Supporting Information
Inhibition of indoleamine2,3-dioxygenase by thielavin derivatives from a soil fungus,Coniochaeta sp. 10F058
Jun-Pil Jang1, Jae-Hyuk Jang1,4, Mijin Oh1,4, Sangkeun Son1,4, Seung Min Kim1, Hye-Min Kim1, Kee-Sun Shin5, Hyuncheol Oh3, Nak Kyun Soung2, Young-Soo Hong1, Bo Yeon Kim2,4, and Jong Seog Ahn1,4
1Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Ochang, Korea, 2WCI Center, KRIBB, Ochang, Korea, 3College of Pharmacy, Wonkwang University, Iksan, Korea, 4Major of Biomolecular Science, University of Science and Technology, Daejeon, Korea, 5Microbial Resources Center, KRIBB, Daejeon, Korea
*Correspondence : Ph. D. Jong Seog Ahn,Chemical Biology Research Center,Korea Research Institute of Bioscience and Biotechnology (KRIBB), 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Republic of Korea,Ph. D. Bo Yeon Kim, WCI Center, KRIBB, 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Republic of Korea, E-mail : , .
Table of Contents
EXPERIMENTAL PROCEDURE
Figure S1.1H NMR spectrum (900 MHz) of thielavin Q (1) in CDCl3
Figure S2. 13C NMR spectrum (225 MHz) of thielavin Q (1) in CDCl3
Figure S3. DEPT-45 spectrum ofof thielavin Q (1) in CDCl3
Figure S4. 1H-1H COSY spectrum ofof thielavin Q (1) in CDCl3
Figure S5. HMQC spectrum of of thielavin Q (1) in CDCl3
Figure S6. HMBCspectrum of of thielavin Q (1) in CDCl3
Figure S7.ROESY spectrum of of thielavin Q (1) in CDCl3
Figure S8. 1H NMR spectrum (400 MHz) of thielavin F (2) in CDCl3
Figure S9. 13CNMR spectrum (100 MHz) ofthielavin F (2) in CDCl3
Figure S10. 1H NMR spectrum (400 MHz) of thielavin B (3) in CDCl3
Figure S11. 13CNMR spectrum (100 MHz) of thielavin B (3) in CDCl3
Figure S12. HRESIMS spectrum of of thielavin Q (1)
Figure S13.IR spectrum of of thielavin Q (1) in CDCl3
EXPERIMENTAL PROCEDURE
General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 polarimeter using a 100 mm glass microcell. UV spectra were obtained on a ShimadzuUV-1601 spectrophotometer. IR spectra were recorded on a Bruker Optic GmbH ALPHA-PFT-IR spectrophotometer. Nuclear magnetic resonance(NMR) spectra were obtained on a Varian Unity Inova 400 MHzspectrometer using as solvent CDCl3with TMS as the internal standard.HMBC NMR spectra were recorded in CDCl3 employing a Bruker Biospin Advance II 900 NMR spectrometer (900 MHz for 1H and 225 MHz for 13C) at Korea Basic Science Institute, Ochang, Korea. Spectra were referenced to residual solvent signals with resonances at δH/C 7.24/77.23 for CHCl3.HRESIMS data were obtained using a ShimadzuLCMS-IT-TOF mass spectrometer. Solvents used for HPLC were analytical grade. Column chromatography was conducted using silica gel 60 (63-200 μm particle size) and RP-18 (40-63 μm particle size)from Merck.Reversed-phase HPLC separations were performed using a semipreparative RS Tech Optima Pak C18 column (10 × 250 mm) at a flow rate of 2.0ml min-1using an Shimadzu SPD-10Avp HPLC system.
