SUPPORTING INFORMATION

Transcription

1 SUPPORTING INFORMATION Simpterpenoid A, a Meroterpenoid with a Highly Functionalized Cyclohexadiene Moiety Featuring gem-propane-1,2-dione and Methylformate Groups, from the Mangrove-Derived Penicillium simplicissimum MA-332 Hong-Lei Li,, Rui Xu,,, Xiao-Ming Li,*, Sui-Qun Yang, Ling-Hong Meng, and Bin-Gui Wang*, Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Laboratory of Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Nanhai Road 7, Qingdao , China Department of Life Sciences, Heze University, Daxue Road 2269, Heze , China H.-L.L. and R.X. contributed equally to this work. Contents General Experimental Method General Instruments...S3 Fermentation, Extraction and Isolation...S3 X-Ray Crystallographic Analysis of Compound 1...S5 Bioassays Influenza neuraminidase inhibitory assay...s5 Antimicrobial assay...s6 Figure S1. HRESIMS spectrum of compound 1...S8 Figure S2. 1 H NMR (500 MHz, CDCl 3 ) spectrum of compound 1...S9 Figure S3. 13 C NMR (125 MHz, CDCl 3 ) and DEPT spectra of compound 1...S10 Figure S4. COSY spectrum of compound 1...S11 S1

2 Figure S5. HSQC spectrum of compound 1...S12 Figure S6. HMBC spectrum of compound 1...S13 Figure S7. NOESY spectrum of compound 1...S14 Figure S8. ECD spectrum of compound 1...S15 Figure S9. Crystal packing of compound 1 at 293(2) K...S16 S2

3 General Experimental Method General Instruments Melting points were determined with an SGW X-4 micro-melting-point apparatus. Optical rotations were measured on an Optical Activity AA-55 polarimeter. UV spectra were measured on a PuXi TU-1810 UV-visible spectrophotometer. CD spectra were acquired on a Chirascan spectropolarimeter. 1D and 2D NMR spectra were recorded at 500 and 125 MHz for 1 H and 13 C, respectively, on a Bruker Avance 500 MHz spectrometer with TMS as internal standard. Mass spectra were determined on a VG Autospec 3000 or an API QSTAR Pulsar 1 mass spectrometer. Analytical and semi-preparative HPLC were performed using a Dionex HPLC system equipped with P680 pump, ASI-100 automated sample injector, and UVD340U multiple wavelength detector controlled by Chromeleon software (version 6.80). Commercially available Si gel ( mesh, Qingdao Haiyang Chemical Co.), Lobar LiChroprep RP 18 (40 63 μm, Merck), and Sephadex LH 20 (Pharmacia) were used for open column chromatography. Solvents for extraction and purification were distilled prior to use. The NA inhibitory activity was assayed by the Neuraminidase Inhibitors Screen Kit (Beyotime Institute of Biotechnology, Jiangsu, China). The fluorescence was read on a Tecan GENios multifunctional microplate reader (Männedorf, Switzerland). Fermentation, Extraction and Isolation The fungal strain P. simplicissimum MA-332 was isolated from the rhizosphere of the marine mangrove plant Bruguiera sexangula var. rhynchopetala, collected at Hainan Island, China, in March The fungal strains were isolated by using a S3

4 protocol as described in our previous report. 1 Fungal identification was performed by analysis of the ITS region of the rdna as described previously. 1 The resulting sequence data of P. simplicissimum MA-332, which was most similar to the sequences of P. simplicissimum MFPX-FS05 (100% compared with KR296878), has been deposited in GenBank (with accession no KU612221). The strains are preserved at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology of the Chinese Academy of Sciences (IOCAS). For chemical investigation, the fresh mycelia of P. simplicissimum MA-332 were grown on PDA medium at 28 C for 4 days and were then inoculated into 60 1 L conical flasks with solid rice medium (each flask contained 70 g rice, 0.1 g corn flour, 0.3 g peptone, 0.1 g sodium glutamate, and 100 ml naturally sourced and filtered seawater, which was obtained from the Huiquan Gulf of the Yellow Sea near near the campus of IOCAS, ph ). The whole fermented cultures were extracted three times with EtOAc, which were evaporated under reduced pressure to afford an extract (5.8 g). The extract was fractionated by Si gel vacuum liquid chromatography (VLC) using different solvents of increasing polarity from petroleum ether (PE) to MeOH to

