerization. Furthermore, alanine
is catabolized to acetate via pyruvate, as seen in the jamaicamide biosynthetic
studies described above. Thus, hectochlorin was isolated from both the S-El13C]alanine and S-[3-13Cjalanine feedings and from a [1-13Cjpyruvate feeding
experiment. 13C NMR spectral data are presented in Figure VI.16 and Table VI.2.
Unfortunately, results from these feedings were inconclusive. For example, both
the S-[1-13C]alanine and [1-13C]pyruvate experiments were expected to label the
Cl 3 and C21 carbon resonances of hectochiorin. While a slight enhancement was
159
observed for these resonances from S-[1-13C]alanine, only C13 appears enriched
by [1-13C]pyruvate. Likewise, S-[3-13C]alanine was expected to label all positions
corresponding to C2 of acetate (carbons 2, 4, 6, 8 and 27) as well as carbon
resonances 16, 17, 24 and 25 of the DHiv moieties. While the C2-acetate carbons
did show enhancement, only one of the four carbons of DHiv (C24) appears
enriched. Additionally, C26 unexplainably appears enhanced in the S-[l13C]alanine, S-[3-13C]alanine and [1-13C]pyruvate feeding experiments. Thus no
consistent or reliable deductions could be drawn from the alanine and pyruvate
data.
160
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
ppm
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
ppm
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
ppm
Figure VLI6. 13C NMR spectra of (A) hectochlorin isolated from cultures provided
with [1-13C]pyruvate, (B) hectochiorin isolated from cultures provided with S- [113C]alanine and (C) hectochiorin isolated from cultures provided with S-[313C]alanine. Bolded/italicized numbers indicate significantly enriched carbon
resonances (see Table Vl.2).
161
Conclusions
The feeding studies described above provide insight into the biosynthetic
assembly of the jamaicamide and hectochlorin classes of marine natural products.
As observed in the 13C NMR spectra of jamaicamide A (12) isolated from [113C]acetate, [2-13C]acetate and [1 ,2-13C2]acetate feedings, the C1-C14 lipid portion
of 12, including the brominated acetylene, derives from a linearly assembled
heptaketide chain of intact acetate units. In addition, C27 originates from C2 of
acetate, likely forming the vinyl chloride functionality via an HMG CoA synthasetype reaction followed by decarboxylation and halogenation. The methoxy carbon
C25 and the lipid chain methyl C26 both occur from SAM methylation. While the
extent of propionate utilization in marine cyanobacteria is unknown, biosynthetic
trends supported in this study demonstrate that pendant methyls bonded to Cl of
acetate derive from an acetate C2 carbon, whereas methyls attached to carbons
from C2 of acetate derive from SAM.
The pyrrolidone ring of jamaicamide A is comprised of a single acetate unit
condensed with S-alanine, as shown by 13C enhancements observed in the [113C]alanine, [3-13CJ-alanine and [1 ,2-13C2]acetate feeding studies. A -alanine
biosynthetic precursor to the NH-C17 section of 12 has been unequivocally
established by coupling patterns observed in the 13C NMR spectrum of
jamaicamide A isolated from cultures provided with exogenous [13C3,15N]3-alanine.
Acetate feeding experiments also demonstrated that the Cl -C8 ketide
portion of hectochlorin and the C26-C27 unit derive from intact acetates while the
pendant methyl C9 derives from SAM. As 13C-labeled glycine feeding experiments
have previously been used as an indirect method for establishing the biosynthetic
precursor of thiazole rings (cysteine and its direct metabolic precursor serine both
proved highly toxic to L. majuscula), origins of the C1O-C12 and C18-C20 sections
of hectochlorin were not investigated. The biosynthesis of the DHiv units were
examined by S-[1-13Cjalanine, S-[3-13C]alanine and [1-13Cjpyruvate feeding
experiments. Unfortunately, the outcome from these latter feedings was
inconclusive.
ir;
EXPERIMENTAL
General Experimental Procedures. All NMR spectra were recorded in
CDCI3 on a Bruker AM400 NMR spectrometer operating at a 13C resonance
frequency of 100.61 MHz. Carbon chemical shifts are reported in ppm and
referenced relative to CDCI3 at öc 77.0. TLC grade (10-40 m) silica gel was used
for vacuum chromatography, and Merck aluminum-backed TLC sheets (Silica gel
60 F2) were used for TLC. HPLC was performed using a Waters model 480 LC
photodiode array spectrophotometer with Waters 515 HPLC pumps. All
chromatography solvents were purchased as HPLC quality, and all stable isotope
substrates and deuterated NMR solvents were purchased from Cambridge
Isotope, Inc.
General Culture Conditions and Isolation Procedure for Isotopelabeled Feeding
Studies.19
Approximately 2-3 g (total) of L. majuscula strain
JHB-22/Aug/ 96-0 1 C2 were inoculated into multiple Erlenmeyer flasks containing
SWBG1 1 medium. Cultures were grown at 28° C under uniform illumination (4.67
tmol photon s m2), aerated and equilibrated three days prior to the addition of
isotopically labeled precursors on days 3, 6, and 8 (substrate was added only on
days 6 and 8 for the [1 -13C]pyruvate feeding). The L. majuscula was harvested 10
days after inoculation, blotted dry, weighed, and repetitively extracted with 2:1
CH2Cl2/MeOH. The filtered lipid extracts were dried in vacuo and weighed, then
applied to silica gel columns (approximately 1.5 x 4.5 cm) and eluted with a
stepped gradient from 50% EtOAc/hexanes to 25% MeOH/EtOAc. The fractions
were analyzed by TLC for hectochiorin and jamaicamide content. Fractions eluting
with 85% EtOAc/hexanes and 100% EtOAc were recombined and purified by
reversed-phase HPLC (17:3 MeOH/H20) using a Phenomenex Sphereclone 5 tm
ODS column (detection at 216 and 254 nm) to yield pure hectochlorin and
jamaicamides A-C. For each feeding experiment, compound purity was
determined by HPLC, 1H and 13C NMR.
