Benzomalvin E, an Indoleamine 2,3-Dioxygenase Inhibitor Isolated from Penicillium Sp. FN070315

Supporting Information

Benzomalvin E, an Indoleamine 2,3-dioxygenase Inhibitor Isolated from Penicillium sp. FN070315

Jun-Pil Jang1,5,#, Jae-Hyuk Jang1,2,#, Nak-Kyun Soung1,2, Hye-Min Kim1,7, Sook-Jung Jeong1, Yukihiro Asami1,3, Kee-Sun Shin4, Mee Ree Kim5, Hyuncheol Oh6, Bo Yeon Kim2 and Jong Seog Ahn1,7

Title and Running head : New benzomalvin derivative from Penicillium sp.

1Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Ochang, Korea, 2World Class Institute, KRIBB, Ochang, Korea, 3Chemical Biology Department, RIKEN Advanced Science Institute, Wakoshi, Saitama, Japan, 4Biological Resources Center, KRIBB, Daejeon, Korea, 5Department of Food and Nutrition, Chungnam National University, Daejeon, Korea, 6College of Medical and Life Sciences, Silla University, Busan, Korea, 7University of Science and Technology, Daejeon, Korea.

#These authors contributed equally.

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, World Class Institute, KRIBB, 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Republic of Korea E-mail : [email protected], [email protected].

1 Table of Contents

1 Figure S1. H NMR spectrum (900 MHz) of benzomalvin E (1) in CDCl3

13 Figure S2. C NMR spectrum (225 MHz) of benzomalvin E (1) in CDCl3

Figure S3. DEPT-45 spectrum of benzomalvin E (1) in CDCl3

1 1 Figure S4. H- H COSY spectrum of benzomalvin E (1) in CDCl3

Figure S5. HMQC spectrum of benzomalvin E (1) in CDCl3

Figure S6. HMBC spectrum of benzomalvin E (1) in CDCl3

Figure S7. NOESY spectrum of benzomalvin E (1) in CDCl3

1 Figure S8. H NMR spectrum (900 MHz) of benzomalvin B (2) in CDCl3

13 Figure S9. C NMR spectrum (225 MHz) of benzomalvin B (2) in CDCl3

1 Figure S10. H NMR spectrum (900 MHz) of benzomalvin C (3) in CDCl3

13 Figure S11. C NMR spectrum (225 MHz) of benzomalvin C (3) in CDCl3

Figure S12. HRESIMS spectrum of benzomalvin E (1)

Figure S13. IR spectrum of benzomalvin E (1) in CDCl3

Figure S14. 1H NMR Spectrum (900 MHz) of (R)-MTPA Ester of benzomalvin E (1a) in

Figure S15. 1H NMR Spectrum (900 MHz) of (S)-MTPA Ester of benzomalvin E (1b) in

2 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 Shimadzu UV-

1601 spectrophotometer. IR spectra were recorded on a Bio-Rad FTS-175C FT-IR

spectrophotometer. All 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 CDCl3. HRESIMS data were obtained using a Shimadzu

LCMS-IT-TOF mass spectrometer. Solvents used for HPLC were analytical grade. Column chromatography was performed with a LiChroprep RP-18 (Merck; 3.5 ´ 20 cm; 40-63 μm).

Reversed-phase HPLC separations were performed using a semipreparative RS Tech Optima

Ò Pak C18 column (10 × 250 mm) at a flow rate of 2.0 mL/min using an Shimadzu SPD-10Avp

HPLC system.

Fungal Material. Strain of a fungus, FN070315 was isolated from the soil sample collected of Daejeon 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 FN070315 indicated Penicillum jensenii (AY443470), Penicillum canescens (AY484896) as the closest matches, showing sequence identities of 100% and 99.98 %, respectively. Therefore, the fungal strain FN070315 was identified and named as a Penicillum sp. FN070315. This strain

3 has been deposited as KCTC1818P at the Korean Collection for Type Culture (KCTC), located within the Biological Resources Center (BRC) in the Korea Research Institute of

Bioscience and Biotechnology (KRIBB), Korea.

