Ansalactams B-D Illustrate Further Biosynthetic Plasticity within the Ansamycin Pathway

Transcription

1 Ansalactams B-D Illustrate Further Biosynthetic Plasticity within the Ansamycin Pathway Tu Cam Le, Inho Yang, Yeo Joon Yoon, Sang-Jip Nam,*, and William Fenical *, Department of Chemistry and Nano Science, Global Top 5 program, Ewha Womans University, Seoul 03760, Korea Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California-San Diego, La Jolla, CA S1

2 Table of Contents General experimental procedures Collection and phylogenetic analysis of strain CNH-189 Cultivation and extraction Isolation of ansalactams B-D S4 S4 S4 S5 Antibacterial Assay S5 Table S1. NMR spectral data for ansalactams B (1), C (2), and D (3) in methanol-d 4.. Figure S1. Key COSY and HMBC correlations for planar structure of ansalactam C (2) Figure S2. Key ROESY correlations for determination of the relative stereochemistry of 2 (a) and 3 (b) Figure S3. 1 H NMR Spectrum (600 MHz) of ansalactam B (1) in methanol-d 4... Figure S4. 13 C NMR Spectrum (75 MHz) of ansalactam B (1) in methanol-d 4. Figure S5. gcosy Spectra (600 MHz) of ansalactam B (1) in methanol-d 4.. Figure S6. ghsqc Spectra (600 MHz) of ansalactam B (1) in methanol-d 4.. Figure S7. ghmbc Spectra (600 MHz) of ansalactam B (1) in methanol-d 4. Figure S8. ROESY Spectra (600 MHz) of ansalactam B (1) in methanol-d 4 Figure S9. 1 H NMR Spectrum (600 MHz) of ansalactam C (2) in methanol-d 4 Figure S C NMR Spectrum (75 MHz) of ansalactam C (2) in methanol-d S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S2

3 Figure S11. gcosy Spectra (600 MHz) of ansalactam C (2) in methanol-d 4.. Figure S12. ghsqc Spectra (600 MHz) of ansalactam C (2) in methanol-d 4.. Figure S13. ghmbc Spectra (600 MHz) of ansalactam C (2) in methanol-d 4. Figure S14. ROESY Spectra (600 MHz) of ansalactam C (2) in methanol-d 4 Figure S15. 1 H NMR Spectrum (600 MHz) of ansalactam D (4) in methanol-d 4... Figure S C NMR Spectrum (75 MHz) of ansalactam D (4) in methanol-d 4. Figure S17. gcosy Spectra (600 MHz) of ansalactam D (4) in methanol-d 4.. Figure S18. ghsqc Spectra (600 MHz) of ansalactam D (4) in methanol-d 4 Figure S19. ghmbc Spectra (600 MHz) of ansalactam D (4) in methanol-d 4... S18 S19 S20 S21 S22 S23 S24 S25 S26 Figure S20. ROESY Spectra (600 MHz) of ansalactam D (4) in methanol-d 4 S27 Figure S21. Energy-minimized model of ansalactam B (1) S28 Figure S22. Energy minimized model of ansalactam D (3) S29 S3

4 General experimental procedures. The optical rotations were measured using a Rudolph Research Autopol III polarimeter with a 10-cm cell. UV spectra were recorded in a Varian Cary UV-visible spectrophotometer with a path length of 1 cm and IR spectra were recorded on a Perkin- Elmer 1600 FT-IR spectrometer. 1 H and 2D NMR spectra data were recorded at 500 MHz in methanol-d 4 solution containing Me 4 Si as internal standard on Varian Inova spectrometers. 13 C NMR spectra were acquired at 75 MHz on a Varian Inova spectrometer. High resolution ESI-MS spectra were measured on a JEOL, JMS-AX505WA mass spectrometer. Low resolution LC/MS data were measured using a Hewlett-Packard series 1100 LC/MS system with a reversed-phase C 18 column (Phenomenex Luna, 4.6 mm 100 mm, 5 m, flow rate of 0.7 ml/min). Collection and phylogenetic analysis of strain CNH-189. The actinobacterium (strain CNH189) was isolated from a nearshore marine sediment collected off Oceanside, California and 16S rrna gene sequence analysis indicated a Streptomyces sp. (accession number HQ214120). Cultivation and extraction. The bacterium (strain CNH189) was cultured in sixty 2.8 L Fernbach flasks each containing 1 L of a natural seawater-based medium (10 g starch, 4 g yeast extract, 2 g peptone, 1 g CaCO 3, 40 mg Fe 2 (SO 4 ) 3 4H 2 O, 100 mg KBr) and shaken at 230 rpm at 27 C. After seven days of cultivation, sterilized XAD-16 resin (20 g/l) was added to adsorb the organic components and the culture and resin were shaken at 215 rpm for 2 hours. The resin was filtered, washed with deionized water, and eluted with acetone. The acetone was evaporated, and the resulting aqueous layer was extracted with ethyl acetate (3 500 ml). The ethyl acetate-soluble layer was dried in vacuo to yield 4.5 g of organic extract. S4