163
Culture Conditions and Isolation Procedure for Bromide Ion
Concentration Studies. Approximately I g of L. majuscula strain JHB-22/Aug/
96-01 C2 was inoculated into each of six 2.5 L Erlenmeyer flasks containing 1.5 L
of SWBG1 1 medium. Two flasks were maintained as controls for the bromide
concentration studies. As the salt formula of Instant Ocean® is proprietary, MBL's
trace mineral solution
recipe20
was used to estimate the bromide ion concentration
of the artificial seawater in the SWBG11 medium; 10-fold NaBr content was then
added to each of two flasks, and 100-fold NaBr content was added to each of the
final two flasks. For the bromide ion depleted medium, Instant Ocean® solution
was replaced by MBL trace mineral solution prepared without sodium bromide. A
MBL medium (containing sodium bromide) was also prepared to ensure culture
viability in this altered artificial seawater. Two 2.5 L flasks containing 1.5 L of MBL
medium and two additional 2.5 L flasks containing 1.5 L of bromide-depleted MBL
medium were also inoculated with approximately 1 g of L. majuscula strain JHB22/Aug/ 96-01C2. All cultures were grown with aeration under the identical light
and temperature conditions specified above. Cultures were harvested on day 28,
and extracted and chromatographed in the same manner as previously described
in the general culture conditions and isolation section. Extracts were examined
qualitatively by RP HPLC using the identical chromatographic procedure described
above.
Quantitative Calculation of 13C Enrichments from Precursor Feeding
Experiments. The relative 13C enrichments from exogenously supplied
isotopically labeled precursors were calculated as follows. All 13C NMR spectra
were recorded using inverse-gated decoupling and processed with 1.0 Hz line
broadening (zgig Bruker pulse program). For each experiment, carbon resonance
intensities for the natural abundance and enriched sample were tabulated and an
average normalization factor was calculated from carbon resonances expected to
be unlabeled. Carbons 16, 22, and 26 were averaged for all jamaicamide A
experiments, with the exception of the S-[methyl-13C]methionine feeding (C24
replaced C26) and the S-[1-13C]alanine feeding (C21 replaced C22). Carbons 14,
19, 22 and 24 of hectochlorin were averaged for the [1-13C]acetate, [2-13C]acetate
164
and S-[methyl-13C]methionine feedings, while carbons 1,4, 12 and 19 were
averaged for the [1-13C]pyruvate, S-[1-13C]alanine and S-[3-13Cjalanine feedings.
An average normalization factor was calculated by dividing the average intensity of
the selected resonances from the natural abundance spectrum by the average
intensity of the same selected resonances from the 13C-enriched spectrum. All
resonances in the 13C-enriched spectrum were then multiplied by this
normalization value and rounded to the nearest tenth.
Although carbon resonances 5 and 27 of hectochlorin reside at an identical
chemical shift (c 20.8), the doubly labeled acetate feeding experiment showed
that C5 and C27 derive from Cl and C2 of acetate, respectively. Thus, intensities
from the singly labeled acetate feedings and the S-[3-13C]alanine could be used to
quantitate their enrichments.
Sodium (1-13C]Acetate Feeding. [1-13C]acetate (150 mg total) was
provided to 3 x 600 mL cultures on days 3, 6, and 8, and the cultures were
harvested on day 10 (3.24 g wet wt., 0.34 g dry wt., 63.5 mg organic extract). A
total of 1.4 mg of 8 and 1.4 mg of 12 were isolated from VLC and HPLC
purification of the crude extract. The 13C NMR spectral data are presented in
Figures Vl.5b and Vl.13b, and relative enrichments are summarized in Tables Vl.1
and Vl.2.
Sodium [2-13C]Acetate Feeding. [2-13Cjacetate (150 mg total) was
provided to 3 x 600 mL cultures on days 3, 6, and 8, and the cultures were
harvested on day 10 (3.64 g wet wt., 0.29 g dry wt., 58.2 mg organic extract). A
total of 0.9 mg of 8 and 0.8 mg of 12 were isolated from VLC and HPLC
purification of the crude extract. The 13C NMR spectral data are presented in
Figures Vl.5c and Vl.13c, and relative enrichments are summarized in Tables Vl.1
and Vl.2.
165
Sodium [1 ,2-13C2]Acetate Feeding. [1 ,2-13C2jacetate (160.8 mg total) was
diluted 1:2 with unlabeled sodium acetate and provided to 2 x 1 L cultures on days
3, 6, and 8, and the cultures were harvested on day 10 (4.24 g wet wt, 0.39 g dry
wt., 64.7 mg organic extract). A total of 1.2 mg of 8 and 1.4 mg of 12 were isolated
from VLC and HPLC of the crude extract. Coupling constants for the intact 13C-13C
units of 8 and 12 are reported in the results and discussion section of this chapter
and shown in Figures Vl.14 and VI.6, respectively.
S-[Methyl-13C]Methionine Feeding. S-[methyl-13C]methionine (54 mg
total) was provided to 3 x 600 mL cultures on days 3, 6, and 8, and the cultures
were harvested on day 10 (2.76 g wet wt., 0.24 g dry wt., 67.2 mg organic extract).
A total of 1.0 mg of 8 and 0.9 mg of 12 were isolated from VLC and HPLC
purification of the crude extract. The 13C NMR spectral data are presented in
Figures Vl.7 and Vl.15, and relative enrichments are summarized in Tables Vl.1
and Vl.2.
