2.3 Extraction and isolation

The fermentation materials were adequately extracted with EtOAc (3 × 25 L), and the organic solvent was evaporated in vacuum to yield ca. 20 g of crude extracts. The crude extracts were subjected to a silica gel vacuum liquid chromatography column, which was eluted with an increasing gradient of EtOAc/petroleum ether (from 30:1 to 1:1) to afford six fractions (Fr. 1-Fr. 6). Fr. 4 (3.2 g), eluting with EtOAc/petroleum ether 5:1, was further fractionated over an ODS reversed-phase silica gel with a mixed solvent system of MeOH/H₂O (from 10 to 100%, v/v). This afforded a total of eight subfractions (Fr. 4.1-Fr. 4.8). Fr. 4.6 was further purified over an open silica gel column chromatography by using the solvent system CH₂Cl₂ and MeOH with the ratio 20:1 to afford 16 mg of compound 2. Fr. 5 (2.5 g), eluting with EtOAc/petroleum ether 2:1, was applied to ODS silica gel with gradient elution of MeOH/H₂O (from 10 to 100%, v/v) to yield eight subfractions (Fr. 5.1-Fr. 5.8). Compound 1 (10.2 mg) was isolated from a two-step purification process, first from Fr. 5.3 over an open silica gel column chromatography using the solvent system CH₂Cl₂ and MeOH with the ratio 20:1, followed by preparative TLC (CH₂Cl₂/MeOH, 15:1, v/v). Compound 1 was further resolved into the pure enantiomers (+)-1 (4.9 mg, t_R = 9.6 min) and (–)-1 (4.7 mg, t_R = 10.9 min) by chiral HPLC using a (R,R). Whelk-O1 chiral column (10 mm; 4.6 × 250 mm; n-hexane-ethanol eluent 6:4, v/v; 1.0 mL/min). Compound 3 (10.2 mg, t_R 7.7 min) was isolated from Fr. 5.4 by semipreparative HPLC (YMC-pack ODS-A, 5 μm; 10 × 250 mm; 55% MeOH/H₂O; flow rate 2 mL/min). Compound 7 (5.6 mg) was isolated from Fr. 5.5 by preparative TLC (CH₂Cl₂/MeOH/acetic acid, 15:1:0.4, v/v). Compound 6 (11.3 mg) was isolated from Fr. 5.6 by preparative TLC (CH₂Cl₂/MeOH/acetic acid, 20:1:0.4, v/v). Fr. 6 (4.0 g), eluting with EtOAc/petroleum ether 1:1, was fractionated by Sephadex LH-20 column chromatography in MeOH to give subfractions Fr. 6.1-Fr. 6.3. Fr. 6.1 was subjected to semipreparative HPLC (65% MeOH/H₂O) to give compounds 4 (20.2 mg, t_R 6.9 min) and 5 (6.2 mg, t_R 8.8 min), respectively. Finally, compound 8 (4.9 mg) was obtained by preparative TLC (CH₂Cl₂/MeOH, 20:1, v/v) from Fr. 6.3.

(±)-Chrysoalide B (1): white amorphous powder; [α]²⁰_D + 9.6 (c 0.10, MeOH) for (+)-1 and [α]²⁰_D –10.2 (c 0.10, MeOH) for (–)-1; UV (MeOH) λ_max (log ε) 213 (2.16), 239 (1.60), 331 (1.49) nm; ¹H and ¹³C NMR data (measured in DMSO-d₆) (see Table 1); HRESIMS m/z 223.0644 [M - H]^– (calcd for C₁₁H₁₁O₅, 223.0606).

NMR data for compounds (±)-1 and 2 in DMSO-d₆ (¹H at 500 MHz and ¹³C at 125 MHz).

Penicidone E (2): colorless oil; [α]²⁰_D + 13.5 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 220 (3.88), 254 (3.26), 309 (2.98); ¹H and ¹³C NMR data (measured in DMSO-d₆) (see Table 1); HRESIMS m/z 390.1547 [M + H]⁺ (C₂₀H₂₄NO₇) and 412.1369 [M + Na]⁺ (C₂₀H₂₃NO₇Na).