Aromatic Compounds from the Halotolerant Fungal Strain of Wallemia ...

Arch Pharm Res Vol 34, No 6, 907-912, 2011 DOI 10.1007/s12272-011-0607-0

Aromatic Compounds from the Halotolerant Fungal Strain of Wallemia sebi PXP-89 in a Hypersaline Medium Xiao-Ping Peng1,*, Yi Wang1,*, Pei-Pei Liu1, Kui Hong2, Hao Chen3, Xia Yin1, and Wei-Ming Zhu1 1 Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China, 2Institute of Tropical Biological Sciences and Biotechnology, Chinese Academy of Tropical Agricultural Science, Haikou 571101, China, and 3First Institute of Oceanography, State Oceanic Administration, Qingdao 266061, China

(Received October 14, 2010/Revised January 4, 2011/Accepted February 7, 2011)

A new cyclopentanopyridine alkaloid, 3-hydroxy-5-methyl-5,6-dihydro-7H-cyclopenta[b]pyridin-7-one (1), together with 11 known aromatic compounds were isolated from the secondary metabolites of the halotolerant fungal strain Wallemia sebi PXP-89 in 10% NaCl. Their structures including the absolute configurations of (2S,3S)-1-(4-hydroxyphenyl)butane-2,3-diol (2), (2R,3S)-1-(4-hydroxyphenyl)butane-2,3-diol (3), and (S)-3-hydroxy-4-(4-hydroxyphenyl)-2-one (4) were elucidated by spectroscopic analysis and a modified Mosher’s method. Compound 1 exhibited antimicrobial activity against Enterobacter aerogenes with a MIC of 76.7 µM. The absolute configurations of compounds 2-4 were determined for the first time. Key words: Halotolerant fungus, Wallemia sebi, Antibacterial activity, Cyclopentanopyridine alkaloid

Selected by Editors INTRODUCTION It was demonstrated that halotolerant marine fungal species have evolved unique metabolic mechanisms that are responsive to high salt concentrations and the marine-derived fungal secondary metabolites could have potential implications for drug discovery (Wang et al., 2007a, 2007b). The extreme conditions that halotolerant fungi live in are usually adverse to the growth of microorganisms due to the high osmolarity and nutrient deprivation. However, this extreme environment might activate some silent genes and induce unique biosynthetic pathways in which structurally unique compounds may be produced (Tamburini and Mastromei, 2000; Kogej et al., 2006). In order to continuously explore the new and bioactive secondary *These authors contributed equally to this work. Correspondence to: Wei-Ming Zhu, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China Tel, Fax: 86-532-82031268 E-mail: [email protected]

metabolites from halotolerant fungi for drug discovery (Wang et al., 2007a, 2007b, 2009; Lu et al., 2008; Zheng et al., 2009, 2010), a halotolerant fungal strain PXP89 was isolated from the surface of the leaves of the mangrove plant, Excoecaria agallocha (Euphorbiaceae) and identified as Wallemia sebi. The W. sebi has been reported to produce several compounds in fungal culture media, such as walleminols A and B (Frank et al., 1999), UCA1064-A and UCA1064-B (Takahashi et al., 1993) and mycosporines that were identified from the metabolites fermented in 10% NaCl (Kogej et al., 2006). When fermented in an actinomycete culture medium containing 10% NaCl, the EtOAc extracts of the fermentation broth of W. sebi PXP-89 showed cytotoxicity against P388 cells. Further, the HPLC profiles of the extracts were distinct from those in 0% and 3% NaCl (Fig. 1). Chemical study on secondary metabolites in 10% NaCl resulted in the isolation and identification of a new cyclopentanopyridine alkaloid, 3-hydroxy-5-methyl-5,6-dihydro-7H-cyclopenta [b]pyridin-7-one (1), together with 11 aromatic compounds, (2S,3S)-1-(4-hydroxyphenyl) butane-2,3-diol (2) (Thornton and Jones, 1964), (2R,3S)-1-(4-hydroxyphenyl)butane-2,3-diol (3) (Thornton and Jones, 1964),

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Fig. 1. Colony picture of Wallemia sebi PXP-89 and the HPLC profiles of secondary metabolites of Wallemia sebi PXP-89 in different salinities

