Natural Occurrence of Organofluorine and other Constituents from Streptomyces sp. TC1

Transcription

1 Natural Occurrence of Organofluorine and other Constituents from Streptomyces sp. TC1 Nanjundan Jaivel, Chokkalingam Uvarani, Ramasamy Rajesh, Devadasan Velmurugan, and Ponnusamy Marimuthu *, Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, , Tamil Nadu, India. Department of Chemistry, School of Chemical Sciences, Bharathiar University, Coimbatore, , Tamil Nadu, India. Department of Crystallography and Biophysics, University of Madras, Chennai, , Tamil Nadu, India. *Corresponding Author Tel: Fax:

2 S1. X-ray crystal data of Compound 1 Crystal data: Data Compound 1 Formula C 17 H 25 FO 2 Formula weight Temperature (K) 293 Crystal system Monoclinic Space group P2 1 /c a (Å) (3) Å b (Å) (5) Å c (Å) (2) Å α ( ) 90 β ( ) (5) γ ( ) 90 Volume (Å 3 ) V = (10) Å 3 Z 4 Density (gcm -3 ) µ (mm -1 ) 0.08 F (000) 608 h min, max -8, 8 k min, max -24, 24 l min, max -19, 19 No. of unique 4229 No.of parameters 181 R_ all, R_ obs 0.093, wr 2 _ all, wr 2 _ obs 0.216, ρ min,max (e Å -3 ) -0.38, 0.30 G.o.F

3 S2. The crystal Packing diagram of the unit cell of compound 1 Crystallographic data of compound 1 have been deposited at the Cambridge Crystallographic Data Centre under the reference number CCDC Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK ( deposit@ccdc.cam.ac.uk). S3. Energy minimized conformer of compound (2S,2'S,4'S)-3 3

4 S4. Changes in the electronic absorption spectra of compounds (1, 2 and 3) (50 µm) with increasing concentrations (0-25 µm) of CT-DNA. The inset shows a fitting of the absorbance data used to obtain the binding constants. 4

5 S5. Emission enhancement spectra of compounds 1, 2 and 3 (50 µm) in the presence of increasing amounts of CT-DNA (0, 5, 10, 15, 20, 25 µm). The arrow shows the emission intensity increases upon increasing the DNA concentration. 5

6 S6. Fluorescence quenching curves of ethidium bromide bound to DNA: [DNA] = 5 µm, [EB] = 5 µm, and [Compound] = 0-50 µm. The inset shows Stern-Volmer plots of the fluorescence titration data used to obtain the quenching constants. 6

7 S7. Synchronous spectra of BSA (2 µm) in the presence of increasing amounts of compounds (1-3) (0-2.5 µm) at wavelength difference of λ = 15 nm. The arrow shows the intensity changes upon increasing in concentration of the compounds. 7

8 S8. Synchronous spectra of BSA (2 µm) in the presence of increasing amounts of compounds (1-3) (0-2.5 µm) at wavelength difference of λ = 60 nm. 8

9 S9. DNA Binding Studies All of the experiments involving the binding of compounds with CT-DNA 1 were carried out in double distilled water with tris(hydroxymethyl)-aminomethane (Tris, 5 mm) and sodium chloride (50 mm) and adjusted to ph 7.2 with hydrochloric acid. A solution of CT-DNA in the buffer gave a ratio of UV absorbance of about 1.9 at 260 and 280 nm, indicating that the DNA was sufficiently free of protein. The DNA concentration per nucleotide was determined by absorption spectroscopy using the molar extinction coefficient value of 6600 M 1 cm 1 at 260 nm. The compounds were dissolved in a mixed solvent of 5% DMSO and 95% Tris-HCl buffer for all of the experiments. Absorption titration experiments were performed with a fixed concentration of the compounds (25 µm) while gradually increasing the concentration of DNA (5-25 µm). While measuring the absorption spectra, an equal amount of DNA was added to both the test solution and the reference solution to eliminate the absorbance of DNA itself. The same experimental procedure was followed for emission studies also. The emission spectra were monitored by keeping the excitation of the test compounds at 380 nm. EB-DNA experiments were conducted by adding our compounds to the Tris-HCl buffer of EB-DNA. The change in the fluorescence intensity was recorded. The excitation and the emission wavelengths were 515 and 607 nm, respectively. S10. In Vitro Cytotoxicity Evaluation by MTT Assay Cytotoxicity studies of the compounds along with cisplatin were carried out on human cervical cancer cell lines (HeLa), stomach adenocarcinoma cell lines (AGS), and colorectal adenocarcinoma cell lines (HCT116), which were obtained from National Centre for Cell Science, Pune, India. Cell viability was carried out using the MTT assay method. 2 The HeLa, AGS and HCT116 cells were grown in Eagles minimum essential medium containing 10% fetal bovine serum (FBS). For the screening experiment, the cells were seeded into 96-well plates in 100 µl of the respective medium containing 10% FBS, at a plating density of 10,000 cells/well, and incubated at 37 C, under conditions of 5% CO 2, 95% air, and 100% relative humidity for 24 h prior to the addition of compounds. The compounds were dissolved in DMSO and diluted in the respective medium containing 1% FBS. After 24 h, the medium was replaced with the respective medium with 1% FBS containing the compounds at various concentrations and incubated at 37 C under conditions of 5% CO 2, 95% air, and 100% relative humidity for 48 h. Triplication was maintained, and the medium not containing the compounds served as the 9

