New tenvermectin analogs obtained by microbial conversion with Saccharopolyspora erythraea

Microbial transformation has been considered a powerful tool to modify the structures of natural bioactive substrates owing to its generally highly regio and stereo selective, simple, safe, environment friendly and relatively mild reaction conditions.^{1, 2, 3} A wide variety of new derivatives of natural products can be obtained by oxidation,⁴ hydrolysis,⁵ isomerization, hydroxylation, esterification,⁶ phosphorylation⁷ and glycosylation⁸ employing different conversion systems. Tenvermectins A and B are a kind of 16-membered macrocyclic lactone antibiotics isolated from the fermentation broth of genetically engineered strain Streptomyces avermitilis MHJ1011 with potent insecticidal properties.^{9, 10} Aiming at obtaining new target derivatives with improved insecticidal activity and lower toxicity, we carried out the transformation study of tenvermectins A and B by several microbial strains and no positive results were obtained. Inspired by literature,^{11, 12} we examined the microbial conversion of tenvermectins A and B by Saccharopolyspora erythraea ATCC 11635. This led to the isolation and identification of two new tenvermectin analogs, 4″-O-glucosyl tenvermectin A (1) and 4″-O-glucosyl tenvermectin B (2) (Figure 1). In this paper, we describe the microbial conversion of tenvermectins A and B, as well as the isolation, structural elucidation, nematocidal and acarcidal activity of the two new compounds.

Figure 1

Structures and key HMBC correlations of compounds 1 and 2.

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 (t_R 16.1 min, 28.6 mg) and 2 (t_R 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).

The physicochemical properties of 1 and 2 are summarized in Table 1. The molecular formulas of 1 and 2 were determined to be C₅₁H₇₈O₁₉ and C₅₂H₈₀O₁₉, respectively, by HRESI-MS analysis, and their molecular masses were 162 mass units higher than the corresponding parent compounds, tenvermectins A and B, respectively. ¹H and ¹³C NMR data for 1 and 2 were shown in Table 2. The NMR data of 1 and 2 were similar to those of tenvermectins A and B except of the additional sugar moiety revealed by the anomeric proton signals at δ_H 4.46 (1H, d, J=7.6 Hz) in 1 and at δ_H 4.46 (1H, d, J=7.5 Hz) in 2. The chemical shift assignments and sugar linkage sites were determined by ¹H–¹H COSY, HMQC and HMBC experiments. By comparison of the chemical shifts and coupling constants of the additional sugar moiety in 1 and 2 with those of β-d-glucose residue that was contained in 2(R)-3-(4′-O-β-d-glucopyranosyl-3′-methoxyphenyl)propane-1,2-diol¹³ and 6-hydroxy-2,2-dimethyl-3,4-dihydro-2H-naphtho[1,2-b]pyran-5-O-β-d-glucopyranoside,¹⁴ it was shown that the additional sugar moiety in 1 and 2 were β-d-glucose. The position of the glucosyl linkage in 1 and 2 were supported by the HMBC correlation (Figure 1) between the anomeric proton H-1‴ and C-4″. The assignment of the terminal glucose residue in 1 and 2 was further confirmed by the fact that the strain S. erythraea ATCC 11635 could transform the avermectin and ivermectin to the corresponding 4″-O-glucosyl derivatives.¹² Therefore, the structures of 1 and 2 (Figure 1) were identified as 4″-O-glucosyl tenvermectin A and 4″-O-glucosyl tenvermectin B, respectively.

Table 1 Physicochemical properties of 1 and 2

Table 2 ¹H and ¹³C NMR data^a of compounds 1 and 2

The nematocidal and acaricidal activity of 1 and 2 against Bursaphelenchus xylophilus and Tetranychus cinnabarinus, respectively, reared in the laboratory were evaluated according to our reported paper.^{9, 10} In addition, for comparison, the nematocidal and acaricidal activity of their parent compounds, tenvermectins A and B, were examined. The bioassays were performed simultaneously on three replicates for each concentration. The bioassay results (Table 3) demonstrated that the two bioconversion products 1 and 2 showed almost the same or a slightly weaker nematocidal activity (LC₅₀s: 1, 6.7984 mg l⁻¹; 2, 5.7980 mg l⁻¹) than tenvermectins A and B against third-instar larvae of B. xylophilus. In addition, compounds 1 and 2 exhibited acaricidal activity against adult mites of T. cinnabarinus with LC₅₀ values of 0.0156 and 0.0113 mg l⁻¹, respectively, nearly as potent as their parent compounds.

Table 3 Biological activity of compounds 1 and 2 against B. xylophilus and T. cinnabarinus