Genetic manipulation of the COP9 signalosome subunit PfCsnE leads to the discovery of pestaloficins in Pestalotiopsis fici

Transcription

1 Supporting Information for: Genetic manipulation of the COP9 signalosome subunit PfCsnE leads to the discovery of pestaloficins in Pestalotiopsis fici Yanjing Zheng,, Ke Ma,, Haining Lyu, Ying Huang #, Hongwei Liu, Ling Liu, Yongsheng Che, Xingzhong Liu, Huixi Zou, Wen-Bing Yin*,, State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, , China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, , China Zhejiang Provincial (Wenzhou) Key Lab for Water Environment and Marine Biological Resources Protection, College of Life and Environmental Science, Wenzhou University, Wenzhou , China # State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, , China State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing *Corresponding author: Wen-Bing Yin, yinwb@im.ac.cn. Tel: S1

2 Table of contents 1. Supplementary methods 2. Supplementary Tables Table S1. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data for 2 in methanol-d 4. Table S2. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data for 3 in DMSO-d 6. Table S3. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data for 4 in DMSO-d 6. Table S4. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data for 5 in methanol-d 4. Table S5. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data for 6 in acetone-d 6. Table S6. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data for 7 in methanol-d 4. Table S7. HR-ESI-MS data of new compounds. 3. Supplementary Figures Figure S1. 1 H NMR spectrum of 1 in methanol-d 4. Figure S2. 13 C NMR spectrum of 1 in methanol-d 4. Figure S3. IR spectrum of 1. Figure S4. 1 H NMR spectrum of 3 in DMSO-d 6. Figure S5. 13 C NMR spectrum of 3 in DMSO-d 6. Figure S6. IR spectrum of 3 Figure S7. ECD spectrum of 3. Figure S8. 1 H NMR spectrum of 4 in DMSO-d 6. Figure S9. 13 C NMR spectrum of 4 in DMSO-d 6. Figure S10. IR spectrum of 4. Figure S11. ECD spectrum of 4. Figure S12. 1 H NMR spectrum of 5 in methanol-d 4. Figure S C NMR spectrum of 5 in methanol-d 4. Figure S14. IR spectrum of 5. Figure S15. 1 H NMR spectrum of 6 in acetone-d 6. Figure S C NMR spectrum of 6 in acetone-d 6. Figure S17. IR spectrum of Supplementary References S2

3 1. Supplementary methods Strains, media and growth conditions. The fungal Pestalotiopsis fici CGMCC and its transformants (Supplementary Table 1) were grown at 25 C on Potato Dextrose Agar (PDA) or Potato Dextrose Broth (PDB) with appropriate antibiotics as required. General analytical methods and equipment overview. HPLC analysis was performed on a Wasters HPLC system (Waster e2695, Waster 2998, Photodiode Array Detector) using a C-18 ODS column ( mm, YMC Pak, 5μm). Semiprepartive HPLC was performed using an ODS column (YMC-Pack ODS-A, mm, 5 μm). JASCO J-815 spectropolarimeter was used to measure CD spectra. Sephadex LH-20 was used to perform the Column chromatography (CC). NMR spectra ( 1 H, 13 C, HMBC, 1 H- 1 H COSY, HSQC) were recorded on a Bruker Avance-500 spectrometer using TMS as internal standard, and chemical shifts were recorded as δ values. HR-ESI-MS utilized on an Agilent Accurate-Mass-QTOF LC/MS 6520 instrument. Chemical analysis and characterization of compounds. PfcsnE mutant 1 and P. fici were grown on PDA and Rice medium for 7 and 25 days, respectively. The culture was extracted with mixed solvent (methanol : acetate ethyl : acetic acid = 10:89:1). After removal of the solvent by vacuum, the residues were dissolved in methanol (5 mg/ml) and then directly injected into the Waters HPLC system. The separation was performed via a linear gradient of acetonitrile in water (0.1% formic acid) from 10% to 100% at a flow rate of 1 ml/min within 30 min. Purification of compounds 1-4 from PfcsnE mutant on PDA. The PfcsnE mutant was cultivated on 10 L PDA at 25 C for 7 days. The fermentation was extracted three times with ethyl acetate. The extract was evaporated under reduced pressure, and the residue (4.0 g) was subjected to a ODS CC using a stepwise gradient of MeOH in H 2 O from 0% to 100% to yield ten fractions (fractions A-J). The fraction B (378.8 mg) and C (343.9 mg) was combined (named as fraction L) and then separated on a Sephadex LH-20 CC eluted with MeOH to give S3

