Supporting Information - images.nature.com fileS2 General procedures: UV spectra were measured using...

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Supporting Information

New 12,8-Eudesmanolides from Eutypella sp. 1-15

Yuezhou Wang1,2,#

, Yue Wang1,2,#

, An-an Wu3, Lei zhang

1,2, Zhiyu Hu

1,2, Huiying

Huang1,2

, Qingyan Xu1,2,*

and Xianming Deng1,2,*

1State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling

Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China. 2State-province Joint Engineering Laboratory of Targeted Drugs from Natural Products,

Xiamen University, Xiamen, Fujian 361102, China. 3State Key Laboratory for Physical Chemistry of Solid Surface, College of Chemistry and

Chemical Engineering, Xiamen University, Xiamen, Fujian, 361005,China. #These authors contributed equally to this work.

*Correspondence: Professor Qingyan Xu or Professor Xianming Deng, E-mail:

xuqingyan@xmu.edu.cn or xmdeng@xmu.edu.cn.

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General procedures: UV spectra were measured using a Jasco V-530

spectrophotometer. Optical rotations were acquired on a PerkinElmer 341 polarimeter

(PerkinElmer Inc.) using a 1 cm cell. IR spectra were acquired on a PerkinElmer 552

spectrophotometer. 1H, 13C, and 2D NMR spectra were obtained on a Bruker

Avance-600 spectrometer (600 MHz) using TMS as the internal standard. Electrospray

ionization (ESI) low-resolution LC/MS data were medsured on a Thermo-Finnigan LCQ

Advantage mass spectrometer. High-resolution electrospray ionization mass spectra

(HR-ESI-MS) was obtained on a Bruker LC-QTOF mass spectrometer. ECD spectrum

was recorded on a Circular Dichroism Spectrometer (JASCO Corporation, Japan). TLC

detection was carried out using precoated silica gel GF254 plates (10−40 μm, Qingdao

Marine Chemical Plant). Column chromatography was performed with silica gel

(200−300 mesh, Qingdao Marine Chemical Plant), reversed-phase RP-18 (40−63 μm,

Merck), and Sephadex LH-20 (Amersham Biosciences).

Fermentation and Extraction: The strain Eutypella sp. 1-15 was inoculated on rice

medium containing rice 1000g, NaCl 100g in 1.0 L water and cultured at 28 ºC for 27

days. A total of 2 L of fungal solid culture was prepared. The fermented agar cakes were

diced and extracted with EtOAc−MeOH− AcOH (v/v/v, 80/15/5, 3 × 4 L). After the

removal of solvents under vacuum, the extract was suspended in EtOAc and washed with

H2O; then the EtOAc layer was concentrated and resuspended in MeOH and petroleum

ether. The MeOH layer was concentrated to give the crude extract (5.6 g).

Isolation of compounds 1−4: The MeOH extract was fractionated by

medium-pressure liquid chromatography over an RP-18 column (170 g) eluting with a

MeOH−H2O gradient (v/v, from 10% to 100% in 4 h, flow rate of 25 mL/min) to afford

fractions a−h. The 30-35% MeOH fraction, b (967.7 mg) , was sequentially subjected to

Sephadex LH-20 (2.5 × 150 cm) eluting with MeOH to get subfractions b-5 (40.1 mg),

then subjected to silica gel chromatography eluting with petroleum ether /acetone (v/v,

15:1) to afford 3 (5.1 mg). The 45-60% MeOH fraction, d (775.3 mg), was subjected to

Sephadex LH-20 (2.5 × 150 cm) eluting with MeOH to afford subfractions d-4 (180.0

mg), d-6 (149.2 mg). d-4 was subjected Sephadex LH-20 (2.5 × 80 cm) eluting with

acetone−MeOH (v/v, 4:1) to affod d-4-2 (75.4 mg), d-4-3 (35.9 mg), d-4-2 was subjected

to silica gel chromatography eluting with petroleum ether/acetone (v/v, 30:1) to afford 5

(49.1 mg). d-4-3 was chromatographied on LH-20 (2.5 × 80 cm) eluting with acetone to

afford 4 (6.1 mg) as a pure compound (elution volume 50-64 mL). d-6 was subjected to

silica gel chromatography eluting with petroleum ether/acetone (v/v, 25:1) to give 2 (81.2

mg). The 60-65% MeOH fraction, e (213.4 mg), was subjected to Sephadex LH-20 (2.5 ×

150 cm) eluting with MeOH to give subfraction e-4 (187.1 mg), then subjected to silica

gel chromatography eluting with petroleum ether/acetone (v/v, 20:1) to give 1 (20.1 mg).

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(1): colorless oil; 20

D +10.4 (c 0.87, MeOH); UV (MeOH) λmax (log ε) 200 (3.47), 281

(2.78) nm; IR (KBr): 790, 1001, 1435, 1762, 2927, 3420 cm-1

; 1H and

13C NMR data,

Table 1; HR-ESI-MS m/z 247.1329 [M + H]+ (calcd for C15H19O3 247.1329).