5 Simpterpenoid A (1): initially obtained as yellowish amorphous powder and crystals were obtained by slow evaporation and slow diffusion of DMSO into a saturated mixture of CHCl 3 MeOH (1:1) after three months; mp C; [α] 25 D (c 0.13, MeOH); UV (MeOH) λ max (log ε) 204 (3.23), 250 (2.89) nm; ECD (2.12 mm, MeOH) λ max (Δε) 215 (+0.49), 264 (+1.01), 339 ( 2.27) nm; 1 H and 13 C NMR data, see Table 1; ESIMS m/z [M + H] +, [M + Na] + ; HRESIMS m/z [M + H] + (calcd for C 27 H 37 O + 7, ), [M + Na] + (calcd for C 27 H 36 O 7 Na +, ). X-Ray Crystallographic Analysis of Compound 1 All crystallographic data were collected on an Agilent Xcalibur Eos Gemini CCD plate diffractometer equipped with a graphite-monochromatic Cu-Kα radiation (λ = Å) at 293(2) K. The data were corrected for absorption by using the program SADABS. 2 The structure was solved by direct methods with the SHELXTL software package. 3 All non-hydrogen atoms were refined anisotropically. The H atoms were located by geometrical calculations, and their positions and thermal parameters were fixed during the structure refinement. The structure was refined by full-matrix least-squares techniques. 4 Bioassays Influenza neuraminidase inhibitory assay The influenza neuraminidase inhibitory assay was carried out in a microplate using a modified procedure of Influenza Neuraminidase Inhibitors Screen Kit. 5 The S5

6 influenza neuraminidase solvent was diluted 100 times with its buffer solution. Each reaction mixture contained 70 μl influenza neuraminidase buffer, 10 μl diluted influenza neuraminidase solvent, 0 10 μl samples solvent (100 nm, DMSO), 0 10 μl milli-q water and 10 μl influenza neuraminidase substrate (2 -(4-methylumbelliferyl)-a-D-acetylneuraminic acid). The homogeneous mixture was incubated at 37 C for 2 min to guarantee that interplay between sample and influenza neuraminidase, before adding the influenza neuraminidase substrate. Then, the catalytic reaction was processed at 37 C for 30 min and a fluorescent product was quantified by a multifunctional microplate reader, using an excitation wavelength at 322 nm and an emission wavelength at 450 nm. The IC 50 values were determined from the Influenza neuraminidase inhibitory curves by plotting inhibition ratio of neuraminidase activity versus inhibitor concentration at 0, 1, 2, 5, 7.5, and 10 nm in the mixture. Antimicrobial assay Antimicrobial evaluation against two human pathogens (E. coli EMBLC-1 and S. aureus EMBLC-2), seven aquatic bacteria (A. hydrophila QDIO-1, E. tarda QDIO-2, M. luteus QDIO-3, P. aeruginosa QDIO-4, V. alginolyticus QDIO-5, V. harveyi QDIO-7, and V. parahemolyticus QDIO-8), and five plant-pathogenic fungi (A. brassicae QDAU-1, C. gloeosprioides QDAU-2, F. oxysporum QDAU-5, G. graminis QDAU-3, and P. piricolav QDAU-6) was performed using a microplate S6

7 assay as described previously. 6 Chloramphenicol and amphotericin B were used as positive controls against bacteria and fungi, respectively. References (1) Wang, S.; Li, X. M.; Teuscher. F.; Li, D. L.; Diesel, A.; Ebel, R.; Proksch, P.; Wang, B. G. J. Nat. Prod. 2006, 69, (2) Sheldrick, G. M. SADABS, Software for Empirical Absorption Correction; University of Gottingen: Germany, (3) Sheldrick, G. M. SHELXTL, Structure Determination Software Programs; Bruker Analytical X-ray System Inc.: Madison, WI, (4) Sheldrick, G. M. SHELXL-97 and SHELXS-97, Program for X-ray Crystal Structure Solution and Refinement; University of Gottingen: Germany, (5) Song, G. P.; Shen, X. T.; Li, S. M.; Si, H. Z.; Li, Y. B.; Luan, H. Y.; Fan, J. H.; Liang, Q. Q.; Liu, S. W. RSC Adv. 2015, 5, (6) Pierce, G. M.; Uppuluri, P.; Tristan, A. R.; Wormley, F. L., Jr.; Mowat, E.; Ramage, G.; Lopez-Ribot, J. L. Nat. Protoc. 2008, 3, S7

8 Figure S1. HRESIMS spectrum of compound 1. S8

9 Figure S2. 1 H NMR (500 MHz, CDCl 3 ) spectrum of compound 1. S9

10 Figure S3. 13 C NMR (125 MHz, CDCl 3 ) and DEPT spectra of compound 1. S10

11 Figure S4. COSY spectrum of compound 1. S11

12 Figure S5. HSQC spectrum of compound 1. S12

13 Figure S6. HMBC spectrum of compound 1. S13

14 Figure S7. NOESY spectrum of compound 1. S14

15 Figure S8. ECD spectrum of compound 1. S15

16 Figure S9. Crystal packing of compound 1 at 293(2) K. S16