Bioassay-Guided Fractionation and
Compound Structure Elucidation of a
Red Sea Strain of Lyngbya majuscula
Elise S. Cowley
September 19, 2011
Mentors: Dr. Kerry McPhail and Dr. Phil Proteau
Department of Pharmaceutical Sciences
College of Pharmacy
Oregon State University
Corvallis, OR 97331
a
Relevance
Relevance
Cyanobacteria are a rich source of natural products that serve as:
oCandidate therapeutic agents
oResearch tools to probe disease mechanisms
oPotential agricultural agents
oPotential industrial agents
Cyanobacteria are classified alongside myxobacteria and Streptomyces as
sources of pharmaceuticals.
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The Red Sea Exploratory Expedition
The Red Sea
 Mangrove Physical Characteristics
 High salinity
 Oligotrophic
 Fine sediment deposition
 OSU Collections
 May 2007
 12 large collections for extraction
 28 small, live collections for
laboratory culture
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Culture of Sample Enq. 004
Culture Methods
Purification Process
oContaminant-brown diatom
oLow light
oOvertook brown diatom
Harvest
o6 months
oYield: 0.85 g
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a
Methods
Methods
May 2007collection from
Red Sea
Large scale
culture in
laboratory
Normal phasevacuum liquid
chromatography
(NP-VLC)
Brine shrimp
assay (BSA)
High performance
liquid
chromatography
(HPLC)
Mass
spectrometry
Reverse phase
solid phase
extraction
(RP-SPE)
Mass
spectrometry
Completed by Chris Thornburg
Nuclear magnetic
resonance (NMR)
techniquesStructure
elucidation
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Cytotoxicity
testing
Completed by Elise Cowley
Current work
a
Lyngbya sp. Enq004
Lyngbya sp RS05
Nabq Mangroves near Sharm el Sheikh, Egypt
 50 liters collected over
12 months
 0.85 g Organic Extract
Active Fractions
NP-VLC
A
37.2 mg
B
62.6 mg
C
108.1 mg
D
144.2 mg
E
12.7 mg
F
131.4 mg
G
165.0 mg
H
190.7 mg
I
88.6 mg
100% Hex
10% EtAc/H
20% E/H
40% E/H
60% E/H
80% E/H
100% E
25% MeOH/E
100% M
Brine Shrimp Assay
1mg/ml: 4 %
0%
100%
100%
89%
100%
100%
85%
0%
BSA
0.1mg/ml: 5 %
0%
96%
97%
7%
85%
94%
7%
0%
Mass Spectrometry
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Slide courtesy of Chris
Thornburg
Lyngbya sp. Enq004
Hypothesized Compounds
7 known compounds & 2 new compounds
1
2
3
4
5
6
7
8 = Unknown: m/z 658
9 = Unknown: m/z 854
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Slide courtesy of Chris
Thornburg
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Hypothesis and Prediction
Hypothesis and prediction
Hypothesis: Based on preliminary chemical and biological profiling
of crude fractions of the extract of cultured black Lyngbya, we
propose that at least two of the targeted nine metabolites detected
are new natural products with significant biological properties.
Prediction: If the hypothesis is correct, then the results from the
spectroscopy methods used on the purified compounds from the
black Lyngbya will demonstrate compounds not previously found in
this particular genus of cyanobacteria.
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a
Lyngbya sp. Enq004
Lyngbya sp RS05
Nabq Mangroves near Sharm el Sheikh, Egypt
 50 liters collected over
12 months
 0.85 g Organic Extract
Active Fractions
NP-VLC
A
37.2 mg
B
62.6 mg
C
108.1 mg
D
144.2 mg
E
12.7 mg
F
131.4 mg
G
165.0 mg
H
190.7 mg
I
88.6 mg
100% Hex
10% EtAc/H
20% E/H
40% E/H
60% E/H
80% E/H
100% E
25% MeOH/E
100% M
BSA
1mg/ml: 4 %
0%
100%
100%
89%
100%
100%
85%
0%
BSA
0.1mg/ml: 5 %
0%
96%
97%
7%
85%
94%
7%
0%
RP-SPE
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a
Lyngbya sp. Enq004-HPLC Fraction F
Lyngbya sp RS05
F
131.4
mg
RP-SPE
1
8.2 mg
2
91.6 mg
3
2.7 mg
4
2.1 mg
70% MeOH/H
90% M/H
100% M
100% DCM
HPLC-90% MeOHSynergiFusion Column
2800
2600
216 nm
230 nm
254 nm
330 nm
2400
2200
3
2000
1800
C
Mass Spec
1600
HPLC-80%MeCNSynergiFusion Column
2000
mAu
1800
1600
1200
1000
1400
800
1200
600
1000
m/z 840Apratoxin A
1400
4
2
400
1
800
200
600
0
0
A
400
200
D
B
6.0
6.5
7.0
7.5
8.0
8.5
9.0
Minutes
10
9.5
10.0
10.5
11.0
11.5
2
3
4
5
6
7
Minutes
0
5.5
1
12.0
12.5
13.0
8
9
10
11
12
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Enq.004 F2C3 Structure Elucidation
EHu5H-2C5A & B 1H Differences
1H
NMR Spectra
for Enq004 F2C3 in
CDCl3 (300 MHz)Experimental
1H
NMR Spectra
for Apratoxin A in
CDCl3 (500 MHz)Published
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Enq.004 F2C3 Structure Elucidation
EHu5H-2C5A & B 1H Differences
13C
NMR Spectra for
Enq004 F2C3 in CDCl3 (300
MHz)-Experimental
13C
NMR Spectra for
Apratoxin A in CDCl3 (300
MHz)-Published
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Fu
Enq004 F2C3-Apratoxin A
Compound Characteristics
•Toxic against KB and LoVo cells
•Induce G1 phase cycle arrest
•No effect on microfilament
network
•No effect on microtubule
polymerization or de-polymerization
•Lack of selectivity limits potential as
antitumor agent, but derivatives are
more effective
13
Fu
Future Goals
Future Goals
 Goals
 Use HPLC to further purify fractions of interest
14
Fu
Future Research Goals-Cont’d
 Spectroscopic analysis of pure
compounds from HPLC
 High interest in obtaining spectroscopic
data for the two unknown compounds
 Characterize the structures of the two
unknown compounds, including absolute
configuration
 Obtain larger quantities of pure
unknowns for biological testing on
human cell lines
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Spectroscopic Methods
oMass Spec
oNMR
HNMR
CNMR
COSY
HSQC
HMBC
ROESY
TOCSY
a
Acknowledgements
Acknowledgements
Funding
•Howard Hughes Medical Institute, facilitated by Dr. Kevin Ahern
•Oregon State University Honors College
•Oregon State College of Pharmacy
•Sigma Xi
•E.R. Jackman Internship Grant
Research Lab
•Dr. Kerry McPhail
•Dr. Phil Proteau
•Christopher Thornburg
•Justyna Sikorska
•Oliver Vining
•Mariko Nonogaki
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