(S)-3-hydroxy-4-(4-hydroxyphenyl)-2-one (4) (Yuki et al., 2009), 3-(hydroxyacetyl)indole (5) (Yang and Cordell, 1997), 1H-indole-3-carboxylic acid (6) (Burton et al., 1986), 5-hydroxy-3-coumaranone (7) (Lee et al., 2010), 4-methylfuran-2-carboxylic acid (8) (Sandham et al., 2004), 3-methylfuran (9) (Hiraoka, 1970), 5-methyluracil (10) (Ellis et al., 1973), 2,5-furandimethanol (11) (Katritzky and Law, 1988), and 5-methylpyridin-3-ol (12) (Boux et al., 1988). The absolute configurations of compounds 2-4 were determined for the first time. Compound 1 showed weak antimicrobial activity against Enterobacter aerogenes with a MIC value of 76.7 µM. The NMR spectral data of compounds 2-4 and 8-10 were also reported for lack of corresponding data in the literature.

MATERIALS AND METHODS General experimental procedures Optical rotations were obtained on a JASCO P-1020 digital polarimeter. IR spectra were taken on a Nicolet NEXUS 470 spectrophotometer in KBr disks. 1D- and 2D-NMR spectra were recorded on a JEOL JNM-ECP 600 spectrometer using TMS as internal standard and chemical shifts were recorded as δ values. ESI-MS was measured on a Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer. Semipreparative HPLC (semiPHPLC) was performed using ODS columns [YMCpack ODS-A, 10 × 250 mm, 5 µm, 4.0 mL/min] on a Waters 600 multisolvent delivery system equipped with a photodiode array detector (Waters 996). Strain The working strain Wallemia sebi PXP-89 was isolated from the surface of the leaves of the mangrove plant, Excoecaria agallocha (Euphorbiaceae) collected in Wenchang, Hainan province of China. It was identified according to its morphological characteristics

by Prof. Hong Kui, Institute of Tropical Biological Sciences and Biotechnology, Chinese Academy of Tropical Agricultural Science, Haikou, China. Working strains were prepared on potato dextrose agar slants containing 30% NaCl and stored at 4oC.

Fermentation The fungus W. sebi PXP-89 was grown at 28oC for 10 days in a 100 L fermenter containing liquid medium (70 L) at pH 7.0 composed of (g/L): glucose (20), yeast extract (10), corn extract (3), soluble starch (10), beef extract (3), peptone (10), K2HPO4 (0.5), MgSO4 · 7H2O (10), KCl (10), NaCl (80), and CaCO3 (2). Extraction The fermented whole broth (70 L) was filtered through cheese cloth to separate filtrate and mycelia. The filtrate was extracted three times with EtOAc, and the EtOAc solution was concentrated under reduced pressure to give a crude extract, while the mycelia were extracted three times with acetone. The acetone fraction was concentrated under reduced pressure to produce an aqueous solution. The aqueous fraction was extracted three times by EtOAc to yield another EtOAc fraction. Both EtOAc fractions were combined and concentrated under reduced pressure to produce a crude extract (60.0 g). Purification The crude extract (60.0 g) was separated into 12 fractions after subjection to vacuum liquid chromatography on a silica gel column using step gradient elution with CHCl3 -petroleum ether (0-100%) and then MeOH-CHCl3 (0-50%). Fraction 3 (923 mg) was separated into 4 subfractions (3-1 to 3-4) by a Sephadex LH-20 column (MeOH-CHCl3, v/v 1:1). The subfraction 3-3 (104 mg) was purified by extensive semi-PHPLC (30% MeOH,

