The strain S. erythraea, purchased from the American Type Culture Collection (Manassas, VA, USA) under the accession number ATCC 11635, was maintained on a yeast-malt-starch (YMS) plate. Three 250 ml Erlenmeyer flasks, each containing 40 ml of seed medium consisted of peptone (Bei Jing Ao Bo Xing, Beijing, China) 0.3%, yeast extract (Bei Jing Ao Bo Xing) 0.2%, beef extract (Bei Jing Ao Bo Xing) 0.1% and glucose (Bei Jing Ao Bo Xing) 1%, pH 7.0, were inoculated with freshly obtained S. erythraea cultured from the YMS plate and cultivated on a rotary shaker with 250 r.p.m. at 28 °C for 40 h. Then, this culture was employed as an inoculum (5%, v/v) for sixty 250 ml Erlenmeyer flasks containing 50 ml of medium comprising dextrose (Bei Jing Ao Bo Xing) 4.4%, beef extract 0.5%, peptone 0.6%, yeast extract 0.4%, tryptone (Oxoid, Basingstoke, England) 0.3%, soybean flour (Ningbo Beilun Jiangnan Grease, Ningbo, China) 1.1%, NaCl 0.15% and K2HPO4 0.05% (pH adjusted to 7.2 prior to sterilization). All the media were sterilized at 121 °C for 20 min. The cultivation was performed on a rotary shaker at 28 °C with 250 r.p.m. After 24 h, the mixture of tenvermectins A and B solution (20 mg of tenvermectins A and B dissolved in 800 μl MeOH) was added to the growing culture and the incubation was continued for a further 120 h.

After a further 5-day incubation, the final 3 l of conversion broth was pooled and filtered. The mycelia cake was extracted with ethanol (1 l). The ethanol extract was then concentrated under reduced pressure to 150 ml at 45 °C and subsequently extracted three times using an equal volume of EtOAc. The combined organic layer was evaporated under reduced pressure to give 5 g of crude residue. The crude residue was applied on a silica-gel column (Qingdao Haiyang Chemical Group, Qingdao, China; 100–200 mesh) eluted with a stepwise gradient of CHCl3/MeOH (100:0–70:30, v/v) to afford two fractions (I and II) based on the TLC profiles. TLC was performed on silica-gel plates (HSGF254, Yantai Chemical Industry Research Institute, Yantai, China), with solvent system of CHCl3/MeOH (9:1). The developed TLC plates were observed under a UV lamp at 254 nm or by heating after spraying with sulfuric acid/ethanol, 5:95 (v/v). The fraction II (220 mg) eluted with CHCl3/MeOH (85:15, v/v) was subjected to a Sephadex LH-20 gel (GE Healthcare, Glies, UK) column using CHCl3/MeOH (50:50, v/v) as eluents and detected by TLC profiles to obtain two subfractions. The second subfraction was further isolated with semipreparative HPLC (Agilent 1100, Zorbax SB-C18, 5 μm, 250 × 9.4 mm inner diameter; 1.5 ml min−1; 245 nm; Agilent, Palo Alto, CA, USA) eluting with a MeOH/CH3CN/H2O mixture (45:45:10) to give compounds 1 (tR 16.1 min, 28.6 mg) and 2 (tR 19.9 min, 27.4 mg). Optical rotation values were measured on a Perkin-Elmer 341 polarimeter (Perkin-Elmer, Fremont, CA, USA). UV spectra were recorded on a Varian CARY 300 BIO spectrophotometer (Varian, Palo Alto, CA, USA), and the IR spectra were conducted on a Nicolet Magna FT-IR 750 spectrometer (Nicolet Magna, Madison, WI, USA); 1H and 13C NMR spectra were measured on a Bruker DRX-400 (400 MHz for 1H and 100 MHz for 13C) spectrometer (Bruker, Rheinstetten, Germany). The ESI-MS and HRESI-MS data were determined on a Q-TOF Micro LC-MS-MS mass spectrometer (Waters, Milford, MA, USA).

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