Trichoderamides A and B, a pair of stereoisomers from the plant endophytic fungus Trichoderma gamsii

The culture of T. gamsii was isolated from the traditional Chinese medicinal plant Panax notoginseng (BurK.) F.H. Chen. The isolate was identified on the basis of the sequence (Genbank Accession No. JF964996) obtained by the analysis of the internal transcribed spacer region of the rDNA. The fungal strain was cultured on slants of potato dextrose agar at 25 °C for 10 days. The agar plugs were used to inoculate in Fernbach flasks (500 ml), each containing 80 g of rice, and incubated at 25 °C for 40 days. The fermented material was extracted with ethyl acetate (5 l for four times). The solution was concentrated to dryness under vacuum to afford a crude extract (50.0 g), which was fractionated by silica gel column chromatography (10 × 100 cm) using CH₂Cl₂–MeOH gradient elution. The fraction (644 mg) eluted with CH₂Cl₂–MeOH 100:4 was separated by Sephadex (LH-20) (Pharmacia, Uppsala, Sweden), column chromatography to afford five fractions (E1–E4). Fraction E3 (20 mg) was purified by RP-HPLC (Lumtech, Berlin, Germany; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 ml min⁻¹, 49% MeOH in H₂O for 40 min) to afford trichoderamide A (1; 2 mg, t_R 28.5 min) and trichoderamide B (2; 2 mg, t_R 33.3 min); fraction E2 (40 mg) was purified by RP-HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 ml min⁻¹, 49% MeOH in H₂O for 20 min) afforded trichodenol A (3; 2 mg, t_R 15.4 min) and trichodenol B (4; 6 mg, t_R 17.3 min); fraction E1 (30 mg) was purified by RP-HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 ml min⁻¹, 51% MeOH in H₂O for 20 min) afforded N-formyl-l-tyrosine methyl ester (5; 4 mg, t_R 13.2 min).

Optical rotations were measured on a Perkin-Elmer 241 Polarimeter (Perkin-Elmer, Bruker, Billerica, MA, USA), ¹H and ¹³C NMR data were acquired using Bruker 600 and Varian Inova 600 spectrometers using solvent signals (DMSO-d₆; δ_H 2.49/δ_C 39.5) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HRESIMS data were acquired using a LTQ Orbitrap XL Mass Spectrometer (Thermo, Waltham, MA, USA).

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