Three Pairs of New Isopentenyl Dibenzo[b,e]oxepinone Enantiomers from Talaromyces flavus, a Wetland Soil-Derived Fungus

Three pairs of new isopentenyl dibenzo[b,e]oxepinone enantiomers, (+)-(5S)-arugosin K (1a), (−)-(5R)-arugosin K (1b), (+)-(5S)-arugosin L (2a), (−)-(5R)-arugosin L (2b), (+)-(5S)-arugosin M (3a), (−)-(5R)-arugosin M (3b), and a new isopentenyl dibenzo[b,e]oxepinone, arugosin N (4), were isolated from a wetland soil-derived fungus Talaromyces flavus, along with two known biosynthetically-related compounds 5 and 6. Among them, arugosin N (4) and 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenz[b,e]oxepin-11(6H)-one (CAS: 160585-91-1, 5) were obtained as the tautomeric mixtures. The structures of isolated compounds were determined by detailed spectroscopic analysis. In addition, the absolute configurations of these three pairs of new enantiomers were determined by quantum chemical ECD calculations.

Since 1 and 2 coexist in the same strain, 2 was also a racemic mixture, and it displayed two peaks in a Phenomenex Lux Cellulose-2 chiral column ( Figure S1    Except for the loss of an oxygenated methyl at δ C 56.9 (5-OCH 3 ) and the appearance of an additional oxygenated methylene at δ C 65.3 (C-1 ) and methyl at δ C 14.8 (C-2 ), the NMR data of 2 (Table 1) were similar to those of 1 (Table 1), which indicated that the 5-OCH 3 in 1 was replaced by 5-OCH 2 CH 3 in 2. This deduction was supported by the 1 H-1 H COSY correlations of Ha-1 /Hb-1 and H 3 -2 and the key HMBC correlations from H-5 to C-1 , from Ha-1 /Hb-1 to C-5/C-2 , and from H 3 -2 to C-1 . On the basis of the exhaustive analysis of 2D NMR data ( 1 H-1 H COSY, HSQC, and HMBC) (Table S2, Supplementary Materials), the planar structure of 2, named arugosin L, was established ( Figure 1).
Since 1 and 3 coexist in the same strain, 3 was also a racemic mixture, and it displayed two peaks in a Phenomenex Lux Cellulose-2 chiral column ( Figure S2 27 D −27.6 (c 0.5, CHCl3)) showed their enantiomeric relationship. The absolute configurations of 3a and 3b were determined by quantum chemical ECD calculations at the APFD/6-311++g(2d,p) level. The predicted ECD curves of (5S)-3 and (5R)-3 were in good accordance with the experimental ECD for 3a and 3b, respectively ( Figure 6). Therefore, the absolute configurations of 3a and 3b were identified as 5S and 5R, respectively.  Table 2). The molecular formula of 4 was fourteen atomic mass units less than 3. Except for the loss of an oxygenated methyl at δC 57.3 (5-OCH3), the 1 H NMR and 13 C-NMR data of 4 ( Table 2) were similar to those of 3 (Table 1), which indicated that the 5-OCH3 in 3 was replaced by the hydroxyl in 4. On the basis of the exhaustive analysis of 2D NMR data ( 1 H-1 H COSY, HSQC, and HMBC) ( Table S4, Supplementary Materials), the planar structure of 4 was established, named arugosin N.
The molecular formula of 5 was fourteen atomic mass units less than 1. Except for the loss of an oxygenated methyl at δC 56.9 (5-OCH3), the 1 H-NMR and 13 C-NMR data of 5 (Table 2) were similar to those of 1 (Table 1), which indicated that the 5-OCH3 in 1 was replaced by the hydroxyl in 5.
The molecular formula of 5 was fourteen atomic mass units less than 1. Except for the loss of an oxygenated methyl at δ C 56.9 (5-OCH 3 ), the 1 H-NMR and 13 C-NMR data of 5 (Table 2) were similar to those of 1 (Table 1), which indicated that the 5-OCH 3 in 1 was replaced by the hydroxyl in 5. On the basis of the exhaustive analysis of 2D NMR data ( 1 H-1 H COSY, HSQC, and HMBC) ( Table S5, Supplementary Materials), the planar structure of 5 was established as the known compound, 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenz[b,e]oxepin-11(6H)-one [7].  13 C-NMR for 100 MHz). The letters of a and b mean that these two potons on C-1 are not chemical equivalence.
The C-5 positions of 4 and 5 were hemiacetal linkage, which were unstable and easy to split. Therefore, 4 and 5 can self-interconvert ( Figure 7) and exist as inseparable tautomeric mixtures in Nature.   13 C-NMR for 100 MHz). The letters of a and b mean that these two potons on C-1' are not chemical equivalence.
The C-5 positions of 4 and 5 were hemiacetal linkage, which were unstable and easy to split. Therefore, 4 and 5 can self-interconvert ( Figure 7) and exist as inseparable tautomeric mixtures in Nature.   [16].

