Mentors: Kerry McPhail, PhD, Phil Proteau, PhD,
and Christopher C. Thornburg, PhD
Undergraduate Honors and Bioresource Research
Thesis Seminar Presentation
May 28, 2013
Roadmap
2
Background and Relevance of Natural Products
Sample Collection by McPhail Lab
Goal of Project
Methods
Culturing
Chromatography
o Compound Hypothesis
Compound Characterization
Results
Known Compounds
New Compounds
Hydrophobic Fraction
Discussion
Relevance of Results
Limitations
Future Work
Acknowledgements
Questions
Roadmap
3
Background and Relevance of Natural Products
Sample Collection by McPhail Lab
Goal of Project
Methods
Culturing
Chromatography
o Compound Hypothesis
Compound Characterization
Results
Known Compounds
New Compounds
Hydrophobic Fraction
Discussion
Relevance of Results
Limitations
Future Work
Acknowledgements
Questions
What are Natural Products?
4
Secondary Metabolites
What produces secondary
metabolites?
Why produce secondary metabolites?
Why are Natural Products Important?
5
www.lipitor.com
www.elucidationimages.com
www.kdna.ucla.edu
www.emory.edu
www.sciencedirect.co
m
Agricultural
Applications
Pharmaceutical Applications
Why Search for More Natural Products?
6
Pharmaceutical Importance: (Newman,
Cragg, 2012)
7
Antibiotic Resistance (Silver, Bostian, 1993)
Anticancer Resistance (Simmons, et al, 2005)
Constant need for new compounds (Strobel,
Daisy, 2003)
Exotic Environments (Baker, et al, 2007)
Why Search for More Natural Products?
Cyanobacteria as Sources of
Natural Products
8
Ancient Phylum
Ubiquitous
Classified alongside Myxobacteria and
Streptomyces as sources of pharmaceuticals (Tan,
2010)
Roadmap
9
Background and Relevance of Natural Products
Sample Collection by McPhail Lab
Goal of Project
Methods
Culturing
Chromatography
o Compound Hypothesis
Compound Characterization
Results
Known Compounds
New Compounds
Hydrophobic Fraction
Discussion
Relevance of Results
Limitations
Future Work
Acknowledgements
Questions
Red Sea Exploratory Expedition
10
OSU Collections
May 2007
12 large collections
28 small live collections
Mangroves
High salinity
Oligotrophic
Fine sediment deposition
Roadmap
11
Background and Relevance of Natural Products
Sample Collection by McPhail Lab
Goal of Project
Methods
Culturing
Chromatography
o Compound Hypothesis
Compound Characterization
Results
Known Compounds
New Compounds
Hydrophobic Fraction
Discussion
Relevance of Results
Limitations
Future Work
Acknowledgements
Questions
Purification Process Brown contaminant
Low light
Overtook brown diatom
Harvest 6 months
Yield: 0.85 g
Culturing Process-Moorea sp.
12 Large-scale cultures of Moorea sp.
Materials and Methods
13
Brine Shrimp
Assay
Neuroblastoma
Assay
Extraction and
Separation
Further Separation to
Pure Compounds Structure
Elucidation
Chromatography Overview
14
NP-VLC
A
C
20% E/H
E
60% E/H
G
100% E
H
25% MeOH/E
I
100% M
F
80% E/H
B
10% EtOAc/H
D
40% E/H
Hydrophobic
Hydrophilic
100% Hex
Chemical Extraction
3
70% MeOH/H
1
RP-SPE
4
2
100% DCM 100% M 90% M/H
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2
mA
u
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
1 0 0 0
1 1 0 0
1 2 0 0
1
2
4 5a
5b
Minutes
3
RP-HPLC
• NP-VLC:Normal Phase
Vacuum Liquid
Chromatography
• RP-SPE: Reversed Phase
Solid Phase Extraction
• RP-HPLC: Reversed
Phase High Performance
Liquid Chromatography
4
Materials and Methods
15
100% Hex
Brine Shrimp Assay (BSA) 1mg/ml: 4 % 0% 100% 100% 89% 100% 100% 85% 0%
Active Fractions
NP-VLC
A 37.2 mg
C 108.1 mg
20% E/H
E 12.7 mg
60% E/H
G 165.0 mg
100% E
H 190.7 mg
25% MeOH/E
I 88.6 mg
100% M
F 131.4 mg
80% E/H
B 62.6 mg
10% EtAc/H
D 144.2 mg
40% E/H
Moorea species
5
Nabq Mangroves near Sharm el Sheikh, Egypt
