SUPPLEMENTARY INFORMATION

Transcription

1 SUPPLEMENTARY INFRMATIN pantimycin A, a new metabolite isolated from Streptomyces sp. RK Toshihiko Nogawa 1, Akiko kano 1, Chung Liang Lim 1,2, Yushi Futamura 1, Takeshi Shimizu 1, Shunji Takahashi 3, Darah Ibrahim 2, Hiroyuki sada 1 1 RIKEN Center for Sustainable Research Science, Chemical biology Research Group, 2-1 Hirosawa, Wako, Saitama , Japan 2 Industrial Biotechnology Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, Minden, Penang, Malaysia 3 RIKEN Center for Sustainable Research Science, Natural Product Biosynthesis Research Unit, 2-1 Hirosawa, Wako, Saitama , Japan Corresponding author Mailing address: RIKEN CSRS, Chemical Biology Research Group, 2-1 Hirosawa, Wako, Saitama , Japan Phone: (+81) Fax: (+81) hisyo@riken.jp CNTENTS EXPERIMENTAL PRCEDURE / page 2 Figure S1. HRTFMS data of compound 1 / page 3 Figure S2. 1 H NMR spectrum of compound 1 / page 4 Figure S3. 1 H NMR spectrum with the addition of D 2 of compound 1 / page 4 Figure S4 13 C NMR spectrum of compound 1 / page 5 Figure S5. 13 C DEPT 135 spectrum of compound 1 / page 5 Figure S6. HSQC spectrum of compound 1 / page 6 Figure S7. DQF-CSY spectrum of compound 1 / page 6 Figure S8. HSQC-TCSY spectrum of compound 1 / page 7 Figure S9. HMBC spectrum of compound 1 / page 7 Figure S10. phase-sensitive NESY spectrum of compound 1 / page 8 1

2 EXPERIMENTAL PRCEDURE General Experimental Procedures. Analytical-grade solvent and reagents were purchased from commercial sources. UV and optical rotations were recorded on a JASC V-630 BI spectrophotometer and a HRIBA SEPA-300 high sensitive polarimeter, respectively. IR spectra were recorded on a HRIBA FT-720 with a DuraSampl IR II ATR instrument. NMR data were obtained at 500 MHz for 1 H NMR and 125 MHz for 13 C NMR on a JEL JNM-ECA-500 spectrometer. Chemical shifts (in ppm) were referenced against the residual undeuterated solvent. Mass spectra were obtained on an AB Sciex Qtrap (ESIMS) and Waters Synapt G2 (HRESIMS). MS/MS experiments were carried out on an AB Sciex API3200 or Waters Synapt G2. LC/MS analysis was performed on a Waters 2965 Alliance system with 2996PDA detector connected to AB Sciex Qtrap by ESI probe on a Waters Xterra C18 column (2.1 mm i.d. x 150 mm, 5 µm) with elution of acetonitrile/0.05% aqueous formic acid linear gradient system (acetonitorile: 5 to 100% in 30 min). Teredyne ISC CombiFlash Companion was used for middle-pressure liquid chromatography (MPLC). Preparative HPLC was performed using a Waters 600E pump system with SenshuPak Pegasil DS column (20 mm i.d. x 250 mm or 10 mm i.d. x 250 mm). Culture Condition and construction of the fraction library and NPPlot. A 30 l of culture broth of Streptomyces sp. RK was used to construct a fraction library. The detail of culture condition and methodology of construction of the fraction library and NPPlot were described in the previous report. 1 Isolation of 1. The 28th fraction of the third MPLC fraction (8.3 mg) was separated by C18-HPLC with acetonitrile/water (45:55) under isocratic elution to afford compound 1 (1.3 mg) as a pale-yellow amorphous solid. Physicochemical properties and 1 H and 13 C NMR chemical shifts of 1 were summarized in Tables 1 and 2, respectively. Cytotoxicity and antimicrobial activity tests. The in vitro cytotoxicity assay methods against human cervix epidermoid carcinoma cell line, HeLa, human promyelocytic leukemia cell line, HL60, and rat kidney cells infected with ts25, a T-class mutant of Rous sarcoma virus Prague strain, src ts -NRK were described in the previous report. 1 The microdilution assay against Staphylococcus aureus 209, Escherichia coli H141, Aspergillus fumigatus Af293, Pyricularia oryzae kita-1, and Candida albicans JCM1542 were described in the previous report. 1 Antimalarial test. Plasmodium falciparum 3D7 were cultured at 37 C under 5% C 2 in human erythrocytes (3% hematocrit) in RPMI1640 (Invitrogen/Life Technologies), supplemented with 10% 2

3 human plasma. To perform the P. falciparum growth assay, 100 µl of 0.3% parasitized red blood cells and 2% hematocrit were dispensed into 96-well plate. Following 72-h exposures to test sample, plates were frozen at -70 C overnight and then thawed at room temperature for at least 4 h. To evaluate LDH activity, 150 µl of freshly made reaction mixture (166 mm sodium L-lactate, 166 µm 3-acetyl pyridine adenine dinucleotide, 208 µm Nitro Blue tetrazolium chloride, 150 µg/ml diaphorase (22.5 U/ml), 0.8% Tween 20, 116 mm Tris-HCl, ph 8.0) was added. Plates were shaken to ensure mixing and absorbance at 650 nm was monitored in a plate reader (PerkinElmer) after 10 min of incubation at room temperature. Reference 1. Lim, CL. et al. RK-1355A and B, novel quinomycin derivatives isolated from a microbial metabolites fraction library based on NPPlot screening. J. Antibiot. 67, (2014). Figure S1. HRTFMS data of compound 1 3

4 H N H H H N Figure S2. 1 H NMR spectrum of compound D 2 disappeared Figure S3. 1 H NMR spectrum with the addition of D 2 of compound 1 4

5 (thousandths) X : parts per Million : Carbon13 Figure S4 13 C NMR spectrum of compound 1 (thousandths) X : parts per Million : Carbon13 Figure S5. 13 C DEPT 135 spectrum of compound 1 5

6 Y : parts per Million : Carbon Figure S6. HSQC spectrum of compound (thousandths) Y : parts per Million : Proton Figure S7. DQF-CSY spectrum of compound 1 6

7 Y : parts per Million : Carbon Figure S8. HSQC-TCSY spectrum of compound (thousandths) Y : parts per Million : Carbon Figure S9. HMBC spectrum of compound (thousandths) 7

8 Y : parts per Million : Proton Figure S10. phase-sensitive NESY spectrum of compound 1 8