S-[1-13C]Alanine Feeding. S-[1-13Cjalanine (300 mg total) was provided to
2 x 1.5 L cultures on days 3, 6, and 8, and the cultures were harvested on day 10
(2.15g wet wt., 0.26 g dry wt., 28.5 mg organic extract). A total of 1.5 mg of 8 and
1.2 mg of 12 were isolated from VLC and HPLC purification of the crude extract.
The 13C NMR spectral data are presented in Figures Vl.8a and Vl.16b, and relative
enrichments are summarized in Tables Vl.1 and Vl.2.
S-[3-13C]Alanine Feeding. S-[3-13C]alanine (195 mg total) was provided to
3 x 600 mL cultures on days 3, 6, and 8, and the cultures were harvested on day
10 (5.09 g wet wL, 0.64 g dry wt., 49.1 mg organic extract). A total of 3.5 mg of 8
and 2.0mg of 12 were isolated from VLC and HPLC purification of the crude
extract. The 13C NMR spectral data are presented in Figures Vl.8b and Vl.16c,
and relative enrichments are summarized in Tables Vl.1 and Vl.2.
[1-13C]Pyruvate Feeding. [1-13Cjpyruvate (100 mg total) was provided to
2 x 600 mL cultures on days 6 and 8, and the cultures were harvested on day 10
(2.99 g wet wt., 0.43 g dry wt., 61.7 mg organic extract). To deter the catabolism
of pyruvate to acetate, 20 mg of unlabeled sodium acetate were provided to each
of the flasks on days 2 through 9. A total of 1.0 mg of 8 and 1.0 mg of 12 were
isolated from VLC and HPLC purification of the crude extract. The 13C NMR
spectrum of 8 is presented in Figure Vl.16a and, and relative enrichments for both
12 and 8 are summarized in Tables Vl.1 and Vl.2, respectively.
[13C3,15N]-Alanine Feeding. [13C3,15N]13-alanine (150 mg total) was
provided to 3 x 600 mL cultures on days 3, 6, and 8, and the cultures were
harvested on day 10 (4.48 g wet wt., 0.49 g dry wt., 34.2 mg organic extract). A
total of 1.4 mg of 12 was isolated from VLC and HPLC purification of the crude
extract. The partial
13C
NMR spectrum of 12 is presented in Figure Vl.10.
Coupling constants for the intact
= 34.5 Hz,
1Jc16-c17
= 47.9 Hz,
13C-13C
JC15-N
and 13C-15N units are as follows:
= 10.5 Hz.
1Jc15-c16
167
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169
CHAPETR SEVEN
CONCLUSION
Although brief in age, the field of marine natural products has been
astoundingly successful in the discovery of new chemistry with potential
pharmaceutical, biochemical and agricultural applications. Among the most
productive sources of such novel and bioactive agents are the marine
cyanobacteria, particularly Lyngbya majuscula. Moreover, recent findings have
demonstrated that much of the unique chemistry isolated from marine
invertebrates is also of cyanobacterial origins.
This research has focused on chemical and biochemical examinations of
five unique L. majuscula collections through the utilization of spectroscopic
methods, predominately high field NMR. Two novel and chemically distinct
lipopeptides, somamide A and somocystinamide A, were isolated from a mixed
cyanobacterial assemblage of Lyngbya majuscula and Schizothrix sp. collected in
1997 from Somo Somo, Fiji. Their planar structures were elucidated through twodimensional NMR techniques and were confirmed by high-resolution mass
spectrometry.
Somamide A is a new cyclic depsipeptide of high structurally homology to
dolastatin 13, a potent cytostatic metabolite first reported in 1989 from the marine
mollusc Dolabella auricularia. Somocystinamide A, however, is a unique linear and
dimeric lipopeptide that displays potent cytotoxicity against mouse neuro-2a
neuroblastoma cells
(IC50
= 1.4 igImL). Interestingly, somocystinamide A is quite
unstable under mildly acidic conditions, rapidly and thoroughly decomposing to a
characterizable, but inactive, derivative. However, the biological activity of the C5
aldehyde alone has not yet been examined.
The sodium channel modulation bioassay was also used to guide the
fractionation of a crude organic extract from a Puerto Rican collection of Lyngbya
majuscula. Remarkably, both the potent sodium channel blocker kalkitoxin and
170
the sodium channel activator antillatoxin were discovered from this extract, as well
as the new secondary metabolite antillatoxin B, an unusual N-methyl-
homophenylalanine analogue of antillatoxin. Similar to antillatoxin, antillatoxin B
exhibits both significant sodium-channel activating (EC50 = 1.77 tM) and
ichthyotoxic (LC = 1 tM) properties.
During this extensive fractionation process several additional nontoxic
metabolites were identified, including a new malyngamide, a new quinoline
alkaloid and a new tryptophan derivative. Their structures were also deduced by
NMR and mass spectral data and, based on interesting structural parallels,
suggest that a convergent biosynthetic pathway exists for most of the enormously
diverse collection of metabolites isolated from this single L. majuscula species.
A L. majuscula collection acquired from Antany Mora, Madagascar in 2000
was discovered to produce a previously described marine molluscan anti-
neoplastic agent, dolastatin 16. Against a minipanel of the U. S. National Cancer
Institute's human cancer cells, dolastatin 16 displayed strong growth inhibition to
lung, colon, brain and melanoma specimens and gave comparable inhibitory
results against five human leukemia cell lines. Along with this medicinally
significant finding, a new collection of lipopeptides, the antanapeptins, were also
discovered. This series possesses high homology to several additional molluscari
compounds, including the kulolides and kulomo'opunalides.