Fungal Material. Strain of a fungus, 10F058 was isolated from the soil sample collected ofOchang in the Korea and was identified on the basis of the rRNA sequences and morphological evaluation. A GenBank search with the 26S rRNA gene of 10F058 indicated Coniochaeta sp. MAB-2010a (HQ829070) andConiochaeta ligniaria (AY198390) as the closest matches, showing sequence identities of 100%,respectively. Therefore, the fungal strain 10F058 was identified and named as a Coniochaeta sp. 10F058.
Fermentation, Extraction and Isolation. Coniochaeta sp. 10F058was grown on the PD agar medium for 7days and was then inoculated into a 500-ml Erlenmeyer flask containing 75 ml of seed culture medium PD broth (24 g l-1 potato dextrose; BD Bioscience, San Jose, CA, USA). Incubation was carried out at 28ºC for 3 days on a rotary shaker operating at 135 rpm. This seed medium (150 ml) was transferred to 9l of the same production medium in a two 14-L jar fermentors. The fermentation was carried out at 28 ºC for 6 dayswith agitation at 165 rpm and an air flow of 10 l min-1.The culture broth (18l) was filtered and extracted three times with an equal volume of EtOAc and the EtOAc layer was concentrated in vacuo.The EtOAc-solubleextract (2.3 g) was separated by silica gel columnchromatography using agradient of CHCl3-MeOH (from 20:1 to 0:1), to yield five fractions (1-5) according to their TLC profiles.Since the fr. 2 [eluted with CHCl3–MeOH (from 20:1 to 10:1)] exhibitedthe strongest inhibitory activity (90% inhibition at30 g/ml), this fraction (315mg) wassubjected to reversed-phase C18flash column chromatography using a stepwise gradient of MeOH-H2O (from 20:80, 40:60, 60:40, 80:20 to 100:0; 700 mL for each step), to seven fractions (Frs.2-12-7). The active fraction 2-7 (81.0 mg) eluted with MeOH/H2O (80/20) was purified by semipreparative reverse phase HPLC using an isocratic solvent system of MeOH/H2O (78/22), to yield compound 1 (tR 25.5 min 3.5 mg), 2 (tR 32.7 min 3.9 mg) and 3 (tR 38.9 min 4.9 mg).
Table Physico-chemical properties of thielavinQ (1)
Appearance / white amorphous powderMolecular formula / C31H34O10
HR-ESI-MS (m/z)
Found / 565.2075 [M - H]-
Calcd. / 565.2074 [M - H]-
UV (MeOH) max (log ε) nm / 288 (3.31)
IR νmax (film) cm-1 / 3426, 1734, 1666, 1451, 1402, 1376, 1172, 1075
[]15D / 6.3 (c 0.1, MeOH)
Thielavin F (2): pale yellow amorphous powder; 1H NMR (400 MHz, CDCl3) δ 6.94 (1H, s, H-3), 6.34 (1H,s H-5), 3.90 (3H, s,2-OCH3), 3.87 (3H, s, 2-OCH3), 2.66 (3H, s,6-CH3), 2.52 (3H, s,3-CH3), 2.40 (3H, s,6-CH3), 2.27 (3H, s,6-CH3), 2.17 (3H,s,5-CH3), 2.15 (3H, s,3-CH3), 2.13 (3H, s,5-CH3); 13C NMR (100 MHz, CDCl3) δ 170.0 (1-carbonyl),168.7 (1-carbonyl), 166.3 (1-carbonyl), 164.3 (C-2), 159.5 (C-4), 157.6 (C-2), 154.0 (C-2), 151.5(C-4), 149.5 (C-4), 140.7 (C-6), 137.6 (C-6), 132.9 (C-5), 126.5 (C-1), 125.7 (C-6), 123.4 (C-1), 122.4 (C-5), 122.1 (C-3), 120.5 (C-3), 111.2 (C-5), 108.9 (C-3), 103.8 (C-1), 62.4 (2-OCH3),62.3 (2-OCH3),24.8 (6-CH3),20.6 (6-CH3),16.9 (6-CH3),13.0 (5-CH3),10.1 (5-CH3),9.6 (3-CH3),7.6 (3-CH3);ESIMS m/z575.7[M+Na]+, 551.7[M-H]-
