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, 100101, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, 100049, China Zhejiang Provincial (Wenzhou) Key Lab for Water Environment and Marine Biological Resources Protection, College of Life and Environmental Science, Wenzhou University, Wenzhou 325035, China # State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850 *Corresponding author: Wen-Bing Yin, E-mail: yinwb@im.ac.cn. Tel: 86-10-64806170 S1
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 S13. 13 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 S16. 13 C NMR spectrum of 6 in acetone-d 6. Figure S17. IR spectrum of 6. 4. Supplementary References S2
1. Supplementary methods Strains, media and growth conditions. The fungal Pestalotiopsis fici CGMCC3.15140 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 (250 4.6mm, YMC Pak, 5μm). Semiprepartive HPLC was performed using an ODS column (YMC-Pack ODS-A, 10 250 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
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 18.09 min, dark yellow solid), 2 3 (2.4 mg, t R 18.35 min, yellow solid), and 4 (3.3 mg, t R 18.71 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 15.529 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 17.341 min, brown solid). Compound 7 (13.2 mg, t R 25.562 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
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) 2 167.0 166.9 3 88.6 5.59, d (2.0) 88.6 5.56, s 4 171.4 171.4 5 98.9 6.14, d (2.0) 98.8 6.11, s 6 162.7 162.7 1 128.3 128.3 1 -CH 3 12.1 1.90, s 12.1 1.87,s 2 131.0 6.65, q (7.0) 131.0 6.26, q (6.9) 3 14.3 1.88, d (7.0) 14.3 1.85, d (7.3) 4-OCH 3 56.9 3.88, s 57.3 3.86, s S5
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) 1 153.7 2 102.9 3 114.9 6.43, d (2.5) 4 150.6 5 113.7 6.63, d (2.5) 6 129.0 7a 30.3 3.11, dd (8.0,16.0) 7b 3.03, dd (8.0,16.0) 8 88.9 4.50, d (8.5) 9 70.0 10 24.8 1.11, s 11 26.3 1.11, s 1 84.9 2 93.0 3 126.4 4a 122.0 5.34, t (1.5) 4b 5.31, t (1.5) 5 23.3 1.92, s 3 S6
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) 1 146.6 2 110.6 3 117.2 6.48, d (3.0) 4 149.6 5 116.6 6.53, d (2.5) 6 121.7 7a 31.3 2.82, dd (5.5,17.0) 7b 2.52, dd (5.5,17.0) 8 67.9 3.60, m 9 77.0 10 20.4 1.25, s 11 25.7 1.12, s 1 86.0 2 93.6 3 126.7 4a 121.6 5.32, t (1.5) 4b 5.29, t (1.5) 5 23.3 1.92, s S7
Table S4. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data for 5 in methanol-d 4. position δ C 2 79.8 5 δ H (J Hz) 3 49.5 2.69 s 4 195.3 4a 121.4 5 108.7 7.01 d (3.1) 6 151.9 7 125.8 6.91 d (3.1) 8 137.2 8a 153.0 9 28.9 3.33 d (7.5) 10 123.9 11 133.2 5.53 t (7.5) 12 68.7 3.96 s 13 13.9 1.77 s 14 26.7 1.43 s 15 26.7 1.43 s S8
Table S5. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data for 6 in acetone-d 6. position δ C 2 172.8 3 129.8 4 161.5 6 δ H (J Hz) 5 97.8 6.11 s 1 68.4 4.67 br 2 36.4 1.77 m 3 26.2 1.36 o 4 29.9 1.36 o 5 32.6 1.36 o 6 23.3 1.36 o 7 14.3 0.88 t (6.8) 8 9.0 1.86 s S9
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) 2 178.1 175.7 3 123.1 122.8 4 164.1 160.5 5 73.2 4.76 br 71.4 4.65 br 1 27.9 2.47 t (7.8) 27.1 2.40 t (7.8) 2 28.7 1.55 m 27.6 1.50 quin (7.8) 3 30.6 1.34 o 29.4 1.28-1.24 m 4 30.1 1.34 o 28.9 1.34-1.28 m 5 32.9 1.34 o 31.6 1.34-1.26 m 6 23.7 1.34 o 22.5 1.31-1.27 m 7 14.4 0.91 t (7.0) 14.0 0.89 t (7.2) 8 8.4 1.79 s 8.5 1.82 s S10
Table S7. HR-ESI-MS data of new compounds compound (+)-HRESIMS Observed (m/z) Ion Calculated Formula Ion Calculated (m/z) 1 475.2093 [M+Na] + C 27 H 32 O 6 Na 475.2091 3 281.1169 [M+Na] + C 16 H 18 O 3 Na 281.1148 4 281.1169 [M+Na] + C 16 H 18 O 3 Na 281.1148 5 277.1438 [M+H] + C 16 H 21 O 4 277.1434 6 229.1439 [M+H] + C 12 H 20 O 4 229.1434 S11
Figure S1. 1 H NMR spectrum of 1 in methanol-d 4. S12
Figure S2. 13 C NMR spectrum of 1 in methanol-d 4. S13
Figure S3. IR spectrum of 1. S14
Figure S4. 1 H NMR spectrum of 3 in DMSO-d 6. S15
Figure S5. 13 C NMR spectrum of 3 in DMSO-d 6. S16
Figure S6. IR spectrum of 3. S17
Figure S7. ECD spectrum of 3. S18
Figure S8. 1 H NMR spectrum of 4 in DMSO-d 6. S19
Figure S9. 13 C NMR spectrum of 4 in DMSO-d 6. S20
Figure S10. IR spectrum of 4. S21
Figure S11. ECD spectrum of 4. S22
Figure S12. 1 H NMR spectrum of 5 in methanol-d 4. S23
Figure S13. 13 C NMR spectrum of 5 in methanol-d 4. S24
Figure S14. IR spectrum of 5. S25
Figure S15. 1 H NMR spectrum of 6 in acetone-d 6. S26
Figure S16. 13 C NMR spectrum of 6 in acetone-d 6. S27
Figure S17. IR spectrum of 6. S28
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, 656-664. (2) Yang, X.-L.; Awakawa, T.; Wakimoto, T.; Abe, I. Tetrahedron Lett. 2013, 54, 5814-5817. (3) Klaiklay, S.; Rukachaisirikul, V.; Tadpetch, K.; Sukpondma, Y.; Phongpaichit, S.; Buatong, J.; Sakayaroj, J. Tetrahedron 2012, 68, 2299-2305. S29