BSA 0.1mg/ml: 5 % 0% 96% 97% 7% 85% 94% 7% 0%
50 liters collected over
12 months
0.85 g Organic Extract
Mass Spectrometry
and RP-SPE
Hydrophobic
Hydrophilic
16
Moorea sp. Hypothesized Compounds
8 = Unknown: m/z 658
9 = Unknown: m/z 854
7 known compounds & 2 new compounds
1 2 3
4
5 6 7
17
100% Hex
Brine Shrimp Assay (BSA) 1mg/ml: 4 % 0% 100% 100% 89% 100% 100% 85% 0%
Active Fractions
NP-VLC
A 37.2 mg
C 108.1 mg
20% E/H
E 12.7 mg
60% E/H
G 165.0 mg
100% E
H 190.7 mg
25% MeOH/E
I 88.6 mg
100% M
F 131.4 mg
80% E/H
B 62.6 mg
10% EtOAc/H
D 144.2 mg
40% E/H
Moorea species
5
Nabq Mangroves near Sharm el Sheikh, Egypt
BSA 0.1mg/ml: 5 % 0% 96% 97% 7% 85% 94% 7% 0%
50 liters collected over
12 months
0.85 g Organic Extract
Mass Spectrometry
and Reversed Phase-
Solid Phase
Extraction (RP-SPE)
Hydrophobic
Hydrophilic
18
Moorea sp. HPLC Fraction F
3 2.7 mg
70% MeOH/H
F 131.4
mg
1 8.2 mg
RP-SPE
4 2.1 mg
2 91.6 mg
100% DCM 100% M 90% M/H
Minutes
5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0
mA
u
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
230 nm
330 nm 254 nm
216 nm
C
A D B Minutes
0 1 2 3 4 5 6 7 8 9 10 11 12 13
mA
u
0
200
400
600
800
1000
1200
1400
1600
1800
2000
HPLC-80%MeCN-SynergiFusion Column
HPLC-90% MeOH-SynergiFusion Column
1 2
4
3
Mass Spec m/z 840-Apratoxin A
19
Moorea sp. F2C3 Structure Elucidation
11
1H NMR Spectra
for Apratoxin A in
CDCl3 (500 MHz)-
Published
1H NMR Spectra
for Enq004 F2C3 in
CDCl3 (300 MHz)-
Experimental
20
Moorea sp. F2C3 Structure Elucidation
12
13C NMR Spectra for
Enq004 F2C3 in CDCl3
(75 MHz)-Experimental
13C NMR Spectra for
Apratoxin A in CDCl3 (75
MHz)-Published
21
Roadmap
22
Background and Relevance of Natural Products
Sample Collection by McPhail Lab
Goal of Project
Methods
Culturing
Chromatography
o Compound Hypothesis
Compound Characterization
Results
Known Compounds
New Compounds
Hydrophobic Fraction
Discussion
Relevance of Results
Limitations
Future Work
Acknowledgements
Questions
Apratoxin A-Known Compound
13
Compound Characteristics Toxic against oral carcinoma (KB)
and human colon adenocarcinoma
(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 (Luesch, et al,
2001)
23
Lyngbyabellin B-Known Compound
13
Compound Characteristics Toxic against oral carcinoma
(KB) and human colon
adenocarcinoma (LoVo) cells
Antifungal properties
Protease inhibitory activity
(Luesch, et al, 2000)
24
Apratoxin A Sulfoxide-New Compound
13
Compound Characteristics
Similar to Apratoxin A except
slower to display toxicity
effects
Toxic against Neuro-2A
blastoma cells and NCI-H460
lung cancer cells
25
Apratoxin H-New Compound
13
Compound Characteristics
Highly toxic against NCI-H460
lung cancer cells and Neuro-2A
neuroblastoma cells
26
Moorea sp. Hypothesized Compounds
8 = Unknown: m/z 658
9 = Unknown: m/z 854
7 known compounds & 2 new compounds
1 2 3
4
5 6 7
27
Roadmap
28
Background and Relevance of Natural Products
Sample Collection by McPhail Lab
Goal of Project
Methods
Culturing
Chromatography
o Compound Hypothesis
Compound Characterization
Results
Known Compounds
New Compounds
Hydrophobic Fraction
Discussion
Relevance of Results
Limitations
Future Work
Acknowledgements
Questions
100% Hex
Brine Shrimp Assay (BSA) 1mg/ml: 4 % 0% 100% 100% 89% 100% 100% 85% 0%
Active Fractions
NP-VLC
A 37.2 mg
C 108.1 mg
20% E/H
E 12.7 mg
60% E/H
G 165.0 mg
100% E
H 190.7 mg
25% MeOH/E
I 88.6 mg
100% M
F 131.4 mg
80% E/H
B 62.6 mg
10% EtOAc/H
D 144.2 mg
40% E/H
Moorea species
5
Nabq Mangroves near Sharm el Sheikh, Egypt
BSA 0.1mg/ml: 5 % 0% 96% 97% 7% 85% 94% 7% 0%
50 liters collected over
12 months
0.85 g Organic Extract
Mass Spectrometry
and RP-SPE
Hydrophobic
Hydrophilic
29
RP-HPLC
4
6.8 mg
2
70.5 mg
0 %
3
33.9 mg
67 %
1
1.3 mg