10 control. After 48 h, 10 µl of MTT (5 mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 37 C for 4 h. The medium with MTT was then flicked off, and the formed formazan crystals were dissolved in 100 µl of DMSO. The absorbance was then measured at 570 nm using a microplate reader. The percentage of cell inhibition was determined using the following formula, and a graph was plotted with the percentage of cell inhibition versus concentration. From this, the IC 50 value was calculated: % inhibition = [mean OD of untreated cells (control)/mean OD of treated cells (control)] 100. S11. Sequence Editing, Submission and Phylogenetic analysis The sequences obtained were edited using DNA STAR offline software (DNASTAR, Madison, WI, USA). The edited contigs were then subjected to BLAST analysis ( The sequences were identified as Streptomyces sp. based on 16S rrna homology and processed for sequence submission to the GenBank, NCBI, USA and the GenBank accession number of strain TC1 is KC In order to understand the phylogenetic placement of the Streptomyces sp, additional sequences belonging to the genus Streptomyces were collected from the GenBank. The sequences were aligned using Clustal W and phylogenetic analysis was performed using MEGA5.0 tool. 3 The Neighbour-Joining analysis 4 was performed using the Jukes Cantor algorithm. 5 The algorithm was chosen by estimating the best DNA model that clearly exhibited the prominent nucleotide substitution pattern. The bootstrap test was conducted by resampling the sites of interior branch of the original tree for 1000 times. 6 10

11 Neighbour-Joining tree showing the relationship between strain TC1 (GenBank KC954629) and related Streptomyces sp. based on the 16S rrna sequence References 1. Raja, D. S.; Bhuvanesh, N. S. P.; Natarajan, K. Inorg. Chem. 2011, 50, Blagosklonny, M.; EI-diery, W. S. Int. J. Cancer 1996, 67, Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Nei, M.; Kumar, S. Mol. Biol. Evol. 2011, 28, Saitou, N.; Nei, M. Mol. Biol. Evol. 1987, 4, Jukes, T. H.; Cantor, C. R. Evolution of protein molecules. In: Munro, H. N. (Ed.), Mammalian Protein Metabolism, Academic Press, New York, 1969; pp Felsenstein, J. Evolution 1985, 39,

12 S12. 1 H NMR (400 MHz, CDCl 3 ) spectrum of compound 1 12

13 S C NMR (100 MHz, CDCl 3 ) spectrum of compound 1 13

14 S14. HSQC (400 MHz, CDCl 3 ) spectrum of compound 1 14

15 S15. HMBC (400 MHz, CDCl 3 ) spectrum of compound 1 15

16 S F NMR (282 MHz, CDCl 3 ) spectrum of compound 1 16

17 S17. HRFAB-MS spectrum of compound 1 17

18 S18. 1 H NMR (400 MHz, CDCl 3 ) spectrum of compound 2 18

19 S C NMR (100 MHz, CDCl 3 ) spectrum of compound 2 19

20 S20. HSQC (400 MHz, CDCl 3 ) spectrum of compound 2 20

21 S21. HMBC (400 MHz, CDCl 3 ) spectrum of compound 2 21

22 S22. COSY (400 MHz, CDCl 3 ) spectrum of compound 2 22

23 S23. HRFAB-MS spectrum of compound 2 23

24 S24. 1 H NMR (400 MHz, CDCl 3 ) spectrum of compound 3 24

25 S25. Expanded views of 1 H NMR spectrum of compound 3 25

26 S C APT (100 MHz, CDCl 3 ) spectrum of compound 3 26

27 S27. HSQC (400 MHz, CDCl 3 ) spectrum of compound 3 27

28 S28. HMBC (400 MHz, CDCl 3 ) spectrum of compound 3 28

29 S29. COSY (400 MHz, CDCl 3 ) spectrum of compound 3 29

30 S30. NOESY (400 MHz, CDCl 3 ) spectrum of compound 3 30

31 S31. HRFAB-MS spectrum of compound 3 31