Fermentation, Extraction and Isolation. Penicillum sp. FN070315 was grown on the PD agar medium for 7 days 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 8 l of the same production medium in a two 14-L jar fermentors. The fermentation was carried out at 28 ºC for 6 days with agitation at

165 rpm and an air flow of 10 l min-1. The culture broth (16 l) was filtered and extracted three times with an equal volume of EtOAc and the EtOAc layer was concentrated in vacuo. The

EtOAc extract (1.3 g) subjected to reversed-phase C18 flash 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 yield five fractions (Frs. 1-5). The active fraction 3 (68.0 mg) eluted with

MeOH/H2O (60/40) was purified by semipreparative reverse phase HPLC using an isocratic

solvent system of MeCN/H2O (50/50), to yield compound 1 (tR 16.3 min 3.5 mg), 2 (tR 23.5

min 5.5 mg) and 3 (tR 26.8 min 5.2 mg).

4 Table Physico-chemical properties of benzomalvin E (1) Appearance pale yellow amorphous powder

Molecular formula C24H19N3O3 HR-ESI-MS (m/z) Found 398.1499 [M + H]+ Calcd. 398.1498 [M + H]+

UV (MeOH) max (log ε) nm 308 (3.60) -1 IR νmax (film) cm 3426, 1734, 1666, 1451, 1402, 1376, 1172, 1075 25 [] D -57.6 (c 0.1, MeOH)

1 Benzomalvin B (2): white amorphous powder ; H NMR (900 MHz, CDCl3) δ 8.45 (1H, d,

8.1, H-12), 7.96 (1H, d, 8.1, H-4), 7.85 (1H, t, 8.1, H-14), 7.73 (1H, d, 8.1, H-15), 7.72 (1H, d, 8.1, H-7), 7.63 (1H, t, 8.1, H-13), 7.61 (1H, t, 8.1, H-6), 7.54 (1H, t, 8.1, H-5), 7.26 (2H, br d, 7.2, H-22, 26), 7.23 (2H, br t, 8.1, H-23, 25), 7.20 (1H, t, 8.1, H-24), 6.80 (1H, s, H-20),

13 3.54 (3H, s, H-27); C NMR (225 MHz, CDCl3) δ 165.9 (C-2), 161.1 (C-10), 151.2 (C-18),

146.8 (C-16), 135.1 (C-14), 133.2 (C-3), 132.9 (C-3), 132.8 (C-6), 132.2 (C-21), 131.7 (C-8),

131.0 (C-5), 131.3 (C-4), 129.4 (C-24), 128.9 (C-23, 25), 128.9 (C-19), 128.8 (C-22, 26),

128.1 (C-13), 127.9 (C-15), 127.6 (C-12), 127.5 (C-7), 121.9 (C-11), 36.2 (C-27); ESIMS m/z

402.4 [M+Na]+ , 382.4 [M+H]+

1 Benzomalvin C (3): white amorphous powder; H NMR (900 MHz, CDCl3) δ 8.32 (1H, d,

8.1, H-12), 8.02 (1H, d, 8.1, H-4), 7.99 (1H, d, 8.1, H-15), 7.90 (1H, t, 8.1, H-14), 7.63 (1H, t,

8.1, H-5), 7.61 (1H, t, 8.1, H-13), 7.49 (1H, t, 8.1, H-6), 7.24 (1H, br t, 8.1, H-24), 7.13 (2H, t,

5 7.2, H-23, 25), 6.95 (1H, d, 8.1, H-7), 6.69 (2H, d, 8.1, H-22, 26), 3.94 (1H, s, H-20), 3.31

13 (3H, s, H-27); C NMR (225 MHz, CDCl3) δ 165.6 (C-2), 160.3 (C-10), 147.9 (C-18), 145.9

(C-16), 135.2 (C-14), 131.9 (C-8), 131.6 (C-3), 130.9 (C-6), 130.2 (C-21), 129.6 (C-4), 129.3

(C-24), 129.0 (C-5), 128.5 (C-7), 128.3 (C-23, 25), 128.2 (C-13), 128.1 (C-15), 127.5 (C-12),

125.9 (C-22, 26), 121.8 (C-11), 72.2 (C-19), 67.9 (C-20), 28.9 (C-27); ESIMS m/z 418.4

[M+Na]+ , 396.4 [M+H]+

(R)-MTPA Ester of 1 (1a). (S)-MTPA chloride (10 μL) was added to a solution of 1 (1.8 mg) in dry pyridine (50 μL) and stirred at room temperature. After 14hr, the reaction mixture

was concentrated to dryness and purified by HPLC (C18 column, 75% aq CH3CN isocratic) to