0.1mg/ml: 0 % *
(70% MeCN, 3.0 mL min-1,
Synergi-Fusion-RP 10 x 250 mm)
0 %
ESI-MS m/z 419, 838
[M+Na]+
RP-HPLC
(98% EtOH, 0.6 mL min-1,
Chirobiotic TAG 4.6 x 250 mm)
0 %
D
C
46 %
B
0.1mg/ml: 0 %
E
100 %
Moorea sp. Fraction C3
6
ESI-MS m/z 431
[M+Na]+
Cytotoxicity
Cell Line
Testing
30
Fraction C-Cytotoxicity Assay-Viability
7
0
20
40
60
80
100
120
140
C2
C2A
C2
B
C2
B3
C2
C2B
C2
D2
C2
E
C2
E2
C2
F2
C2
F3
C2
G
C3
C
C3
D
C3
E
% o
f co
ntr
ol r
esp
on
se (
Ne
uro
-2
A C
ells
)
Fraction
Moorea sp. Fraction C Neuro2A Blastoma Assay-Viable Cells
p<0.01
31
Hypothesized Masses and Applications
8
Mass Spectrometry
Determination Structural Components
No published compounds
with same masses
o MarinLit
ESI-Mass Spectrometry-Direct Inject C2C2B
o m/z 403, [M+Na]+
C2E2
o m/z 431 [M+Na]+
C2F2
o m/z 471, [M+Na]+
0
20
40
60
80
100
120
140
C2
C2A
C2
B
C2
B3
C2
C2B
C2
D2
C2
E
C2
E2
C2
F2
C2
F3
C2
G
C3
C
C3
D
C3
E
% o
f co
ntr
ol r
esp
on
se
(N
eu
ro-2
A c
ells
)
Fraction
Moorea sp. Fraction C Neuro2A Blastoma Assay-Viable Cells
32
Results Summary
33
2 Known Compounds Characterized
Apratoxin A
Lyngbyabellin B
2 New Compounds Characterized
Apratoxin A Sulfoxide
Apratoxin H
New Hydrophobic Fractions
0
50
100
150
C2
C2A
C2
B
C2
B3
C2
C2B
C2
D2
C2
E
C2
E2
C2
F2
C2
F3
C2
G
C3
C
C3
D
C3
E
%o
f co
ntr
ol r
esp
on
se
(Ne
uro
-2A
ce
lls)
Fraction
Moorea sp. Fraction C Neuro2A Blastoma Assay-Viable Cells
Roadmap
34
Background and Relevance of Natural Products
Sample Collection by McPhail Lab
Goal of Project
Methods
Culturing
Chromatography
o Compound Hypothesis
Compound Characterization
Results
Known Compounds
New Compounds
Hydrophobic Fraction
Discussion
Relevance of Results
Limitations
Future Work
Acknowledgements
Questions
9
Abundance of Apratoxin Production
Gene Cluster
Regulated by Environment
More Knowns
Thornburg classified two more
apratoxins and heptachlorin
Conclusions-Known Compounds
35
(Chen, Liu, Luesch, 2011)
9
New apratoxins
Medicinal Applications
New Chemical Motifs
More Unknowns?
More separation
Conclusions-Unknown Compounds
36
9
Predicted new
hydrophobic
compounds
Applications
Gerwick Group
Curacin A
FDA Clinical
Trials
www.ucsd.edu
Conclusions-Hydrophobic Fraction
37
9
Prolific secondary metabolite producer
Source of potent and selective analogues
for synthetic scaffolds
Regulated by Environment
Culturable
Produces secondary metabolites in
absence of environmental stresses
More with stress?
Conclusions-Moorea sp. Overall
38
Roadmap
39
Background and Relevance of Natural Products
Sample Collection by McPhail Lab
Goal of Project
Methods
Culturing
Chromatography
o Compound Hypothesis
Compound Characterization
Results
Known Compounds
New Compounds
Hydrophobic Fraction
Discussion
Relevance of Results
Limitations
Future Work
Acknowledgements
Questions
15
Limitations of the Project
40
Relying on slow growing organism
Sensitive to culture media
Low resolution LC-MS can’t definitively
identify compounds
Not suitable for untargeted compound
screening
Roadmap
41
Background and Relevance of Natural Products
Sample Collection by McPhail Lab
Goal of Project
Methods
Culturing
Chromatography
o Compound Hypothesis
Compound Characterization
Results
Known Compounds
New Compounds
Hydrophobic Fraction
Discussion
Relevance of Results
Limitations
Future Work
Acknowledgements
Questions
15
Spectroscopic analysis of
pure compounds from
HPLC
High interest in obtaining
enough of the unknown m/z
658 for characterization
Obtain larger quantities of
pure unknowns for
biological testing on
human cell lines
Future Directions of the Project
42
10
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
Acknowledgements
43
Support Wanda Crannell
Perakis Lab
15
Questions?
44