The oxazole-containing dodecapeptide wewakazole was isolated from an
L.
majuscula collected from Wewak Bay, Papua New Guinea in 1998. Partial
structure identification and sequencing of this large and complex molecule
required detailed analyses of mass spectra and multiple two-dimensional NMR
experimental data including TOCSY, ROESY and HMBC experiments optimized
for 4, 8 and 12 Hz coupling. Final structure confirmation of a three amino acid
fragment in wewakazole also involved an interesting application of I D HMBC.
Stable isotope-labeled precursors and altered culturing conditions were
utilized in biosynthetic investigations of hectochlorin and jamaicamide A, two
chemically dissimilar secondary metabolites produced by a cultured strain of
Lyngbya majuscula, originally collected in 1996 from Hector Bay, Jamaica.
Results from these studies have demonstrated that both molecules arise from
171
mixed PKS and NRPS origins through the assembly of malonyl-derived acetate
units in combination with a variety of amino acids. Particularly noteworthy is the
incorporation of the atypical residue -alanine in the biosynthesis of jamaicamide A
as well as the demonstration that acetate and alanine condense and cyclize to
form its pyrrolidone ring.
It is evident that marine organisms like Lyngbya majuscula, the subject of
this research thesis, are capable of synthesizing compounds with immense
potential for the treatment of human disease. However, these prospective drug
resources are clearly limited and rapidly disappearing as human activities
(pollution, overfishing) deteriorate the fragile and complex marine ecosystems in
which they reside. While improvements in collection and conservation practices
are also necessary, future marine exploration is dependent upon the development
of both aquaculture/fermentation and genetic engineering methodologies. These
practices will not only preserve the oceans as a viable and continuous resource,
but also will aid in meeting supply demands for these novel therapeutics.
172
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APPENDICES
185
APPENDIX A
ISOLATION AND STRUCTURE ELUCIDATION OF DESMETHOXY-
MAJUSCULAMIDE C FROM A FIJIAN LYNGBYA MAJUSCULA
In conjunction with our collaborators at DOW Agro, the organic extract of a
Lyngbya majuscula collection from Kauviti Reef, Fiji was found to strongly inhibit
the growth of several plant fungal pathogens. Assay-guided fractionation of this
extract led to the discovery of a new majusculamide C analogue (1) as the
bioactive
constituent.1
0
17
18
)L
8 N''"
O31
R7
-30
/
52
0
37
HN
39
N)50
47
R6
Desmethoxymajusculamide c (1)
Majusculamide c (2)
Normajusculamide c (3)
Lyngbyastatin 1 (4)
Dolastatin 11(5)
Dolastatin 12 (6)
iO
H
0
1'
R3R2
'S 5
R1
R2
R3
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
cH3
H
CH3
H
H
R4
H
OCH3
OCH3
0CH3
OCH3
H
R5
CH3
CH3
CH3
H
H
H
R6
R7
H
CH3
CH3
H
H
H
CH3
CH3
CH3
cH3
cH3
cH3
While the IR spectrum displayed absorption bands at 1739 and 1641 cm'
for both ester and amide functionalities, the HRFABMS of compound I analyzed
for
C49H78N8011,
indicating a molecule of peptide origin. 1H and 13C NMR spectra
1E;I:
of I were well dispersed in CDCI3, allowing for construction of nine partial
structures by 2D NMR and accounting for all atoms in the molecular formula of 1.
Six standard amino acids were deduced as alanine (Ala), N-methyl-valine (NMeVal), N-methyl-phenylalanine (N-MePhe), N-methyl-isoleucine (N-Melle) and
two glycine (Gly) units. Two additional residues were assembled to form the ahydroxy acid, 2-hydroxy-3-methylpentanoate (Hmpa) and the 13-amino acid, 3-
amino-2-methylpentanoate (Map). The ninth and final moiety, deduced mainly
from H MBC, was constructed as 4-amino-2,2-dimethyl-3-oxo-pentanoate (Dmop).
Amino acid sequencing was accomplished by HMBC connectivities (Table A.1)
and was supported by mass spectral fragmentation of the base hydrolysis product
of I (Figure A.1). Interestingly, both desmethoxymajusculamide C (I) and its
linearized form display equally potent cytotoxicity (0.3 tg/mL) against neuro-2a
neuroblastoma
cells.2
m1z630
m/z469
m/z803
m/z 356
m/z299
0
OH
ooIHI
mlz 342
nilz 675
m/z 505
Figure A.1. Mass fragments observed by FABMS of the base hydrolysis product
of I. With the exception of peaks at m/z 342 and 356, all mass fragments were
also present in the corresponding collision induced dissociation mass spectrum
(CIDMS).
187
Table A.1. NMR spectral data for desmethoxymajusculamide C (1) in CDCI3.
