Aurachin SS, a new antibiotic from Streptomyces sp. NA04227

Transcription

1 Supporting Information Aurachin SS, a new antibiotic from Streptomyces sp. NA04227 Mei Zhang, Cheng Long Yang, Yong Sheng Xiao, Bo Zhang, Xin Zhao Deng, Li Yang, Jing Shi, Yi Shuang Wang, Wei Li, Rui Hua Jiao, Ren Xiang Tan, Hui Ming Ge* State Key Laboratory of Pharmaceutical Biotechnology, Institute of Functional Biomolecules, School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing, , China. * hmge@nju.edu.cn (H.M.G.) Table of Contents Experimental section General experimental procedures Strain isolation and identification Strain cultivation Isolation of compounds 1-3 Antimicrobial bioassay Figure S1 1 H NMR spectrum for compound 1 in acetone-d 6 Figure S2 13 C NMR spectrum for compound 1 in acetone-d 6 Figure S3 DEPT NMR spectrum for compound 1 in acetone-d 6 Figure S4 HSQC NMR spectrum for compound 1 in acetone-d 6 Figure S5 1 H- 1 H-COSY NMR spectrum for compound 1 in acetone-d 6 Figure S6 HMBC NMR spectrum for compound 1 in acetone-d 6 Figure S7 NOESY NMR spectrum for compound 1 in acetone-d 6

2 Experimental section General experimental procedures High resolution mass spectra were acquired on an Agilent 6250 TOF LC-MS instrument. NMR experiments were recorded in acetone-d6 or DMSO-d6 on Bruker Avance-600 spectrometer operating at 600 MHz for 1 H and 150 MHz for 13 C NMR. Semipreparative RP-HPLC (reverse phas-high performance liquid chromatography) was perfomed on an Agilent 1200 HPLC with an ODS-2 Hypersil column (5µ, mm). Silica gel ( mesh) for CC (column chromatography) and GF254 (10-20 µm) for TLC (thin layer chromatography) were produced by Qingdao Marine Chemical Company, China. Sephadex LH-20 from GE Biotechnology, USA. Resin is XAD 16N (20-60 mesh). All chemicals used in the study were of analytical grade. Strain isolation and identification The earwig Forficula auricularia sample collected from the Qixia Mountain in the suburb of Nanjing in October 2015 was used for isolation of insect endosymbiont. After surface sterilization of earwig, it was ground, diluted by sterilized water, and then spread on ISP4 agar medium supplemented with cycloheximide. After 10 days of incubation at 28 C, the strain NA04227 was isolated, purified and identified as Streptomyces sp. based on the 16S rrna gene sequence analysis (accession number: KY465886). Strain cultivation The strain NA04227 was cultivated in 500 ml flasks containing 100 ml of TSB medium at 28 o C for 2 days at 140 rpm. Then 10 ml of the seed culture was transferred into 200 ml of YEME medium (yeast extract 4 g, malt extract 10 g, Glucose 4 g in 1 L distilled water, ph 7.2) in 1000 ml flask, further incubation was carried out at 28 o C for 10 days shaking at 140 rpm. In total, 20 L

3 of fermentation liquid was obtained, and after filtration, the broth was adsorbed by XAD-16N resin for 3 hours. The resin was eluted three times by methanol. The methanol extract was concentrated under reduced pressure to yield 8.0 g crude extract. Isolation of compounds 1-3 The crude extract was fractionated upon silica gel column chromatography with petroleum ether/etoac and CH2Cl2/MeOH to give 30 fractions. Fr.5 was further partitioned by gel filtration over Sephedax LH20 eluted with MeOH to give 5 subfractions. Fr5.2 was purified by HPLC with MeOH/H2O (v/v, 69:31) to afford new compound 1 (10.8 mg; retention time: 15.1 min; Rf value: 0.5, DCM/MeOH: 40/1). Fr.5-3 was purified by HPLC with CH3CN /H2O (v/v, 34:66) to yield compounds 2 (18.0 mg; retention time: 23.1 min; Rf value: 0.4, DCM/MeOH: 40/1) and 3 (5.0 mg; retention time: 25.3 min; Rf value: 0.45, DCM/MeOH: 40/1). Compound 1: brown oil, UV (CH2Cl2) λmax (log ɛ) 248 (4.1), 346 (3.7) nm; HRESIMS: m/z [M+H] + and [M+Na] + (calcd. for C21H28NO2, ; C21H27NO2Na, ). Antimicrobial bioassay Minimum inhibitory concentration (MIC). MIC was determined by broth microdilution method according to CLSI guidelines and loaded onto 96-well sterile polypropylene microtiter plates (LabServ, Fisher Scientific, China). The medium used for the bacteria was Mueller-Hinton broth (MHB). All test compounds were dissolved in dimethyl sulfoxide (DMSO) to obtain stock solutions ranging from 0.05 to 12.8 mm, and then tested for their antimicrobial activity in vitro to five bacteria: Staphylococcus aureus CMCC(B) 26003, Methicillin-resistant Staphylococcus

4 aureus (MRSA) ATCC 43300, Streptococcus pyogenes ATCC 19615, Bacillus subtilis CICC and Micrococcus luteus. A bacterial cell suspension (50 μl) at CFU/mL was added in all wells except the negative control (broth only), normal growth (broth plus inoculum). The final bacterial cell density in the assay was CFU/mL with 1% DMSO (v/v). Subsequently, the plates were evenly mingled by shaking for 10 min at 150 rpm/min, followed by standing at 37 C for 20 h. After incubation, the MIC values were determined as the lowest sample concentration, at which no bacterial growth could be discerned. Rifampicin and Ampicillin were co-assayed as positive controls for the pathogenic bacteria. The antibacterial activity was based on at least triplicate experimentation.

5 Figure S1 1 H NMR spectrum for compound 1 in acetone-d 6 Figure S2 13 C NMR spectrum for compound 1 in acetone-d 6

6 Figure S3 DEPT NMR spectrum for compound 1 in acetone-d 6 Figure S4 HSQC NMR spectrum for compound 1 in acetone-d 6

7 Figure S5 1 H- 1 H-COSY NMR spectrum for compound 1 in acetone-d 6 Figure S6 HMBC NMR spectrum for compound 1 in acetone-d 6

8 Figure S7 NOESY NMR spectrum for compound 1 in acetone-d6