A New Cyclopentanopyridine Alkaloid

4.0 mL/min) to yield compound 8 (40 mg). Fraction 5 (2.1 g), eluted with CHCl3 -MeOH (v/v 50:1, 40:1, 30:1, 20:1, 10:1, 1:1), was purified into 3 subfractions (51~5-3). The subfraction 5-2-1 (89 mg) yielded from the fraction 5-2 on a Sephadex LH-20 column (MeOH– CHCl3, 3:1) was further purified by extensive semiPHPLC (45% MeOH, 4.0 mL/min) to yield 4 (4.2 mg). Another subfraction 5-2-2 (175 mg) was separated by a Sephadex LH-20 column (MeOH) to yield subfraction 5-2-2-1 (63 mg) that was further purified by extensive semi-PHPLC (40% MeOH, 4.0 mL/min) to yield 5 (1.6 mg). Fraction 6 (549 mg) was separated into two subfractions (6-1 and 6-2) by a Sephadex LH-20 column (MeOH–CHCl3, v/v 1:1). Fraction 6-1 (230 mg), eluted with petroleum ether–acetone (v/v 5:1, 5:2, 5:3, 5:4, 1:1), was purified into four subfractions on silica gel column. The fraction 6-1-2 (50 mg) was purified by extensive semi-PHPLC (20% MeOH, 4.0 mL/min) to yield 1 (4.3 mg). The subfraction 6-2 (120 mg) was purified by extensive semi-PHPLC (30% MeOH, 4.0 mL/min) to yield 9 (7.6 mg). Fraction 7 (604 mg) was separated into 4 subfractions (7-1~7-4) by a Sephadex LH-20 column (MeOH–CHCl3, v/v 1:1). The subfraction 7-2-1 (90 mg) isolated from subfraction 7-2 (223 mg) on a silica gel column eluted with petroleum ether– acetone (v/v 5:2, 5:3, 1:1) and was further purified by extensive semi-PHPLC (30% MeOH, 4.0 mL/min) to yield 11 (60 mg) and 12 (11 mg). The subfraction 7-3 (78 mg) was purified by extensive semi-PHPLC (5% MeOH, 4.0 mL/min) to yield 10 (9.1 mg) and the subfraction 7-4 (68 mg) was purified by extensive semi-PHPLC (40% MeOH, 4.0 mL/min) to yield 6 (4.4 mg). Fraction 8 (1.2 g) was separated into 4 subfractions (8-1~8-4) by silica gel column eluted with petroleum ether-acetone (v/v 2:1, 3:2, 1:1). The fraction 8-2 (160 mg) was further separated on a Sephadex LH-20 column (MeOH-CHCl3, v/v 3:1) to afford 3 subfractions (8-2-1~8-2-3). The subfraction 8-2-1 (50 mg) was purified by extensive semi-PHPLC (25% MeOH, 4.0 mL/min) to yield 2 (19.9 mg), and subfraction 8-2-3 (67 mg) was purified by extensive semiPHPLC (10% MeOH, 4.0 mL/min) to yield 7 (4.6 mg). The subfraction 8-3-3 (57 mg), obtained from the fraction 8-3 (233 mg) on a LH-20 column (MeOHCHCl3, v/v 1:1), was purified by extensive semiPHPLC (25% MeOH, 4.0 mL/min) to yield 3 (4.6 mg).

Preparation of the (S)-and (R)-MTPA esters of 2 by modified Mosher’s method Compound 2 (2.0 mg) was transferred into a clean NMR tube and was dried completely under vacuum. Deuterated pyridine (0.5 mL) and S(+)-α-methoxy-α(trifluoromethyl)phenylacetyl chloride (10 µL) were

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added into the NMR tube quickly under a N2 gas stream. The reaction NMR tube was allowed to stand at room temperature for 48 h. 1H-NMR and 1H-1H COSY of the (R)-MTPA esters 2b and 2d were measured directly. Similarly, another portion of compound 2 (2.0 mg) was reacted in a second NMR tube with R(–)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (10 µL) at room temperature for 72 h in deuterated pyridine (0.5 mL) to afford the corresponding (S)-MTPA esters 2a and 2c.