5 Isolation of ansalactams B-D. The organic extract (4.5 g) was fractionated by open column chromatography on silica gel (25 g), eluted with a step gradient of dichloromethane and methanol. The dichloromethane/methanol 50:1 fraction contained a mixture of ansalactams, which were further purified by reversed-phase HPLC (Phenomenex Luna C-18 (2), mm, 2.0 ml/min, 5 m, 100 Å, UV = 210 nm, H 2 O:CH 3 CN = 35:65) to afford ansalactams B (2, 5.0 mg), C (3, 6.0 mg), D (4, 2.0 mg), and A (4, 70.0 mg). Ansalactam B (1): pale yellow oil; [ ] D (c 0.7, methanol); IR (KBr) ν max 3402, 2955, 1684, 1567, 1277, 1031 cm -1 ; UV (methanol) max (log ) 238 (4.3), 278 (4.2) nm; 1 H and 2D-NMR (500 MHz, methanol-d 4 ), see Table 1; HRESITOFMS [M+H] + m/z (C 33 H 40 NO 6, calcd [M+H] ). Ansalactam C (2): pale yellow oil; [ ] 21 D 33 (c 0.05, methanol); IR (KBr) ν max 3354, 2955, 1698, 1451, 1293, 736 cm -1 ; UV (methanol) max (log ) 250 (4.5), 295 (4.1), 315 (3.8) nm; 1 H and 2D-NMR (500 MHz, methanol-d 4 ), see Table 2; HRESITOFMS [M+H] + m/z (C 33 H 42 NO 7, calcd [H+H] ). Ansalactam D (3): pale yellow oil; [ ] 21 D 86 (c 0.3, methanol); IR (KBr) ν max 3402, 2935, 1670, 1451, 1272 cm -1 ; UV (methanol) max (log ) 250 (4.4), 290 (3.9), 315 (3.5) nm; 1 H and 2D-NMR (500 MHz, methanol-d 4 ), see Table 2; HRESITOFMS [M+H] + m/z (C 33 H 40 NO 6, calcd [M+H] ). Antibacterial Assay: Methicillin-resistant Staphylococcus aureus (MRSA) was inoculated in Mueller-Hinton agar media for 24 h at 37 C. The bacterial colony was cultivated in a 15 ml round tube containing 6 ml of Mueller-Hinton broth media at 37 C and 225 rpm for 24 h. Test compounds and a positive control (vancomycin) were dissolved in DMSO and 100 μl were added to a 96-well microtiter plate having 50 μl of Mueller-Hinton broth in each well. The samples were serially diluted and 50 μl of bacterial Mueller-Hinton broth media adjusted to concentration S5

6 of 1/100 diluted McFarland 0.5% standard. The 96-well was incubated for 24 h at 37 C. The minimum inhibitory concentration (MIC) values were determined as the concentration of compound that inhibits 50% of the growth of bacteria. The positive control showed the MIC values in the range of 0.1 to 1 μg/ml. S6

7 Table S1. NMR spectral data for Ansalactams B (1), C (2), and D (3) in methanol-d 4 a no. C, mult. b H (mult, J (Hz)) C, mult. b H (mult, J (Hz)) COSY HMBC C, mult. b H (mult, J (Hz)) COSY HMBC , qc 197.3, qc 191.9, qc , qc 69.3, qc 67.5, qc , CH , CH , d (11.4) 1, 2, 4, 10, 20, , CH , d (14.0) 1, 2, 3, 4, 10, , d (11.4) 2.23, d (14.0) , qc 84.4, qc 82.1, qc , qc 112.0, CH 6.88, s 121.4, qc , qc 162.0, qc 165.8, qc , qc 126.3, qc 130.8, qc , CH 7.79, s 131.0, CH 7.72, s 1, 6, 7, 9, , CH 7.88, s 1, 6, 7, 9, , qc 122.3, qc 124.6, qc , qc 151.0, qc 150.8, qc , qc 171.7, qc 201.6, qc , qc 130.1, qc 88.1, qc , CH 6.39, s 140.3, CH 7.27, s 11, 12-Me, 14-Me 68.6, CH 3.81, s 3, 4, 10, 11, 12, 12-Me, 14, , qc 132.3, qc 136.3, qc , CH 5.41, br s 134.6, CH 5.42, d (11.0) 16 13, 14-Me, 16-Me, , CH 5.77, d (4.2) , CH 2.97, m 35.0, CH , 16-Me, Me 34.0, CH 3.41, m 15, 17 15, 16-Me , CH 4.77, d (8.0) 143.8, CH 6.73, d (11.0) 16 15, 16, 18-Me, , CH 6.24, d (10.0) 16 15, , qc 135.0, qc 130.8, qc , qc 207.5, qc 202.7, qc , CH 2.47, qd (6.8, 4.3) 63.3, qc 47.5, CH 3.23, qd (6.8, 3.4) 20-Me, Me , CH 2.53, dd (9.0, 4.3) 53.4, CH , 2, 3, 4, , CH 2.43, dd (6.8, 3.4) 20, 22 3, 20, , CH 2.87, dt (9.0, 4.5) 43.1, CH 2.53, m 21, , CH 2.59, ddd (14.0, 4.5, 2.4) 21, 24 23, , qc 181.3, qc 186.4, qc , CH , m 43.6, CH , m 22, 25 22, 23, , CH , m 22, 25 22, , m 1.16, m 1.69, m , CH 1.78, m 25.1, CH 1.41, m 24, 26, , CH 1.99, m 24, 26, 27 22, 24, 26, , CH , d (6.8) 23.7, CH , d (6.8) 25 24, 25, , CH , d (6.8) 25 24, 25, , CH , d (6.8) 22.1, CH , d (6.8) 25 24, 25, , CH , d (6.8) 25 24, 25, 26 7-Me 17.5, CH , s 16.0, CH , s 6, 7, , CH , s 6, 7, 8 12-Me 13.3, CH , s 14.3, CH , s 11, , CH , s 11, 12, Me 23.3, CH , s 23.0, CH , s 13, , CH , s 13, 14, Me 21.9, CH , d (6.8) 20.7, CH , d (7.0) 16 15, 16, , CH , d (6.8) 16 15, 16, Me 14.8, CH , s 13.9, CH , s 17, , CH , s 17, 18, Me 11.0, CH , d (6.8) 20.3, CH , s 4, 19, 20, , CH , d (6.9) 20 19, 20, 21 a 600 MHz for 1 H NMR and 75 MHz for 13 C NMR. b Numbers of attached protons were determined by analysis of 2D spectra. S7

