Supporting Information
Cinatrin D and E, and virgaricin B, three novel compounds
produced by a fungus, Virgaria boninensis FKI-4958
Takahiro Ishii,†,‡ Kenichi Nonaka, †,‡ Akihiro Sugawara,†,‡ Masato Iwatsuki,†,‡ Rokuro
Masuma,†,‡ Tomoyasu Hirose,†,‡ Toshiaki Sunazuka,†,‡ Satoshi Ōmura,*,† and Kazuro
Shiomi*,†,‡
†
Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku,
Tokyo 108-8641, Japan
‡
Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane,
Minato-ku, Tokyo 108-8641, Japan
Corresponding Author
*Tel: +81-3-5791-6439. Fax: +81-3-5791-6131. E-mail: shiomi@lisci.kitasato-u.ac.jp (K.S.).
omuras@insti.kitasato-u.ac.jp (S.Ō).
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Content
S1. Fermentation, extraction, and isolation of the known compounds produced by FKI-6621
strain.
Figure S1. Structures of the known compounds produced by FKI-6621 strain.
Figure S2. 1H NMR spectrum of compound 1 (pyridine-d5, 600 MHz).
Figure S3.
13
C NMR spectrum of compound 1 (pyridine-d5, 150 MHz).
Figure S4. 1H NMR spectrum of compound 2 (pyridine-d5, 600 MHz).
Figure S5.
13
C NMR spectrum of compound 2 (pyridine-d5, 150 MHz).
Figure S6. 1H NMR spectrum of compound 3 (DMSO-d6, 400 MHz).
Figure S7.
13
C NMR spectrum of compound 3 (DMSO-d6, 100 MHz).
Figure S8. 1H NMR spectrum of methyl ester of 1 (1a) (DMSO-d6, 400 MHz).
Figure S9. 1H NMR spectrum of methyl ester of 2 (2a) (DMSO-d6, 400 MHz).
Figure S10. 1H NMR spectrum of (R)-PGME amide 1 (1b) (pyridine-d5, 500 MHz).
Figure S11. 1H NMR spectrum of (S)-PGME amide 1 (1c) (pyridine-d5, 500 MHz).
Figure S12. 1H NMR spectrum of (R)-MTPA ester of 3 (3a) (CD3OD, 500 MHz).
Figure S13. 1H NMR spectrum of (S)-MTPA ester of 3 (3b) (CD3OD, 500 MHz).
Figure S14. 1H NMR spectrum of (R)-MTPA ester of 4 (4a) (CD3OD, 500 MHz).
Figure S15. 1H NMR spectrum of (S)-MTPA ester of 4 (4b) (CD3OD, 500 MHz).
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S1. Fermentation, extraction, and isolation of the known compounds from FKI-6621.
Strain FKI-6621 was grown and maintained on a LcA agar slant consisting of 0.1%
glycerol, 0.08% KH2PO4, 0.02% K2HPO4, 0.02% MgSO4·7H2O, 0.02% KCl, 0.2% NaNO3,
0.02% yeast extract and 1.5% agar (adjusted to pH 6.0 before sterilization). A loop of spores
of Virgaria sp. FKI-6621 was inoculated into 10 ml of seed medium consisting of 2.0%
glucose, 0.2% yeast extract, 0.5% Polypepton, 0.05% MgSO4·7H2O, 0.1% KH2PO4 and 0.1%
agar (adjusted to pH 6.0 before sterilization), in a test tube. The inoculated tube was
incubated on a reciprocal shaker (300 rpm) at 27°C for three days. A 1-ml portion of the seed
culture was transferred to each of five 500-ml Erlenmeyer flasks containing 100 ml of the
production medium, consisting of 3.0% sucrose, 3.0% soluble starch, 1.0% malt extract,
0.3% Ebios, 0.5% KH2PO4 and 0.05% MgSO4·7H2O (adjusted to pH 6.0 before sterilization).
Fermentation was carried out statically at 22°C for 14 days.
The whole culture broth (500 mL) was subsequently added to an equal amount of ethanol
and then filtered. The filtrate was concentrated, under reduced pressure, to remove the
ethanol and then extracted with ethyl acetate. The organic layer was concentrated to dryness
in vacuo to afford a crude extract (139 mg). The ethyl acetate extract was chromatographed
on a silica gel column using a chloroform/methanol gradient solvent system of increasing
polarity, to yield four fractions (Fr. I-IV). Fr. I (22.1 mg) eluted with chloroform/methanol
(95/5) was further separated by a combination of preparative TLC with chloroform/methanol
(98/2) and reversed-phase HPLC (Senshu Pak Pegasil ODS, 40% acetonitrile, flow rate 8
ml/min, UV at 210 nm detection) to give 2,3-dihydro-2-hydroxy-2,4-dimethyl-5-transpropenylfuran-3-one (2.4 mg), isoochracein (0.3 mg), and mellein (0.7 mg). Fr. II (41.8 mg)
eluted with chloroform/methanol (9/1) was further purified by a combination of preparative
TLC with chloroform/methanol (9/1) and reversed-phase HPLC (Senshu Pak Pegasil ODS,
30% acetonitrile, flow rate 8 ml/min, UV at 210 nm detection) to 2,3-dihydro-2-hydroxy-2,4-
3
dimethyl-5-trans-propenylfuran-3-one
(1.5
mg)
and
2-(hydroxylmethyl)-3-(1-
hydroxypropyl)phenol (1.6 mg).
O
OH
O
2,3-Dihydro-2-hydroxy-2,4-dimethyl-5-trans-propenyl-furan-3-one
OH
OH
OH
OH
O
O
2-(Hydroxymethyl)-3-(1-hydroxypropyl)phenol
Isoochracein
O
OH
O
Mellein
Figure S1. Structures of the known compounds produced by FKI-6621 strain.
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Figure S2. 1H NMR spectrum of compound 1 (pyridine-d5, 600 MHz).
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Figure S3.
13
C NMR spectrum of compound 1 (pyridine-d5, 150 MHz).
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Figure S4. 1H NMR spectrum of compound 2 (pyridine-d5, 600 MHz).
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Figure S5.
13
C NMR spectrum of compound 2 (pyridine-d5, 150 MHz).
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Figure S6. 1H NMR spectrum of compound 3 (DMSO-d6, 400 MHz).
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Figure S7.
13
C NMR spectrum of compound 3 (DMSO-d6, 100 MHz).
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Figure S8. 1H NMR spectrum of methyl ester of 1 (1a) (DMSO-d6, 400 MHz).
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Figure S9. 1H NMR spectrum of methyl ester of 2 (2a) (DMSO-d6, 400 MHz).
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Figure S10. 1H NMR spectrum of (R)-PGME amide 1 (1b) (pyridine-d5, 500 MHz).
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Figure S11. 1H NMR spectrum of (S)-PGME amide 1 (1c) (pyridine-d5, 500 MHz).
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Figure S12. 1H NMR spectrum of (R)-MTPA ester of 3 (3a) (CD3OD, 500 MHz).
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Figure S13. 1H NMR spectrum of (S)-MTPA ester of 3 (3b) (CD3OD, 500 MHz).
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Figure S14. 1H NMR spectrum of (R)-MTPA ester of 4 (4a) (CD3OD, 500 MHz).
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Figure S15. 1H NMR spectrum of (S)-MTPA ester of 4 (4b) (CD3OD, 500 MHz).
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