General Experimental Procedures
Optical rotations were measured on a P1020 digital polarimeter (Jasco International Co. Ltd., Tokyo, Japan). UV data were recorded using a Jasco V-550 UV/Vis spectrometer. IR data were recorded on a Jasco FT/IR-480 plus spectrometer. ECD spectrum was recorded on a Jasco J-810 spectrophotometer using MeOH as the solvent. The ESI-MS spectra were recorded on a Finnigan LCQ Advantage MAX mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The HR-ESI-MS spectra were obtained on a Micromass Q-TOF mass spectrometer (Waters Corporation, Milford, MA, USA). The NMR spectra were measured with Bruker AV-400 and Bruker AV-600 spectrometers (Bruker BioSpin Group, Faellanden, Switzerland) using the solvent signals (CDCl3: δH 7.26/δC 77.0) as internal standard. The analytical HPLC was performed on a Dionex HPLC system equipped with an Ultimate 3000 pump, an Ultimate 3000 diode array detector (DAD), an Ultimate 3000 Column Compartment, an Ultimate 3000 autosampler (Thermo Fisher Scientific, Inc.) using a Gemini C18 column (4.6 mm × 250 mm, 5 μm) (Phenomenex Inc., Los Angeles, CA, USA). The semi-preparative HPLC was performed on a Shimadzu LC-6-AD Liquid Chromatography with an SPD-20A Detector using a Phenomenex Gemini C18 column (Phenomenex Inc.) (10.0 mm × 250 mm, 5 μm). The chiral HPLC was performed on a Shimadzu LC-6-AD Liquid Chromatography with an SPD-20A Detector using a Phenomenex Lux Cellulose-2 chiral column (4.6 mm × 250 mm, 3 μm). Scheme 1. The plausible biosynthetic pathways of 1-6.

General Experimental Procedures
Optical rotations were measured on a P1020 digital polarimeter (Jasco International Co. Ltd., Tokyo, Japan). UV data were recorded using a Jasco V-550 UV/Vis spectrometer. IR data were recorded on a Jasco FT/IR-480 plus spectrometer. ECD spectrum was recorded on a Jasco J-810 spectrophotometer using MeOH as the solvent. The ESI-MS spectra were recorded on a Finnigan LCQ Advantage MAX mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The HR-ESI-MS spectra were obtained on a Micromass Q-TOF mass spectrometer (Waters Corporation, Milford, MA, USA). The NMR spectra were measured with Bruker AV-400 and Bruker AV-600 spectrometers (Bruker BioSpin Group, Faellanden, Switzerland) using the solvent signals (CDCl 3 : δ H 7.26/δ C 77.0) as internal standard. The analytical HPLC was performed on a Dionex HPLC system equipped with an Ultimate 3000 pump, an Ultimate 3000 diode array detector (DAD), an Ultimate 3000 Column Compartment, an Ultimate 3000 autosampler (Thermo Fisher Scientific, Inc.) using a Gemini C 18 column (4.6 mm × 250 mm, 5 µm) (Phenomenex Inc., Los Angeles, CA, USA). The semi-preparative HPLC was performed on a Shimadzu LC-6-AD Liquid Chromatography with an SPD-20A Detector using a Phenomenex Gemini C 18 column (Phenomenex Inc.) (10.0 mm × 250 mm, 5 µm). The chiral HPLC was performed on a Shimadzu LC-6-AD Liquid Chromatography with an SPD-20A Detector using a Phenomenex Lux Cellulose-2 chiral column (4.6 mm × 250 mm, 3 µm).

Fungus Material
The strain AHK07-3 was isolated from the soil, collected at the wetland of Ahongkou, Sinkiang Province, China. The strain was identified as Talaromyces flavus based on the morphological characters and gene sequence analysis. The ribosomal internal transcribed spacer (ITS) and the 5.8S rRNA gene sequences (ITS1-5.8S-ITS2) of the strain have been deposited at GenBank as KX011167. The fungus was cultured on slants of potato dextrose agar at 25 • C for 5 days. Agar plugs were used to inoculate four Erlenmeyer flasks (250 mL), each containing 100 mL of potato dextrose broth. Four flasks of the inoculated media were incubated at 25 • C on a rotary shaker at 200 rpm for 5 days to prepare the seed culture. Fermentation was carried out in 20 Erlenmeyer flasks (500 mL), each containing 70 g of rice. Distilled H 2 O (105 mL) was added to each flask, and the rice was soaked overnight before autoclaving at 120 • C for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the seed culture containing mycelia and incubated at room temperature for 50 days.