8 Figure S1. Key COSY and HMBC correlations for planar structure of ansalactam C (2) S8

9 Figure S2. Key ROESY correlations for determination of the relative stereochemistry of 2 (a) and 3 (b) S9

10 Figure S3. 1 H NMR Spectrum (600 MHz) of Ansalactam B (1) in Methanol-d 4 S10

11 Figure S4. 13 C NMR Spectrum (75 MHz) of Ansalactam B (1) in Methanol-d 4 S11

12 Figure S5. gcosy Spectra (600 MHz) of Ansalactam B (1) in Methanol-d 4 S12

13 Figure S6. ghsqc Spectra (600 MHz) of Ansalactam B (1) in Methanol-d 4 S13

14 Figure S7. ghmbc Spectra (600 MHz) of Ansalactam B (1) in Methanol-d 4 S14

15 Figure S8. ROESY Spectra (600 MHz) of Ansalactam B (1) in Methanol-d 4 S15

16 Figure S9. 1 H NMR Spectrum (600 MHz) of Ansalactam C (2) in Methanol-d 4 S16

17 Figure S C NMR Spectrum (75 MHz) of Ansalactam C (2) in Methanol-d 4 S17

18 Figure S11. gcosy Spectra (600 MHz) of Ansalactam C (2) in Methanol-d 4 S18

19 Figure S12. ghsqc Spectra (600 MHz) of Ansalactam C (2) in Methanol-d 4 S19

20 Figure S13. ghmbc Spectra (600 MHz) of Ansalactam C (2) in Methanol-d 4 S20

21 Figure S14. ROESY Spectra (600 MHz) of Ansalactam C (2) in Methanol-d 4 S21

22 Figure S15. 1 H NMR Spectrum (600 MHz) of Ansalactam D (3) in Methanol-d 4 S22

23 Figure S C NMR Spectrum (75 MHz) of Ansalactam D (3) in Methanol-d 4 S23

24 Figure S17. gcosy Spectra (600 MHz) of Ansalactam D (3) in Methanol-d 4 S24

25 Figure S18. ghsqc Spectra (600 MHz) of Ansalactam D (3) in Methanol-d 4 S25

26 Figure S19. ghmbc Spectra (600 MHz) of Ansalactam D (3) in Methanol-d 4 S26

27 Figure S20. ROESY Spectra (600 MHz) of Ansalactam D (3) in Methanol-d 4 S27

28 Figure S21. Energy-minimized model of ansalactam B (1) (calculated by Chem3D ver 15.0, red = oxygen, white = hydrogen, gray = carbon, pink = lone pair, double solid arrow = ROESY correlation), Upper arrow depicted ROESY correlation between H-22 and 20 Me at beta side of the ring. Lower arrow depicts ROESY between H-3 / H-21 / H-20 at alpha side of the ring S28

29 Figure S22. Energy minimized model of ansalactam D (3) a) model of beta O ether, b) model of alpha O ether between C-4 / C-20 (calculated by Chem3D ver 15.0, red = oxygen, white = hydrogen, gray = carbon, pink = lone pair, double solid arrow = ROESY correlation) S29