position
172.8
42.7
51.4
1
2
3
4 (NH)
5
6
7
8
9
1H (J in Hz)
HMBCa
2.76, qd (7.0, 1.6)
1, 3
4.52, m
7.07, d (10.1)
3, 8
10.0
1.10,d(7.0)
1,2,3
26.3
11.2
173.0
48.5
1.56, 1.48
2, 3, 7
0.93, t (7.4)
3, 6
4.45
8, 11
10(NH)
7.76, d(7.9)
9,11,12
1.07, d (7.9)
8, 9
4.92
14, 19
7.32, d (6.4)
14, 15,20
1.53,s
12,13,14,18
1.48, s
12, 13, 14, 17
1.16, d (6.8)
14, 15
21
22.5
21.7
19.3
168.0
61.0
5.23
20, 23, 24, 30
22(N)
23
35.7
3.31, dd (14.0, 6.3)
20, 21, 24, 25/29
11
12
13
14
15
16 (NH)
17
18
19
20
15.7
172.3
55.1
210.3
52.0
2.89, dd (14.0, 8.2)
24
25/29
26/28
27
30
31
32
137.0
129.7
129.0
127.2
29.5
170.4
58.3
7.25
7.28
23, 24, 26/28, 27
24, 25/29
7.20, t (6.9)
25/29
2.99, $
21,31
4.79, d (10.6)
31, 34, 35, 36,37,38
2.22, m
2.97, s
32,
32,
32,
32,
4.62, dd (17.6, 7.9)
38, 41
33 (N)
34
35
36
37
38
39
27.3
18.7
18.6
29.5
169.5
40.8
0.33, d (6.4)
0.74, d (6.5)
35, 36
34, 36
34, 35
38
3.47, d (17.6)
40 (NH)
41
42
43(N)
44
45
46
47
48
49
50
171.3
61.4
33.0
25.2
10.3
15.8
30.6
170.2
41.0
7.58 d (7.9)
41
4.91
41,44,45,47,48,49
2.11
1.46, 1.11
44
0.89
44, 45
1.03, d (7.3)
42, 44,45
3.22, s
42, 49
4.44
49,52
3.57, dd (15.9,4.6)
51 (NH)
52
53
54
55
56
57
a
170.7
78.6
37.6
24.0
11.9
15.36
7.36, t (5.5)
49, 50
5.21
1, 52, 54, 55, 57
2.07
1.47, 1.24
53, 54, 56
0.90
0.90
54,55
Proton showing correlation to indicated carbon resonance.
53. 54
188
The structure of I proved nearly identical to that of the known metabolite
majusculamide C
(2),1
originally reported in 1984 as an antifungal agent from an L.
majuscula collected from Enewetak Atoll in the Marshall Islands. Various
bioassay-guided fractionations have also directed the isolations of compounds
and
44
33
from additional cyanobacterial collections and dolastatins 11 and 12 (5, 6)
from the marine mollusc
Do/abe/Ia
auricularia. However, this structurally
homologous (but confusingly designated!) series of metabolites has unfortunately
proved quite cytotoxic, precluding their further development as useful antibiotics.
An
L
configuration for the Ala, N-MeVal, N-MePhe and N-Melle residues of
I was deduced by Marley's
analysis,6
while the remaining stereocenters for the
Hmp, Map and Dmop units are presumed identical to this series of metabolites,
whose absolute stereochemistries were established or confirmed by total
synthesis.7
189
EXPERIMENTAL
General Experimental Procedures. The IR spectrum was recorded on a
Nicolet 510 Fourier transform lR spectrophotometer and optical rotation was
measured on a Perkin-Elmer 243 polarimeter. All NMR spectral data were
recorded on a Bruker AM400 spectrometer, with the solvent CDCI3 used as an
internal standard (c 77.23 E, 7.27). Chemical shifts are reported in ppm and
coupling constants (J) are reported in Hz. The FAB mass spectrum and CID mass
spectrum were recorded on Kratos MS5OTC and Perkin Elmer Sciex API3 mass
spectrometers, respectively. HPLC isolation of I was performed using a Waters
Millipore Lambda-Max model 480 LC spectrophotometer with Waters 515 HPLC
pumps.
Cyanobacterial Collection. The marine cyanobacterium Lyngbya
majuscula
(voucher specimen available as collection number VKR-16/Feb/00-05)
was collected by B. Marquez and T. Okino from the Kauviti Reef of Yanuca Island,
Fiji on February 16, 2000. The material was stored in 2-propanol at reduced
temperature until extraction.
Extraction and Isolation. Approximately 300 grams dry weight of the
cyanobacterium was repetitively extracted with 2:1 CH2Cl2/MeOH to yield 5.5 g of
crude extract. A portion of the extract (5.3 g) was fractionated by VLC over silica
gel. The fraction eluting with 100% EtOAc was further chromatographed by C18
SPE (7:3 MeOH/H20) followed by RP HPLC (Phenomenex Sphereclone 5tm ODS
column, 17:3 MeOH/H20) to yield 27.5mg of pure 1.
Desmethoxymajusculamide C (1): glassy oil;
[ctJ22D
-1 04.1° (c 1.86,
CH2Cl2); IR (neat) 3315, 2966, 2934, 2877, 1739, 1641, 1519, 1460, 1410, 1286
cm; 1H and 13C NMR data, see Table A.1; HRFABMS m/z EM + HJ 955.5867
(calculated for C49H79N8011, 955.5868).
190
Base Hydrolysis of 1: Approximately 6 mg of I was suspended in 2 mL
of a 1:1 CH3OH/0.5 M NaOH solution and allowed to stand overnight at room
temperature. The CH3OH was removed by evaporating under N2, and the mixture
was neutralized with HCI and extracted with EtOAc. The organic layer was dried
under N2 and purified using the above HPLC conditions to give the base hydrolysis
product. Hydrolysate of 1: FAB MS (3-nitrobenzylalcohol) m/z 973 (35), 803 (25),
675(41), 630 (52), 505 (30), 469 (100), 356 (66), 342 (54), 299 (82), 244 (31).
Absolute Configuration of the Standard Amino Acid Residues in
Desmethoxymajusculamide C (1). Approximately 1.0 mg of I was hydrolyzed
with 6N HCI (Ace high pressure tube, microwave, 1.0 mm), and the hydrolysate
was evaporated to dryness and resuspended in H20 (50 tL). A 0.1% 1-fluoro-2,4dinitrophenyl-5-L-alaninamide (Marfey's reagent) solution in acetone (50 ML) and
20 ML of 1 N NaHCO3 were added, and the mixture was heated at 40°C for 1 hour.
The solution was cooled to room temperature, neutralized with 10 ML of 2N HCI
and evaporated to dryness. The residue was resuspended in H20 (50 ML) and
analyzed by reversed-phase HPLC [LiChrospher 100 C18, 5 t, UV detection at 340
nm] using a linear gradient of 9:1 50 mM triethylammonium phosphate (TEAP)
buffer (pH 3.1)/CH3CN to 1:1 TEAP/CH3CN over 60 mm.