4 two subfractions. The fraction L1 (72.4 mg) was further purified by semi-preparative HPLC (MeCN/H 2 O:40/60, 0.1% formic acid 3 ml/min) to afford 1 (4.4 mg, t R 17.8 min, brown solid). Compounds 2 (2.7 mg, t R min, dark yellow solid), 2 3 (2.4 mg, t R min, yellow solid), and 4 (3.3 mg, t R min, brown solid) was obtained from fraction L2 (47.9 mg) by applying the same isolation procedure. Purification of compounds 5-7 from PfcsnE mutant on rice medium. Similarly, The PfcsnE mutant was cultivated on 2 kg rice at 25 C for 25 days. Then, the fermentation was extracted three times with ethyl acetate. The organic solvent was evaporated to dryness under vacuum to obtain 9.0 g of crude extract. The extract was fractionated on a ODS CC using a stepwise gradient elution of MeOH-H 2 O (0:1 1:0) to give 10 fractions (fractions A-J). The subfraction F (1.35 g) was further purified by Sephadex LH-20 eluted with MeOH to give two subfractions (Fr. F1.and Fr. F2). The Fr. F1 (53.8 mg) was isolated by semi-preparative HPLC (55:45 MeCN/H 2 O, 0.1% formic acid 2.5 ml/min) to afford 5 (3.6 mg, t R min, yellow solid), and Fr. F2 (24.6 mg) was purified by semi-preparative HPLC (45:55 MeCN/H 2 O, 0.1% formic acid 2.5 ml/min) to yield 6 (1.9 mg, t R min, brown solid). Compound 7 (13.2 mg, t R min, yellow solid) 3 was isolated via semi-preparative HPLC (65:35 MeCN/H 2 O, 0.1% formic acid 2.5 ml/min) from fraction H. S4

5 Table S1. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data for 2 in methanol-d 4. 2 reference 2 position δ C δ H (J Hz) δ C δ H (J Hz) , d (2.0) , s , d (2.0) , s CH , s ,s , q (7.0) , q (6.9) , d (7.0) , d (7.3) 4-OCH , s , s S5

6 Table S2. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data for 3 in DMSO-d 6. position δ C δ H (J Hz) , d (2.5) , d (2.5) a , dd (8.0,16.0) 7b 3.03, dd (8.0,16.0) , d (8.5) , s , s a , t (1.5) 4b 5.31, t (1.5) , s 3 S6

7 Table S3. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data for 4 in DMSO-d 6. position δ C 4 δ H (J Hz) , d (3.0) , d (2.5) a , dd (5.5,17.0) 7b 2.52, dd (5.5,17.0) , m , s , s a , t (1.5) 4b 5.29, t (1.5) , s S7

8 Table S4. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data for 5 in methanol-d 4. position δ C δ H (J Hz) s a d (3.1) d (3.1) a d (7.5) t (7.5) s s s s S8

9 Table S5. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data for 6 in acetone-d 6. position δ C δ H (J Hz) s br m o o o o t (6.8) s S9

10 Table S6. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data for 7 in methanol-d 4. Pos. 7 Pestalolide 3 δ C δ H (J Hz) δ C δ H (J Hz) br br t (7.8) t (7.8) m quin (7.8) o m o m o m o m t (7.0) t (7.2) s s S10

11 Table S7. HR-ESI-MS data of new compounds compound (+)-HRESIMS Observed (m/z) Ion Calculated Formula Ion Calculated (m/z) [M+Na] + C 27 H 32 O 6 Na [M+Na] + C 16 H 18 O 3 Na [M+Na] + C 16 H 18 O 3 Na [M+H] + C 16 H 21 O [M+H] + C 12 H 20 O S11

12 Figure S1. 1 H NMR spectrum of 1 in methanol-d 4. S12

13 Figure S2. 13 C NMR spectrum of 1 in methanol-d 4. S13

14 Figure S3. IR spectrum of 1. S14

15 Figure S4. 1 H NMR spectrum of 3 in DMSO-d 6. S15

16 Figure S5. 13 C NMR spectrum of 3 in DMSO-d 6. S16

17 Figure S6. IR spectrum of 3. S17

18 Figure S7. ECD spectrum of 3. S18

19 Figure S8. 1 H NMR spectrum of 4 in DMSO-d 6. S19

20 Figure S9. 13 C NMR spectrum of 4 in DMSO-d 6. S20

21 Figure S10. IR spectrum of 4. S21

22 Figure S11. ECD spectrum of 4. S22

23 Figure S12. 1 H NMR spectrum of 5 in methanol-d 4. S23

24 Figure S C NMR spectrum of 5 in methanol-d 4. S24

25 Figure S14. IR spectrum of 5. S25

26 Figure S15. 1 H NMR spectrum of 6 in acetone-d 6. S26

27 Figure S C NMR spectrum of 6 in acetone-d 6. S27

28 Figure S17. IR spectrum of 6. S28

29 4. Supplementary References (1) Zheng, Y.; Wang, X.; Zhang, X.; Li, W.; Liu, G.; Wang, S.; Yan, X.; Zou, H.; Yin, W. B. Sci. China Life Sci. 2017, 60, (2) Yang, X.-L.; Awakawa, T.; Wakimoto, T.; Abe, I. Tetrahedron Lett. 2013, 54, (3) Klaiklay, S.; Rukachaisirikul, V.; Tadpetch, K.; Sukpondma, Y.; Phongpaichit, S.; Buatong, J.; Sakayaroj, J. Tetrahedron 2012, 68, S29