3-Hydroxy-5-methyl-5,6-dihydro-7H-cyclopenta [b]pyridin-7-one (1) Yellow amorphous powder; [α]D25 − 2.7 (c 0.10, MeOH); EIMS (70 eV) m/z (%): 163 (M+, 95), 162 (14), 148 ([MCH3]+, 100), 134 ([M-29]+, 59), 120 ([M-43]+, 19), 84 (22), 66 (24); HR-EIMS m/z 163.0635 [M]+ (calcd. for C9H9NO2 163.0633 ); IR νmax cm-1 (KBr) 3410, 2925, 1701, 1592, 1484, 1243, 1069. 1H (DMSO-d6, 600 MHz) and 13C (DMSO-d6, 150 MHz) see Table I. (2S,3S)-1-(4-Hydroxyphenyl)butane-2,3-diol (2) Yellow crystal; [α]D25 − 23.6 (c 0.10, MeOH); ESI-MS: m/z 183.1 [M+H]+; 1H-NMR (DMSO-d6, 600 MHz): δ 9.08 (1H, s, HO-4'), 6.99 (2H, d, J = 8.2 Hz, H-2', H-6'), 6.63 (2H, d, J = 8.2 Hz, H-3', H-5'), 4.36 (1H, br s, HO3), 4.30 (1H, br s, HO-2), 3.46 (1H, dq, J = 8.6, 6.6 Hz, H-3), 3.34 (1H, ddd, J = 8.8, 8.6, 4.4 Hz, H-2), 2.65 (1H, dd, J = 13.7, 4.4 Hz, H-1a), 2.39 (1H, dd, J = 13.7, 8.8 Hz, H-1b), 1.02 (3H, d, J = 6.6 Hz, H-4); 13C-NMR (DMSO-d6, 150 MHz): δ 155.2 (s, C-4'), 130.2 (s, C-1'), 130.0 × 2 (d, C-2' & C-6'), 114.7 × 2 (d, C-3' & C-5'), 75.7 (d, C-2), 68.4 (d, C-3), 37.5 (t, C-1), 18.8 (q, C-4). (2R,3S)-1-(4-Hydroxyphenyl)butane-2,3-diol (3) Yellow crystal; [α]D25 + 13.2 (c 0.27, MeOH); ESI-MS: m/z 183.1 [M+H]+; 1H-NMR (DMSO-d6, 600 MHz): δ 9.06 (1H, s, HO-4'), 6.99 (2H, d, J = 8.2 Hz, H-2', H-6'), 6.64 (2H, d, J = 8.2 Hz, H-3', H-5'), 4.41 (1H, d, J = 5.5 Hz, HO-3), 4.30 (1H, d, J = 5.5 Hz, HO-2), 3.36 (1H, ddq, J = 2.3, 5.5, 6.1 Hz, H-3), 3.32 (1H, dddd, J = 3.8, 8.4, 2.3, 5.5 Hz, H-2), 2.71 (1H, dd, J = 13.7, 3.8 Hz, H1a), 2.37 (1H, dd, J = 13.7, 8.4 Hz, H-1b), 1.05 (3H, d, J = 6.1 Hz, H-4); 13C-NMR (DMSO-d6, 150 MHz): δ 155.2 (s, C-4'), 130.2 (s, C-1'), 130.1 × 2 (d, C-2' & C-6'), 114.6 × 2 (d, C-3' & C-5'), 76.3 (d, C-2), 69.2 (d, C-3), 38.3 (t, C-1), 18.9 (q, C-4 ). (S)-3-Hydroxy-4-(4-hydroxyphenyl)butan-2-one (4) Pale yellow solid; [α]D25 + 12.5 (c 0.30, MeOH); ESI-MS: m/z 181.0 [M+H]+; 1H-NMR (DMSO-d6, 600 MHz): δ 9.23 (1H, br s, HO-4'), 6.95 (2H, d, J = 8.2 Hz, H-2', H-

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6'), 6.68 (2H, d, J = 8.2 Hz, H-3', H-5'), 5.37 (1H, d, J = 5.5 Hz, HO-3), 4.11 (1H, dq, J = 5.5, 6.6 Hz, H-3), 3.73 (2H, s, H-1), 1.18 (3H, d, J = 6.6 Hz, H-4); 13C-NMR (DMSO-d6, 150 MHz): δ 211.5 (s, C-2), 155.8 (s, C-4'), 130.6 × 2 (d, C-2' & C-6'), 124.8 (s, C-1'), 115.0 × 2 (d, C-3' & C-5'), 72.0 (d, C-3), 42.7 (t, C-1), 19.5 (q, C-4).

4-Methylfuran-2-carboxylic acid (8) Red solid; ESI-MS: m/z 127.0 [M+H]+; 1H-NMR (CDCl3, 600 MHz): δ 7.73 (1H, s, H-5), 6.45 (1H, s, H3), 2.38 (3H, s, CH3-4); 13C-NMR (CDCl3, 150 MHz): δ 174.1 (s, CO2H-2), 154.3 (d, C-5), 149.2 (s, C-2), 143.4 (s, C-4), 113.3 (d, C-3), 14.4 (q, CH3-4). 3-Methylfuran (9) Yellow solid; ESI-MS: m/z 83.0 [M+H]+; 1H-NMR (DMSO-d6, 600 MHz): δ 7.35 (1H, d, J = 8.3 Hz, H-5), 7.33 (1H, s, H-2), 6.81 (1H, d, J = 8.3 Hz, H-4), 2.44 (3H, s, CH3-3). 5-Methyluracil (10) Colorless crystal; ESI-MS: m/z 127 [M+H]+; 1H-NMR (DMSO-d6, 600 MHz): δ 11.01 (1H, s, NH-3), 10.59 (1H, s, NH-1), 7.25 (1H, s, H-6), 1.73 (3H, s, CH3-5).

