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