Mentors: Kerry McPhail, PhD, Phil Proteau, PhD,
and Christopher C. Thornburg, PhD
Undergraduate Honors and Bioresource Research
Thesis Seminar Presentation
May 28, 2013
Roadmap
 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
2
Roadmap
 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
3
What are Natural Products?
Secondary Metabolites
What produces secondary
metabolites?
Why produce secondary metabolites?
4
Why are Natural Products Important?
Pharmaceutical Applications
www.elucidationimages.com
www.kdna.ucla.edu
www.sciencedirect.co
m
Agricultural
Applications
www.lipitor.com
www.emory.edu
5
Why Search for More Natural Products?
Pharmaceutical Importance: (Newman,
Cragg, 2012)
6
Why Search for More Natural Products?
 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)
7
Cyanobacteria as Sources of
Natural Products
 Ancient Phylum
 Ubiquitous
 Classified alongside Myxobacteria and
Streptomyces as sources of pharmaceuticals (Tan,
2010)
8
Roadmap
 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
Red Sea Exploratory Expedition
 OSU Collections
 May 2007
 12 large collections
 28 small live collections
Mangroves
 High salinity
 Oligotrophic
 Fine sediment deposition
10
Roadmap
 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
11
Culturing Process-Moorea sp.
Purification Process
 Brown contaminant
 Low light
 Overtook brown diatom
Harvest
 6 months
 Yield: 0.85 g
Large-scale cultures of Moorea sp. 12
Materials and Methods
Brine Shrimp
Assay
 Extraction and
Separation
Neuroblastoma
Assay
 Further Separation to
Pure Compounds
 Structure
Elucidation
13
Chromatography Overview
NP-VLC
Chemical Extraction
Hydrophobic
B
100% Hex 10% EtOAc/H
•
•
•
C
D
E
F
G
H
I
20% E/H
40% E/H
60% E/H
80% E/H
100% E
25% MeOH/E
100% M
NP-VLC:Normal Phase
Vacuum Liquid
Chromatography
RP-SPE: Reversed Phase
Solid Phase Extraction
RP-HPLC: Reversed
Phase High Performance
Liquid Chromatography
RP-SPE
1
2
3
4
70%
MeOH/H
90% M/H
100% M
100% DCM
5b
1200
1100
1000
RP-HPLC
2
900
mAu
A
Hydrophilic
800
5a
4
700
600
500
400
1
300
200
3
100
0
0
1
2
3
4
5
6
7
8
9
10
11
Minutes
12
13
14
15
16
17
18
19
20
21
22
14
Materials and Methods
4
15
Moorea species
Nabq Mangroves near Sharm el Sheikh, Egypt
 50 liters collected over
12 months
 0.85 g Organic Extract
Active Fractions
NP-VLC
Hydrophobic
Hydrophilic
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 (BSA)
1mg/ml: 4 %
0%
100%
100%
89%
BSA
0.1mg/ml: 5 %
0%
96%
97%
7%
100%
85%
100%
94%
85%
7%
0%
0%
Mass Spectrometry
and RP-SPE
5
16
Moorea sp. 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
17
Moorea species
Nabq Mangroves near Sharm el Sheikh, Egypt
 50 liters collected over
12 months
 0.85 g Organic Extract
Active Fractions
NP-VLC
Hydrophobic
Hydrophilic
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% EtOAc/H
20% E/H
40% E/H
60% E/H
80% E/H
100% E
25% MeOH/E
100% M
Brine Shrimp Assay (BSA)
1mg/ml: 4 %
0%
100%
100%
89%
BSA
0.1mg/ml: 5 %
0%
96%
97%
7%
5
100%
85%
100%
94%
Mass Spectrometry
and Reversed PhaseSolid Phase
Extraction (RP-SPE)
85%
7%
0%
0%
18
Moorea sp. HPLC Fraction F
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
1800
C
Mass Spec
1600
HPLC-80%MeCNSynergiFusion Column
2000
mAu
1800
1600
mAu
3
2000
1400
m/z 840Apratoxin A
1400
1200
1000
4
800
1200
600
2
1000
400
800
1
200
600
400
A
200
B
0
5.5
6.0
6.5
7.0
7.5
0
D
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
0
1
2
3
4
5
6
7
8
9
10
11
12
13
Minutes
12.0
12.5
13.0
Minutes
19
Moorea sp. F2C3 Structure Elucidation
1H
NMR Spectra
for Enq004 F2C3 in
CDCl3 (300 MHz)Experimental
1H
NMR Spectra
for Apratoxin A in
CDCl3 (500 MHz)Published
11
20
Moorea sp. F2C3 Structure Elucidation
13C
NMR Spectra for
Enq004 F2C3 in CDCl3
(75 MHz)-Experimental
13C
NMR Spectra for
Apratoxin A in CDCl3 (75
MHz)-Published
12
21
Roadmap
 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
22
Apratoxin A-Known Compound
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 depolymerization
13