(2): colorless solid; 20

D +70.2 (c 1, MeOH); UV (MeOH) λmax (log ε) 200 (3.46), 329

(2.57) nm; IR (KBr): 994, 1076, 1231, 1614, 1758, 2926, 3419 cm-1

; 1H and

13C NMR

data, Table 1; HR-ESI-MS m/z 245.1172 [M + H]+ (calcd for C15H17O3, 245.1172).

(3): yellow powder;20

D +162.1 (c 0.5，MeOH); UV (MeOH) λmax (log ε) 200 (3.45),

335 (2.31) nm; IR(KBr): 1247, 1374, 1663, 1762, 2359, 2926, 3415 cm-1

; 1H and

13C

NMR data, Table 1; HR-ESI-MS m/z 259.0965 [M + H]+ (calcd for C15H15O4, 259.0965).

(4): colorless oil; 20

D -40.9 (c 0.6，MeOH); UV (MeOH) λmax (log ε) 225(3.48) nm;

IR(KBr): 1010, 1436, 1734, 2918, 3450 cm-1

; 1H and

13C NMR data, Table 1;

HR-ESI-MS m/z [M+H]+

265.1435 (calcd for C15H21O4, 265.1434).

Computational Analysis: In this work, all calculations were performed with the

Gaussian 09 package.1 For the geometry optimization, the calculations were performed

by B3LYP2-3

in conjunction with the basis set of 6-31+G(D,P)4. Analytical frequencies

were calculated in order to confirm that a local minimum has no imaginary frequency.

The ECD data were calculated using TDDFT(CAM-B3LYP)5 with the basis set

6-311+G(D,P)6 for all atoms. As far as the solvent is concerned, its effects were included

by the PCM7 model for all calculations. The ECD spectra were obtained by the following

equation:

where is the width of the band at 1/e height (fixed at 0.30 eV), Ei and Ri are the

excitation energies and rotatory strengths for transition i, respectively.

The specific rotation was calculated using B3LYP in conjunction with the

6-311+G(D,P) basis set.

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Figure S1.1H NMR spectrum of 1 in acetone-d6

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Figure S2.13C NMR spectrum of 1 in acetone-d6

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Figure S3. 1H-1H COSY spectrum of 1 in acetone-d6

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Figure S4. HSQC spectrum of 1 in acetone-d6

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Figure S5. HMBC spectrum of 1 in acetone-d6

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Figure S6. NOESY spectrum of 1 in acetone-d6

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Figure S7. HRESIMS spectrum of 1

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Figure S8.1H NMR spectrum of 2 in Acetone-d6

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Figure S9.13C NMR spectrum of 2 in Acetone-d6

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Figure S10. 1H-1H COSY spectrum of 2 in acetone-d6

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Figure S11.HSQC spectrum of 2 in Acetone-d6

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Figure S12. HMBC spectrum of 2 in Acetone-d6

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Figure S13. HRESIMS spectrum of 2

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Figure S14. 1H NMR spectrum of 3 in acetone-d6

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Figure S15. 13C NMR spectrum of 3 in acetone-d6

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Figure S16. HSQC spectrum of 3 in Acetone-d6

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Figure S17.HMBC spectrum of 3 in Acetone-d6

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Figure S18. HRESIMS spectrum of 3

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Figure S19. 1H NMR spectrum of 4 in Acetone-d6

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Figure S20. 13C NMR spectrum of 4 in Acetone-d6

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Figure S21. HSQC spectrum of 4 in Acetone-d6

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Figure S22. HMBC spectrum of 4 in Acetone-d6

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Figure S23. NOESY spectrum of 4 in Acetone-d6

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Figure S24. Enlarged NOESY spectrum of 4 in Acetone-d6

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Figure S25.HRESIMS spectrum of 4

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Figure S26.1H NMR spectrum of 5 in Acetone-d6

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Figure S27.13C NMR spectrum of 5 in Acetone-d6

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References

1. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.;

Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.;

Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.;

Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.;

Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.;

Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R.

E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth,

G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox,

D. J. Gaussian 09, Revision A. 01;Gaussian, Inc.: Wallingford, CT, 2009.

2. Becke, A.D. Phys. Rev. B 1988, 38, 3098.

3. Lee, C.; Yang, W.; Par, R. G. Phys. Rev. B 1988, 37, 785.

4. Peterson, G.A.; Al-Laham, M.A. J. Chem.Phys. 1991, 94, 6081.

5. Yanai, T.; Tew, D.; Handy, N. Chem. Phys. Lett. 2004, 393.51.

6. Krishman, R.; Binkley, J. S.; Seeger, R.; Pople, J. A. J. Chem. Phys. 1980, 72, 650.

7. Tomasi, J.; Mennucci, B.;Cammi, R. Chem. Rev. 2005, 105, 2999.