The retention times
(tR,
mm) of the derivatized residues in the hydrolysate
of I matched L-Ala (22.1; D-Ala, 26.9), N-Me-L-Val (36.5; N-Me-D-Val, 39.7), and
N-Me-L-Phe (38.5; N-Me-D-Phe, 39.2). Given that only N-Me-L-lle and N-Me-L-
a/b-lIe were commercially available, the o-Marfey's reagent (synthesized by L. T.
Tan) was used to make the N-Me-D-lle and N-Me-D-a//o-lIe chromatographic
equivalents. The retention time of the hydrolysate lie derivative matched that of NMe-L-Ile (40.7; N-Me-L-a//o-Ile, 41.2; N-Me-o-lle, 44.2; N-Me-o-a//o-lle, 44.7).
191
REFERENCES
Carter, D. C.; Moore, R. E.; Mynderse, J. S.; Niemczura, W. P.; Todd, J. S.
J. Org. Chem. 1984, 49, 236-241.
2.
Manger, R. L.; Leja, L. S.; Lee, S. Y.; Hungerford, J. M.; Hokama, Y.;
Dickey, R. W.; Granade, H. R.; Lewis, R.; Yasumoto, T.; Wekell, M. M. J.
AOAC Intern. 1995, 78, 521-527.
3.
Mynderse, J. S.; Hunt, H. J. Nat. Prod. 1988, 51, 1299-1301.
4.
Harrigan, G. G.; Yoshida, W. Y.; Moore, R. E.; Nagle, D. G.; Park, P. U.;
Biggs, J.; Paul, V. J.; Mooberry, S. L.; Corbett, T. H.; Valeriote, F. A. J. Nat.
Prod. 1998, 61, 1221-1225.
5.
Pettit, G. R.; Kamano, Y.; Kizu, H.; Dufresne, C.; Herald, C. L.; Bontems,
R. J.; Schmidt, J. M.; Boettner, F. E.; Nieman, R. A. Heterocycles 1989, 28,
553-558.
6.
Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596.
7.
(a) Bates, R. B.; Gangwar, S. Tetrahedron: Asymmetry 1993, 4, 69-72. (b)
Bates, R. B.; Brusoe, K. G.; Burns, J. J.; Caldera, S.; Gui, W.; Gangwar, S.;
Gramme, M. R.; McClure, K. J.; Rouen, G. P.; Schadow, H.; Stessman, C.
C.; Taylor, S. R.; Vu, V. H.; Yarick, G. V.; Zhang, J.; Pettit, G. R.; Bontems,
R.J.Am. Chem. Soc. 1997, 119, 2111-2113.
192
APPENDIX B
ISOLATION AND STRUCTURE ELUCIDATION OF JAMAICAMIDE C FROM A
CULTURED JAMAICAN LYNGBYA MAJUSCULA
Jamaicamide C (3) was discovered as a minute constituent of the cultured
L. majuscula that produces both hectochiorin and jamaicamides A (1) and B (2)
(see chapter six for culture conditions and chromatographic details).
1
N)(L
0
H
Jamaicamide A (1) R = Br
Jamaicamide B (2) R = H
0
27Cl
22
Jamaicamide C (3)
The HR FABMS of 3 produced an [M+H] of 491.2674 for the molecular
formula C27H39O4N2C1 (9 degrees of unsaturation). As observed in jamaicamide B,
the molecular ion isotope pattern (m/z 491/493 in an approximate 1:0.4 ratio)
supported the presence of a single chlorine atom. The high structural homology of
3 to compound 2 was demonstrated by nearly identical 1H and 13C chemical shifts
(Table
B.1).2
However, jamaicamide C lacked the characteristic acetylene carbon
resonances of 2, instead containing new methine (oc 138.4,
methylene (c 114.6,
H
6H
5.82) and
5.01, 4.96) signals indicative of a mono-substituted olefir,.
193
2D NMR spectroscopy was then used to clearly establish that jamaicamide C was
the alkene equivalent of jamaicamide B.
Table B.1. 1H and 13C NMR comparison of jamaicamides C (3) and B (2).
position
13C NMR of 3a
1H NMR of 3 (J in Hz)
'C NMR of 2a
1H NMR of 2 (J in Hz)
1
114.6 (t)
5.01, dd (17.0, 1.8)
68.6 (s)
1.97, s
4.96, dd (10.2, 1.8)
2
138.4 (d)
5.82, m
84.1 (s)
3
33.5 (t)
2.11, m
18.3 (t)
4
26.3 (t)
1.50
26.1 (t)
1.64, m
5
6
29.6 (t)
2.18, m
29.3 (t)
2.26, m
7
32.6 (t)
1.99, m
32.6 (t)
8
34.7 (t)
1.33, m
34.7 (t)
1.33, m
9
36.3 (d)
2.01, m
36.3 (d)
2.01, m
10
136.6 (d)
5.26, dd (15.1, 7.5)
136.6 (d)
5.26, dd (15.1, 7.8)
11
127.5 (d)
5.35, dt (15.3, 6.0)
127.5 (d)
5.35, dt (1 5.1, 6.5)
12
28.5 (t)
2.28, m
28.5 (t)
2.28, m
13
14
36.7 (t)
2.17, m
36.7 (t)
2.17, t (7.8)
15
38.2 (t)
3.51, m
38.2 (t)
3.5, m
16
32.2 (t)
2.98, 2.83, m
32.2 (t)
2.98, 2.83, m
17
175.4 (s)
18
94.9 (d)
6.72, s
94.9 (d)
19
166.0 (s)
20
170.1 (s)
21
125.9 (d)
6.07, dd (6.2, 1.6)
125.9 (d)
6.07, dd (6.2, 1.0)
22
23
24
153.1 (d)
7.21, dd (6.2, 2.0)
153.1 (d)
7.21, dd (6.2, 1.9)
58.1 (d)
4.87, m
58.1 (d)
4.87, m
17.9 (q)
1.45, d (6.8)
17.9 (q)
1.45, d (6.6)
56.1 (q)
3.74, s
56.1 (q)
3.74, s
20.8 (q)
0.93, d (6.8)
20.8 (q)
0.93, d (6.7)
112.0(d)
5.76,s
112.7(d)
5.81,s
25
26
27
142.5(d)
2.19, m
141.9(d)
172.4 (s)
1.99, m
172.4 (s)
175.4 (s)
6.72, s
166.0 (s)
170.1 (s)
NH
6.73
Multiplicity deduced by mutiplicity edited HSQC.