1 afford 1a (1.2 mg); H NMR (CDCl3, 900 MHz)  8.29 (1H, d, J = 8.1 Hz, H-12), 8.05 (1H, d,

J = 8.1 Hz, H-4), 7.84 (1H, t, J = 8.1 Hz, H-6), 7.76 (1H, t, J = 8.1 Hz H-14), 7.74 (1H, t, J =

8.1 Hz H-5), 7.70 (1H, d, J = 8.1 Hz, H-7), 7.53 (1H, d, J = 8.1 Hz H-15), 7.52 (1H, t, J = 8.1

Hz, H-13), 7.29 (2H, d, J = 8.1 Hz H-23 and 25), 7.27 (1H, d, J = 8.1 Hz H-24), 7.04 (2H, t, J

= 8.1 Hz H-22 and 26), 5.35 (1H, d, J = 12.6 Hz H-19), 5.09 (1H, d, J = 11.7 Hz, H-20), 3.45

(3H, s, H-27).

(S)-MTPA Ester of 1 (1b). In the same manner as described for 1a, compound 1 (1.5 mg)

1 was reacted with (R)-MTPA chloride to give 1b (1.1 mg); H NMR (CDCl3, 900 MHz)  8.27

6 (1H, d, J = 8.1 Hz, H-12), 8.10 (1H, d, J = 8.1 Hz, H-4), 7.83 (1H, t, J = 8.1 Hz, H-6), 7.78

(1H, t, J = 8.1 Hz H-5), 7.75 (1H, t, J = 8.1 Hz H-14), 7.63 (1H, d, J = 8.1 Hz, H-7), 7.57 (1H, d, J = 8.1 Hz H-15), 7.52 (1H, t, J = 8.1 Hz, H-13), 7.31 (2H, d, J = 8.1 Hz H-23 and 25), 7.30

(1H, d, J = 8.1 Hz H-24), 7.16 (2H, t, J = 8.1 Hz H-22 and 26), 5.48 (1H, d, J = 11.7 Hz H-

19), 5.10 (1H, d, J = 12.6 Hz, H-20), 3.40 (3H, s, H-27).

IDO assay Procedures. The enzymatic inhibition assays were performed as described by

Takikawa et al. with some modifications.19 Briefly, the reaction mixture (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 to 0.1 μM pure DMSO or, if not soluble at 1000 μM, by four orders of magnitude from their highest soluble concentration. The reaction was conducted at 37 C for 60 min and stopped by addition of 30% (w/v) trichloroacetic acid (40 μL). To convert N-formylkynurenine to kynurenine, the tubes were incubated at 60C for 30 min, followed by centrifugation at 20000 g for 20 min. Finally, 150

μL of supernatant is added to 150 μL of p-dimethylaminobenzaldehyde (pDMAB) (2%, v/v) in acetic acid to generate a Schiff base with kynurenine that was detected at a wavelength of

480 nm.

7 Figure

1 S1. H NMR spectrum (900 MHz) of benzomalvin E (1) in CDCl3

8 Figure S2. 13C

NMR spectrum (225 MHz) of benzomalvin E (1) in CDCl3

9 Figure S3. DEPT-45 spectrum of benzomalvin E (1) in CDCl3

10 Figure

S4. 1H-1H COSY

spectrum of

benzomal

vin E (1) in CDCl3

Figure

S5. HMQC

spectrum of

benzomal

vin E (1) in CDCl3

11 Figure

S6. HMBC spectrum of

benzomal

vin E (1) in CDCl3

Figure

S7. NOESY spectrum of

benzomalvin E (1) in CDCl3

12 Figure S8. 1H

NMR spectrum of benzomalvin B (2) in CDCl3

14 15 13 Figure S9. C NMR spectrum of benzomalvin B (2) in CDCl3

16 Figure

S10. 1H NMR spectrum of

benzomalvin C (3) in CDCl3

13 Figure S11. C NMR spectrum of benzomalvin C (3) in CDCl3

17 Figure S12. HRESIMS spectrum of benzomalvin E (1)

18 Figure

S13. IR spectrum of

benzom alvin E (1)

Figure

S14. 1H NMR

Spectrum (900 MHz) of (R)-

MTPA Ester of benzomalvin E (1a) in CDCl3

19 Figure

S15. 1H NMR

Spectrum (900

MHz) of (S)-MTPA

Ester of

benzomal

vin E (1b) in CDCl3