RESULTS AND DISCUSSION Structure determination Compound 1 was obtained as a yellow amorphous powder. Its molecular formula was determined as C9H9NO2 according to the HR-EIMS and 1H- and 13CNMR spectral data (Table I). The diagnostic IR peaks at 1701 cm−1 and 3410 cm−1 were observed corresponding to a ketone and hydroxyl groups, respectively. Nine carbon atoms were further classified by DEPT experiments as a methyl, a methylene, three methines, and four quaternary carbons including a ketone group (Table I). The two sp2 methines at δH/C 7.29/117.4 and 8.24/140.6 and three sp2 quaternary carbons at δC 157.7, 157.5 and 144.2 suggested a 2,3,5-trisubstituted Table I. 1H- and Position

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pyridine nucleus that was further supported by HMBC correlations from H-2 (δH 8.24) to C-3 (δC 144.2) and C-7a (δC 155.7), and from H-4 (δH 7.29) to C-3 (δC 144.2) and C-4a (δC 157.5). 1H-1H COSY from H-8 (δH 1.29) to H-6 (δH 2.17/2.84) through H-5 (δH 3.31) and HMBC correlations between H-6 and C-7 (δC 202.2) indicated a CH3-CH-CH2-C=O moiety in the molecule. These data indicated a cyclopentano[b] pyridine skeleton (Westerwelle et al., 1991) that was further supported by the key HMBC correlations between H-8 with C-4a, and between H-5 with C-4a and C-7a, and the hydroxyl, methyl and carbonyl were linked to position 3, 5 and 7, respectively (Fig. 3). Compared with 5,6-dihydro-7H-cyclopenta[b]pyridin7-one (Westerwelle et al., 1991), the C-4a (δC 157.5) and C-7a (δC 157.7) of 1 were shifted downfield resulting from the electron-withdrawing effects and the resonance effects of C3-OH. Thus, the structure of 1 was elucidated as 3-hydroxy-5-methyl-5,6-dihydro7H-cyclopenta[b]pyridin-7-one (1). The constitutions of compounds 2-4 were elucidated as 1-(4-hydroxyphenyl)butane-2,3-diol (2, 3) and 3hydroxy-4-(4-hydroxyphenyl)butan-2-one (4) by analysis of their ESI-MS, 1D- and 2D-NMR spectra (Fig. 3). The large coupling constant of J2,3 (8.6 Hz) of 2 corresponds to threo- configuration, while the small J2,3 (2.3 Hz) of 3 means erythro- configuration. The absolute configuration of 2 was determined by a modified Mosher’s method (Kusumi and Ohtani, 1999). When reacted with (R)- and (S)-MTPA chloride, compound 2 gave the corresponding (S)- and (R)- MTPA esters 2a, 2b and 2c, 2d, respectively. The 1H-NMR chemical shifts of 2a-2d were assigned by analysis of 1H-1H COSY correlations. The observed chemical shift differences ∆δS-R (Fig. 3) clearly defined the absolute configuration of both C-2 and C-3 as S-. The absolute configuration of erythro-3 was elucidated as (2R,3S)by comparing [α]D (+13.2) to those of (2R,3S)-1phenylbudane-2,3-diol and (2S,3R)-1-phenylbudane2,3-diol (+41.9 and −41.9, respectively) (Awano et al.,

C-NMR (600 and 150 MHz) Data of 1 in DMSO-d6 1

H (J in Hz)

2 8.24 (s) 3 / 4 7.29 (s) 4a / 5 3.31 (m) 6 2.17 (dd, 18.7, 2.8), 2.84 (dd, 18.7, 7.7) 7 / 7a / 8 1.29 (3H, d, 7.1) a could be interchanged.

