Structure elucidation of a80915 a from streptomyces cp54 7
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Transcript of Structure elucidation of a80915 a from streptomyces cp54 7
Structure elucidation of A80915A from Streptomyces CP54-7
SCOTT CAMPIT, 08/03/15
Outline:
Why look at marine derived bacteria for future medicine?
Streptomyces CP54-7 and isolating A80915A Significance and future goals
Actinomyces as a source of medicine
Olano, C., Méndez, C., & Salas, J. A. (2009). Antitumor Compounds from Marine Actinomycetes. Marine Drugs, 7(2), 210–248. doi:10.3390/md7020210
Doxorubicin – Chemotherapeutic
Miyaoka, R., Hosokawa, M., Ando, M., Mori, T., Hamaguchi, H., & Takeyama, H. (2014). In Situ Detection of Antibiotic Amphotericin B Produced in Streptomyces nodosus Using Raman
Microspectroscopy. Marine Drugs, 12(5), 2827–2839. doi:10.3390/md12052827
Amphotericin B – Antifungal
Why look towards the ocean?
Multidrug resistant organisms
Huge diversity
Unexplored potential
http://www.f.u-tokyo.ac.jp/~fukuyama/project2000.htm
http://barcelona-ocean.com/training/course/ecology-marine-microorganismshttps://www.superstock.com/stock-
photos-images/824-94770
MethodologyHypothesis: Marine obligate actinomycetes have the potential to produce novel compounds that can be further developed to treat cancer and other human diseases.
1. Isolate bacteria from sediment
samples
2. Culture isolated strains in growth
media
3. Extract chemical
compounds produced by
bacteria in growth media
4. Separate the crude extract into pure compounds
5. Analyze the data
CP54-7 1. High yielding strain 2. Interesting
chemical profile
1L Media Study of CP54-7
Media A
Media C
Media S
Media F
15L Media Study of CP54-7A
15L crude extract: 4.79838g Flash column chromatography
separated compounds further: 20:80% MeOH/H2O -
429.86mg 40:60% MeOH/H2O -
62.950mg 60:40% MeOH:H2O -
133.26mg 80:20% MeOH/H2O - 1.4762g 100% MeOH - 703.14mg
http://www.sorbeadindia.com/flash-chromatography.html
Purification of CP54-7A 100% Divided into 7
FractionsF6 - 210.94mg
6100% MeOH
Purification of CP54-7A 100% Divided into 7
FractionsF6 - 210.94mg
80% Divided into 11 fractions
F11 - 88.12mg
6
11
100% MeOH
80% MeOH
Isolation of A80915AThese appeared to be the same compound based on
previous H-NMR and RT-HPLC chemical profile100% F6 - 10.18mg80% F11 - 3.2mg
100% MeOH crude profile50-100% 20 min ACN
80% MeOH purification80-100% 20 min MeOH
1D NMR Analysis – Structure elucidation
1H-NMR 13C-NMR / DEPT
Cl
Primary
Secondary
Tertiary
Quaternary
C-NMR
2D NMR Analysis - COSY Correlations
Shows how nearby protons are interacting with each
other A.
A.
B.
B.
Click to add text
H-NMR 1
H-N
MR
2
2D NMR Analysis - HSQC Correlations
Shows how adjacent C-H are interacting with each
other
H-N
MR
C-NMR
C.
D.
C.
D.
2D NMR Analysis - HMBC Correlations
Shows you how neighboring C-H bonds are interacting from multiple
bond lengths
C-NMR
H-N
MR
E.
E2. E1.
E3-4
F.
F1
F2F3
Figure 3B - A80915A induces apoptosis of HCT-116 cells,
analyzed using FACS
Future goals: Continue separation of other organic
compounds in crude extract If novel compounds are found, elucidate
the structure and subject the compound to biochemical assays
Figure out the mechanism of action for these novel compounds
AcknowledgementsSpecial thanks to: Dr. Taro Amagata, Dr. Carmen Domingo, Dr. Raymond
Esquerra, Dr. Tester Baird and Dr. Frank Bayliss My lab mates: Stephanie Gee, Eric Yip, Peishan Li,
Juliana Thai, Duyen Vo, Jorge Rivas and John Flores The Department of Chemistry & Biochemistry and
Department of Biology NIH-MARC Grant No: 5T34GM008574-19