6.68, m
Interestingly, recent screening efforts in our laboratory have revealed a
potent sodium channel blocking
activity3
for jamaicamides A (1) and C (3), but not
B (2). Further testing is currently underway.
194
REFERENCES
Jamaicamide C (3): pale yellow oil; [a]250 490 (c. 0.39, MeOH); UV
(MeOH) Xmax 273 nm (log c = 3.8); IR (neat) 3303, 2928, 2857, 1721, 1660,
1601, 1550, 1437, 1397,1202,1171, 1082, 823 cm1; 1H and 13C NMR
data, see Table B.1; LRFABMS (positive ion, 3-nitrobenzylalcohol) [M + H]
cluster at m/z 49 1/493 (1:0.4); HRFABMS m/z [M + HJ 491.2674
(calculated for C27H4004N2C1, 491.2677).
2.
Marquez, B. L. Structure and Biosynthesis of Marine Cyanobacterial
Natural Products: Development and Application of New NMR Methods.
Ph.D. Thesis, Oregon State University, Corvallis, OR, 2001, pp. 45-79.
3.
Manger, R. L.; Leja, L. S.; Lee, S. Y.; Hungerford, J. M.; Hokama, Y.;
Dickey, R. W.; Granade, H. R.; Lewis, R.; Yasumoto, T.; Wekell, M. M. J.
AOAC intern. 1995, 78, 521-527.
195
APPENDIX C
ISOLATION OF KNOWN METABOLITES FROM A PUERTO RICAN
COLLECTION OF LYNGBYA MAJUSCULA
A chemically prolific Lyngbya majuscula (voucher specimen available as
collection number PRLP-16/Sept/97-03) was obtained near Collado Reef, Puerto
Rico, on September 16, 1997. Detailed examination of the organic extract from
this collection led to the discovery of four new secondary metabolites (see chapter
three) and the isolation of twelve previously reported
structures.15
The crude extract was first subjected to Si VLC, producing 14 chemically
distinct fractions. The fraction eluting with 25% EtOAc in hexanes was purified by
a C18 solid phase extraction (SPE) cartridge (7:3 MeOH/H20) to yield 280.2 mg of
lyngbic acid (1). The VLC fraction eluting with 35% EtOAc was subjected to C18
SPE (7:3 MeOH/H20) and repetitive reversed-phase HPLC (9:1 MeOH/H20,
Phenomenex Sphereclone 5
Spherisorb 10
t
t
ODS, followed by 17:3 MeOH/H20, Phenomenex
ODS) to obtain 2.8 mg of hermitamide A (8) and 1.0 mg of
kalkitoxin (12). Purification of the fraction eluting with 40% EtOAc by C18 SPE (4:1
MeOH/H20) and RP HPLC (9:1 MeOH/H20, Phenomenex Sphereclone 51.1 ODS)
provided 6.3 mg of malyngamide C-acetate (3). The 45-50% EtOAc VLC fraction
was also subjected to C18 SPE (7:3 MeOH/H20) and RP HPLC (4:1 MeOH/H20,
Phenomenex Spherisorb 10 1.t ODS) to produce 3.6 mg of antillatoxin (11) and
15.1 mg of malyngamide F-acetate (5). Further RP HPLC purification of this
subfraction (7:3 MeOH/H20, Phenomenex Spherisorb 10 j.t ODS) led to the
isolation of 0.3 mg of hermitamide B (9) and 1.2 mg of malyngamide K (6). The
55-65% VLC fraction was crudely chromatographed over LH-20 using 100%
MeOH (3
x
40 cm). The first fraction was purified by C18 SPE (7:3 MeOH/H20)
and RP HPLC (4:1 MeOH/H20, Phenomenex Spherisorb 10 p. ODS), yielding an
additional 7.0 mg of malyngamide F-acetate (RP HPLC, 9:1 MeOH/H20,
Phenomenex Sphereclone 5 p. ODS).