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C

140.6 CH 144.2 qC 117.4 CH 157.5a qC 29.5 CH 44.0 CH2 202.2 qC 157.7a qC 20.7 CH3

HMBC (H→C) C-3, C-7a / C-3, C-4a / C-4a, C-7a C-5, C-7 / / C-6, C-5, C-4a

1

H-1H COSY 4 / 2 / 6, 8 5 / / 5

A New Cyclopentanopyridine Alkaloid

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Fig. 2. Structures of compounds 1-4

Fig. 3. Key HMBC and 1H-1H COSY correlations of 1 and 4, and ∆δ values (= δS − δR) for (S)- and (R)-MTPA esters of 2

1995). The absolute configuration of compound 4 was assigned as 3S- by comparison of [α]D (+12.5) with that of (R)-3-hydroxy-1-phenylbutan-2-one (−23.6) (Guo et al., 1999). Thus, the structures of 2-4 were determined as (2S,3S)-1-(4-hydroxyphenyl)butane-2,3diol, (2R,3S)-1-(4-hydroxyphenyl)butane-2,3- diol, and (S)-3-hydroxy-4-(4-hydroxyphenyl)-2-one, respectively (Fig. 2).

Cytotoxicity and antimicrobial activity Compound 1 was assayed for the cytotoxic effect in HL-60 cell lines by the MTT assay (Mosmann, 1983) and A-549 cell lines using the SRB method (Skehan et al., 1990). The antimicrobial activities against Staphylococcus aureus, Escherichia coli, Enterobacter aerogenes, Bacillus subtilis, Pseudomonas aeruginosa, and Candida albicans were evaluated by an agar dilution method (Zaika, 1988). Compound 1 exhibited weak antibacterial activity against E. aerogenes with a MIC value of 76.7 µM, while no cytotoxicity against HL-60 and A-549 cell lines was observed (IC50 > 100 µM).

ACKNOWLEDGEMENTS This work was supported by grants from National Basic Research Program of China (No. 2010CB833800), from Special Fund for Marine Scientific Research in the Public Interest of China (No. 2010418022-3), from the National Natural Science Foundation of China (No. 30670219) and from PCSIRT (No. IRT0944). The cytotoxic assays were performed at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

REFERENCES Awano, K.-I., Yanai, T., Watanabe, I., Takagi, Y., Kitahara, T., and Mori, K., Volatile components of Wisteria flowers. Part II. Synthesis of all four possible stereoisomers of 1phenyl-2,3-butanediol and both enantiomers of 3-hydroxy -4-phenyl-2-butanone to determine the absolute configuration of the natural constituents. Biosci. Biotechnol. Biochem., 59, 1251-1254 (1995). Boux, L. J., Milligan, J. R., and Archer, M. C., Base-catalyzed decomposition of N-nitrosobis(2-oxopropyl)amine. Chem. Res. Toxicol., 1, 32-34 (1988). Burton, G., Ghini, A. A., and Gros, E. G., 13C NMR spectra of substituted indoles. Magn. Reson. Chem., 24, 829-831 (1986). Ellis, P. D., Dunlap, R. B., Pollard, A. L., Seidman, K., and Cardin, A. D., Carbon-13 nuclear magnetic resonance of 5substituted uracils. J. Am. Chem. Soc., 95, 4398-4403 (1973). Frank, M., Kingston, E., Jeffery, J. C., Moss, M. O., Murray, M., Simpson, T. J., and Sutherland, A., Walleminol and walleminone, novel caryophyllenes from the toxigenic fungus Wallemia sebi. Tetrahedron Lett., 40, 133-136 (1999). Guo, Z., Goswami, A., Mirfakhrae, K. D., and Patel, R. N., Asymmetric acyloin condensation catalyzed by phenylpyruvate decarboxylase. Tetrahedron Asymmetry, 10, 4667-4675 (1999). Hiraoka, H., Photochemistry of methylfurans. Selectivities of ring contraction reactions. J. Phys. Chem., 74, 574-581 (1970). Katritzky, A. R. and Law, K. W., A 13C study of hydroxymethyl derivatives of five-membered ring heterocycles. Magn. Reson. Chem., 26, 129-133 (1988). Kogej, T., Gostinˇc ar, C., Volkmann, M., Gorbushina, A. A., and Gunde-Cimerman, N., Mycosporines in extremophilic