Thielavin B (3): yellowish powder; 1H NMR (400 MHz, CDCl3) δ 6.34 (1H, s, H-5), 3.86 (3H, s,2-OCH3), 3.85 (3H, s,2-OCH3), 2.66 (3H, s,6-CH3), 2.41 (3H, s,6-CH3), 2.35 (3H, s,3-CH3), 2.23 (3H, s,5-CH3), 2.22 (3H,s,6-CH3), 2.18 (3H, s,3-CH3), 2.14 (3H, s,3-CH3),2.13 (3H, s,5-CH3); 13C NMR (100MHz, CDCl3) δ 171.1 (1-carbonyl),170.0 (1-carbonyl), 166.0 (1-carbonyl), 164.3 (C-2), 159.0 (C-4), 154.4 (C-2), 153.8 (C-2), 149.9(C-4), 149.5 (C-4), 140.6 (C-6), 133.7 (C-6), 133.6 (C-6), 126.4 (C-1), 126.2 (C-), 125.9 (C-5), 125.8 (C-5), 122.3 (C-3), 122.1 (C-3), 111.2 (C-5), 109.0 (C-3), 103.7 (C-1), 62.3 (2-OCH3),62.2 (2-OCH3),24.8 (6-CH3),17.2 (6-CH3),17.0 (6-CH3),13.1 (5-CH3),13.1 (5-CH3),10.3 (3-CH3),10.2 (3-CH3),7.6 (3-CH3);ESIMS m/z589.7[M+Na]+, 565.7[M-H]-
IDO assay Procedures. The enzymatic inhibition assays were performed as describedby Takikawa et al. with some modifications.19 Briefly, the reactionmixture (200 mL) contained potassium phosphate buffer(50 mM, pH 6.5), ascorbic acid (10 mM), methylene blue (5 mM),purified recombinant IDO (43 mM), L-Trp (100 mM), and DMSO(10 μL). The compounds were serially diluted 10-fold from 1000 to0.1 μM pure DMSO or, if not soluble at 1000 μM, by four orders ofmagnitude from their highest soluble concentration. The reactionwas conducted at 37 C for 60 min and stopped by addition of 30%(w/v) trichloroacetic acid (40 μL). To convert N-formylkynurenineto kynurenine, the tubes were incubated at 60C for 30 min, followedby centrifugation at 20000 g for 20 min. Finally, 150 μL ofsupernatant is added to 150 μL of p-dimethylaminobenzaldehyde(pDMAB) (2%, v/v) in acetic acid to generate a Schiff base withkynurenine that was detected at a wavelength of 480 nm.
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Figure S1.1H NMR spectrum (900 MHz) of thielavin Q (1) in CDCl3
Figure S2. 13C NMR spectrum (225 MHz) of thielavin Q (1) in CDCl3
Figure S3. DEPT-45 spectrum ofthielavin Q (1) in CDCl3
Figure S4. 1H-1H COSY spectrum of thielavin Q (1) in CDCl3
Figure S5. HMQC spectrum of thielavin Q (1) in CDCl3
Figure S6. HMBC spectrum of thielavin Q (1) in CDCl3
Figure S7. ROESY spectrum of of thielavin Q (1) in CDCl3
Figure S8. 1H NMR spectrum (400 MHz) of thielavin F (2) in CDCl3
FigureS9. 13C NMR spectrum (100 MHz) of thielavin F (2) in CDCl3
Figure S10. 1H NMR spectrum (400 MHz) of thielavin B (3) in CDCl3
FigureS11. 13C NMR spectrum (100 MHz) of thielavin B (3) in CDCl3
Figure S12. HRESIMS spectrum of thielavin Q (1)
Figure S13. IR spectrum of thielavin Q (1)
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