196
0
OCH3
OH
7(S)-methoxytetradec-4(E)-enoic acid (= lyngbic acid, 1)
0
OCH3
R
H
iI°..J
Malyngamide C (2) R = OH
Malyngamide C acetate (3) R = OAc
0
OCH3
Malyngamide F (4) R = OH
CI 'j(
HI
Cl
R
0
Malyngamide F acetate (5) R OAc
Malyngamide K (6) R = H
0
QCH3
Malyngamide J (7)
0
ii
o'I
i-i
H3CO\_
OCH3
0
OCH3
Hermitamide A (8)
H
NH
a
OCH3
Hermitamide B (9)
HO
OMe
I
Quinoline alkaloid (10)
H
0
Antillatoxin (11)
N-
0
Kalkitoxin (12)
197
The second LH-20 fraction was also purified by
C18
and RP HPLC (4:1 MeOH/H20, Waters SymmetryShield 5
SPE (7:3 MeOH/H20)
t
ODS), affording 1.3
mg of quinoline alkaloid 10 and 2.1 mg of malyngamide C (2). Purification of the
fraction eluting with 80-90% EtOAc by
C18
SPE (7:3 MeOH/H20) and RP HPLC
(4:1 MeOH/H20, Phenomenex Spherisorb 10
t
ODS) yielded 1.1 mg of
malyngamide F (4). Lastly, the VLC fraction eluting between 100% EtOAc and 5%
MeOH in EtOAc was purified by LH-20 (100% MeOH), C18 SPE (4:1 MeOH/H20)
and RP HPLC (7:3 MeOH/H20, Phenomenex Spherisorb 10
mg of malyngamide J (7).
t
ODS), to give 11.9
1EI;]
REFERENCES
(a) Cardellina, J. H., II; Dalietos, D.; Marner, F.-J.; Mynderse, J. S.; Moore,
R. E. Phytochemistiy 1978, 17, 2091-2095. (b) Ainslie, R. D.; Barchi, J. J.,
Jr.; Kuniyoshi, M.; Moore, R. E.; Mynderse, J. S. J. Org. Chem. 1985, 50,
2859-2862. (C) Gerwick, W.H.; Reyes, S.; Alvarado, B. Phytochemistry
1987, 26, 1701-1704. (d) Orjala, J.; Nagle, D.; Gerwick, W. H. J. Nat. Prod.
1995, 58, 764-768. (e) Wu, M.; Milligan, K. E.; Gerwick, W. H. Tetrahedron
1997, 53, 15983-15990. (f) MiHigan, K. E.; Marquez, B.; Williamson, R. T.;
Davies-Coleman, M.; Gerwick, W. H. J. Nat. Prod. 2000, 63, 965-968.
2.
Tan, L. T.; Okino, T.; Gerwick, W. H. J. Nat. Prod. 2000, 63, 952-955.
3.
Orjala, J.; Gerwick, W. H. Phytochemistty 1997, 45, 1087-1 090.
4.
Orjala, J.; Nagle, D. G.; Hsu, V. L.; Gerwick, W. H. J. Am. Chem. Soc.
1995, 117, 8281-8282.
5.
Wu, M.; Okino, T.; Nogle, L. M.; Marquez, B. L.; Williamson, R. T.;
Sitachitta, N.; Berman, F. W.; Murray, T. F.; McGough, K.; Jacobs, R.;
Colsen, K.; Asano, T; Yokokawa, F.; Shioiri, T.; Gerwick, W. H. J. Am.
Chem. Soc. 2000, 122, 12041-12042.
199
APPENDIX D
ISOLATION OF CURACIN D FROM A FIJIAN ASSEMBLAGE OF MARINE
CYANOBACTERIA
The organic extract of a Lyngbya majuscula/Schizothrix sp. marine
cyanobacterial assemblage collected from Somo Somo, Fiji in 1997 (voucher
specimen available as collection number VSO-8/Feb/97-02) displayed toxicity in
the brine shrimp bioassay (100 ppm). The extract was chromatographed over
silica gel, with fractions eluting between 10%-15% EtOAc in hexanes possessing
the most potent activity (10 ppm). Further assay-guided fractionation by additional
silica VLC (5% EtOAc in hexanes) and HPLC (9:1 hexanes/EtOAc, Alltech
Versapack Si 10
t)
led to the isolation of 0.7 mg of curacin D (1) as the active
constituent.
OCH3
N
CuracinD(1)R=H
Curacin A (2) R = CH3
Originally isolated in low yield as a brine shrimp
H'
toxin,1
'H
the structure of I
was nearly identical to that of curacin A (2), an antimitotic agent discovered from a
Curacao L. majuscula in 1994.
REFERENCES
Marquez, B.; Verdier-Pinard, P.; Hamel, E.; Gerwick, W. H. Phytochemistry
1998, 49, 2387-2389.
2.
Gerwick, W. H.; Proteau, P. J.; Nagle, D. G.; Hamel, E.; Blokhin, A.; Slate,
D. L. J. Org. Chem. 1994, 59, 1243-1245.
200
APPENDIX E
ISOLATION OF MALYNGOLIDE FROM A PAPUA NEW GUINEA COLLECTION
OF
LYNGBYA MAJUSCULA
The organic extract of a
L.
majuscula collected from Hermit Village, Papua
New Guinea in 1998 (voucher specimen available as collection number PNHV1 1/SeptJ98-03) displayed toxicity in the brine shrimp bioassay (100 ppm). The
extract was chromatographed over silica gel, with fractions eluting between 30%-
40% EtOAc in hexanes possessing the most potent activity (10 ppm). Further
assay-guided fractionation by LH-20 chromatography (100% MeOH),
C18
MeOH/H20) and RP HPLC (9:1 MeOH/H20) Phenomenex Sphereclone 5
VLC (3:1
t
ODS)
led to the isolation of malyngolide (1, 23.6 mg) as the active constituent.
Malyngolide (1)
Originally isolated for its antibiotic
properties,1
malyngolide has been and
continues to be of great interest to synthetic chemists, having been synthesized
numerous times since its discovery in 1979.2,3
REFERENCES
1.
Cardellina, J. H., II; Moore, R. E.; Arnold, E. V.; Clardy, J. J. Org. Chem.
1979, 44, 4039-4042.
2.
Ghosh, A. K.; Shirai, M.
3.
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