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fungi-novel complementary osmolytes? Environ. Chem., 3, 105-110 (2006). Kusumi, T. and Ohtani, I., Determination of the absolute configuration of biologically active compounds by the modified Mosher’s method. In Cooper, R. and Snyder, J. K. (Eds.). The Biology-Chemistry Interface. Marcel Dekker, New York, pp. 103, (1999). Lee, C. Y., Chew, E. H., and Go, M. L., Functionalized aurones as inducers of NAD(P)H: quinone oxidoreductase 1 that activate AhR/XRE and Nrf2/ARE signaling pathways: synthesis, evaluation and SAR. Eur. J. Med. Chem., 45, 2957-2971 (2010). Lu, Z. Y., Lin, Z. J., Wang, W. L., Du, L., Zhu, T. J., Fang, Y. C., Gu, Q, Q., and Zhu, W. M., Citrinin dimers from the halotolerant fungus Penicillium citrinum B-57. J. Nat. Prod., 71, 543-546 (2008). Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65, 55-63 (1983). Sandham, D. A., Barker, L., Beattie, D., Beer, D., Bidlake, L., Bentley, D., Butler, K. D., Craig, S., Farr, D., FfoulkesJones, C., Fozard, J. R., Haberthuer, S., Howes, C., Hynx, D., Jeffers, S., Keller, T. H., Kirkham, P. A., Maas, J. C., Mazzoni, L., Nicholls, A., Pilgrim, G. E., Schaebulin, E., Spooner, G. M., Stringer, R., Tranter, P., Turner, K. L., Tweed, M. F., Walker, C., Watson, S. J., and Cuenoud, B. M., Synthesis and biological properties of novel glucocorticoid androstene C-17 furoate esters. Bioorg. Med. Chem., 12, 5213-5224 (2004). Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S., and Boyd, M. R., New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst., 82, 11071112 (1990). Takahashi, I., Maruta, R., Ando, K., Yoshida, M., Iwasaki, T., Kanazawa, J., Okabe, M., and Tamaoki, T., UCA1064B, a new antitumor antibiotic isolated from Wallemia

X.-P. Peng et al.

sebi: production, isolation and structural determination. J. Antibiot., 46, 1312-1314 (1993). Tamburini, E. and Mastromei, G., Do bacterial cryptic genes really exist? Res. Microbiol., 151, 179-182 (2000). Thornton, D. A. and Jones, M. E. B., GB 948494 (1964). Wang, W. L., Lu, Z. Y., Tao, H. W., Zhu, T. J., Fang, Y. C., Gu, Q. Q., and Zhu, W. M., Isoechinulin-type alkaloids, variecolorins A-L, from halotolerant Aspergillus variecolor. J. Nat. Prod., 70, 1558-1564 (2007a). Wang, W. L., Zhu, T. J., Tao, H. W., Lu, Z. Y., Fang, Y. C., Gu, Q. Q., and Zhu, W. M., Three novel, structurally unique spirocyclic alkaloids from the halotolerant B-17 fungal strain of Aspergillus variecolor. Chem. Biodivers., 4, 2913-2919 (2007b). Wang, W., Wang, Y., Tao, H., Peng, X., Liu, P., and Zhu, W., Cerebrosides of the halotolerant fungus Alternaria raphani isolated from a sea salt field. J. Nat. Prod., 72, 1695-1698 (2009). Westerwelle, U., Esser, A., and Risch, N., β-Aminoketone als schlusselverbindungen zur synthese von pyridinen: Einneuartiger, leistungsfahiger zugang zu kondensierten bi- und terpyridinen. Chem. Ber., 124, 571-576 (1991). Yang, S. W. and Cordell, G. A., Metabolism studies of indole derivatives using a staurosporine producer, Streptomyces staurosporeus. J. Nat. Prod., 60, 44-48 (1997). Yuki, K., Yasuo, Y., David, B., and Justin B. S., WO 2009046370 (2009). Zaika, L. L., Spices and herbs: their antimicrobial activity and its determination. J. Food Saf., 9, 97-118 (1988). Zheng, J., Zhu, H., Hong, K., Wang, Y., Liu, P., Wang, X., Peng, X., and Zhu, W., Novel cyclic hexapeptides from marine-derived fungus, Aspergillus sclerotiorum PT06-1. Org. Lett., 11, 5262-5265 (2009). Zheng, J., Xu, Z., Wang, Y., Hong, K., Liu, P., and Zhu, W., Cyclic tripeptides from the halotolerant fungus Aspergillus sclerotiorum PT06-1. J. Nat. Prod., 73, 1133-1137 (2010).