 Lack of selectivity limits potential
as antitumor agent, but derivatives
are more effective (Luesch, et al,
2001)
23
Lyngbyabellin B-Known Compound
Compound Characteristics
 Toxic against oral carcinoma
(KB) and human colon
adenocarcinoma (LoVo) cells
 Antifungal properties
 Protease inhibitory activity
(Luesch, et al, 2000)
13
24
Apratoxin A Sulfoxide-New Compound
Compound Characteristics
 Similar to Apratoxin A except
slower to display toxicity
effects
 Toxic against Neuro-2A
blastoma cells and NCI-H460
lung cancer cells
13
25
Apratoxin H-New Compound
Compound Characteristics
 Highly toxic against NCI-H460
lung cancer cells and Neuro-2A
neuroblastoma cells
13
26
Moorea sp. 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
27
Roadmap
 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
28
Moorea species
Nabq Mangroves near Sharm el Sheikh, Egypt
 50 liters collected over
12 months
 0.85 g Organic Extract
Active Fractions
NP-VLC
Hydrophobic
Hydrophilic
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% EtOAc/H
20% E/H
40% E/H
60% E/H
80% E/H
100% E
25% MeOH/E
100% M
Brine Shrimp Assay (BSA)
1mg/ml: 4 %
0%
100%
100%
89%
BSA
0.1mg/ml: 5 %
0%
96%
97%
7%
100%
85%
100%
94%
85%
7%
0%
0%
Mass Spectrometry
and RP-SPE
5
29
Moorea sp. Fraction C3
RP-HPLC
0.1mg/ml:
0%
1
1.3 mg
0%
2
70.5 mg
(70% MeCN, 3.0 mL min-1,
Synergi-Fusion-RP 10 x 250 mm)
67 %
*
3
33.9 mg
0%
4
6.8 mg
RP-HPLC
0.1mg/ml:
B
0%
(98% EtOH, 0.6 mL min-1,
Chirobiotic TAG 4.6 x 250 mm)
46 %
0%
C
D
ESI-MS
m/z 419, 838
[M+Na]+
100 %
E
ESI-MS
m/z 431
[M+Na]+
Cytotoxicity
Cell Line
Testing
6
30
Fraction C-Cytotoxicity Assay-Viability
7
C3E
C3D
C3C
C2G
C2F3
C2F2
C2E2
C2E
C2D2
C2C2B
C2B3
C2B
140
120
100
80
60
40
20
0
C2C2A
% of control response (Neuro2A Cells)
Moorea sp. Fraction C Neuro2A
Blastoma Assay-Viable Cells
Fraction
p<0.01
31
Hypothesized Masses and Applications
ESI-Mass Spectrometry-Direct Inject
Mass Spectrometry
Determination
 C2C2B
o m/z 403, [M+Na]+
 C2E2
o m/z 431 [M+Na]+
 C2F2
o m/z 471, [M+Na]+
 Structural Components
 No published compounds
with same masses
o MarinLit
Moorea sp. Fraction C Neuro2A Blastoma Assay-Viable
Cells
% of control response
(Neuro-2A cells)
140
120
100
80
60
40
20
C3E
C3D
C3C
C2G
C2F3
C2F2
C2E2
C2E
C2D2
C2C2B
C2B3
C2B
C2C2A
0
Fraction
8
32
Results Summary
 2 Known Compounds Characterized
 Apratoxin A
 Lyngbyabellin B
 2 New Compounds Characterized
 Apratoxin A Sulfoxide
 Apratoxin H
 New Hydrophobic
Fractions
Moorea sp. Fraction C Neuro2A
150
100
50
0
C2C2A
C2B
C2B3
C2C2B
C2D2
C2E
C2E2
C2F2
C2F3
C2G
C3C
C3D
C3E
%of control response
(Neuro-2A cells)
Blastoma Assay-Viable Cells
Fraction
33
Roadmap
 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
34
Conclusions-Known Compounds
(Chen, Liu, Luesch, 2011)
Abundance of Apratoxin Production
Gene Cluster
 Regulated by Environment
 More Knowns
 Thornburg classified two more
apratoxins and heptachlorin
9
35
Conclusions-Unknown Compounds
New apratoxins
 Medicinal Applications
 New Chemical Motifs
 More Unknowns?
 More separation
9
36
Conclusions-Hydrophobic Fraction
Predicted new
hydrophobic
compounds
 Applications
www.ucsd.edu
Gerwick Group
 Curacin A
 FDA Clinical
Trials
9
37
Conclusions-Moorea sp. Overall
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?
9
38
Roadmap
 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
39
Limitations of the Project
Relying on slow growing organism
Sensitive to culture media
Low resolution LC-MS can’t definitively
identify compounds
 Not suitable for untargeted compound
screening
15
40
Roadmap
 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
41
Future Directions of the Project
 Spectroscopic analysis of
pure compounds from
HPLC


15
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
42
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
Support
 Dr. Kerry McPhail
 Dr. Phil Proteau
 Christopher Thornburg
 Justyna Sikorska
 Oliver Vining
 Mariko Nonogaki
 Wanda Crannell
 Perakis Lab
10
43
Questions?
15
44