8′-epimer of herbicidin F and its congeners from Streptomyces sp. YIM 66142

Herbicidins are isolated from different strains of Streptomyces, and they exhibit several interesting biological activities.^{1, 2, 3, 4, 5} Herbicidins A and B indicated no activity at a concentration of 100 μg mL⁻¹ against Xanthomonas oryzae in vitro, but protection of the leaves of rice plant from bacterial leaf blight disease was caused by X. oryzae.¹ Herbicidins A and B have also been shown to reduce seed germination¹ and although they exhibit selective toxicity toward dicotyledonous plants, no toxicity against animals is observed.¹ In our effort to search for new biological compounds from endophytes, a new antibiotic 8′-epimer of herbicidin F (1), along with four known compounds, herbicidin F (2),⁵ herbicidin A (3),⁵ herbicidin B (4)^{5, 6, 7} and 2′-O-demethylherbicidin F (5)⁸ were isolated from the broth of Streptomyces sp. YIM 66142, a Dendrobium chrysotoxum endophyte (Figure 1). The absolute configuration of the 8′-epimer of herbicidin F (1) was established using its electronic CD (ECD) spectrum, density functional theory (DFT)-ECD calculations and NOESY spectrum. An antibiotic activity assay showed that herbicidin B (4) and 2′-O-demethylherbicidin F (5) had antibacterial activity against Bacillus subtilis as well as their inhibition of germination of the plant seeds, such as cabbage.

Figure 1

Herbicidin derivatives from the Streptomyces sp. YIM 66142.

Streptomyces sp. YIM 66142 was cultivated in 250 mL Erlenmeyer flasks containing 50 mL of seed medium (0.4% yeast extract, 0.4% glucose, 0.5% malt extract, decavitamin, 0.03% alanine, pH 7.2). The flasks were shaken at 300 r.p.m. at 28 °C for 2 days using a rotary shaker. The seed culture (10%) was transferred into 1 L Erlenmeyer flasks containing 250 mL of fermentation medium (0.3% soybean powder, 2% glucose, 0.2% yeast extract, 0.5% starch, 0.2% peptone, 0.4% NaCl, 0.05% K₂HPO₄, 0.05% MgSO₄ and 0.2% CaCO₃, pH 7.2). The flasks were shaken at 300 r.p.m. for 6 days at 28 °C using a rotary shaker. The fermented whole broth (60 L) was centrifuged to separate the mycelia, and the fermented solution was extracted three times by an equivalent volume of EtOAc, to yield the EtOAc extract (29.1 g). The mycelia were extracted three times with acetone, yielding the acetone extract (8.0 g). The two extracts were then suspended in distilled H₂O and extracted three times with EtOAc, to concentrate the total sample in solution (13 g). This sample was separated into seven fractions (A1–A7) using a silica gel column with petroleum ether to MeOH. The fifth fraction, A5 (CHCl₃-MeOH 20:1), was separated using a Sephadex LH-20 column and eluted with methanol to yield six fractions (A5-I to A5-VI). Fraction A5-III was again fractionated with a Sephadex LH-20 column and eluted with methanol to obtain compound 2 (100 mg). Fraction A5-IV was subsequently separated into nine fractions (fractions A5-IV–I to A5-IV–IX) with a Sephadex LH-20 column. Fraction A5-IV–VI was further fractionated on silica gel with chloroform/acetone elution solvent (12:1–12:4, v/v) to obtain 1 (1.2 mg), and fraction A5-IV–VII was separated on silica gel with chloroform/methanol (45:1, v/v) to obtain 4 (1.5 mg). The A6 part was separated into four fractions (A6-I to A6-IV) using a Sephadex LH-20 column and eluted with methanol. The fraction A6-IV was again fractionated with a Sephadex LH-20 column and eluted with methanol to obtain 3 (4.2 mg) and 5 (5.1 mg).

The molecular mass was determined by electrospray ionization mass spectrometry (ESI-MS) in positive mode, and the molecular formula C₂₃H₂₉N₅O₁₀ was obtained by high-resolution electron ionization mass spectrometry (HREIMS) of compound 1 (m/z 535.1939; calcd for C₂₃H₂₉N₅O₁₀ at m/z 535.1914). The ¹H NMR (Table 1) spectrum of 1 displayed two three-proton signals at δ1.83 (3H, d, J=6.9 Hz), 1.87 (3H, s) for methyl groups attached to a double bond and 3.50 (3H, s) and 3.74 (3H, s) for the methyloxy groups; for one methylene group at δ 2.24 (2H, m); and for 11 methine groups at δ 3.82 (1H, m), 3.83 (1H, d, 3.9 Hz), 4.23 (1H, s), 4.47 (1H, s), 4.55 (1H, s), 4.58 (1H, s), 5.63 (1H, d, 4.0 Hz), 6.12 (1H, s), 7.06 (1H, d), 8.16 (1H, s) and 8.22 (1H, s). The ¹³C NMR spectrum (Table 1) and DEPT spectra of 1 showed the following 23 carbon signals: 2 methyls, 1 methylene, 11 methines, 2 methyloxy and 7 quaternary carbons. The 1D NMR data and molecular formula of 1 demonstrated that the structure of 1 has the backbone of the undecose antibiotic herbicidin. Comprehensive NMR spectral data showed that the structure of 1 was similar to herbicidin F (2) except for the chemical shift differences at C-6′, C-7′, C-8′, C-11′, C-1′′, C-2′′ and C-3′′. An analysis of COSY spectroscopic data revealed the C_4′-C_5′-C_6′ structural connection. The connection of C-1′ to N-9 was determined by the HMBC H-1′ to C-8 correlation. The C_1′-C_2′-C_3′ fragment was determined by the HMBC correlations from H-1′ to C-2′ and H-2′ to C-4′, H-3′ to C-2′ and C-4′. The C_7′-C_8′-C_9′-C_10′-C_11′ segment was determined by the HMBC correlations between H-10′ and C-6′, C-8′, C-9′ and C-11′. The (E)-2-methyl-2-butenoic acid moiety was determined by the following HMBC correlations: 4′′-H to 2′′-C and 3′′-C, 5′′-H to 2′′-C and 1′′-C. Further structural data were obtained using 2D NMR (Figure 2). ROESY correlations were found between H-1′ and H-2′, H-3′ and H-4′, H-2′ and 8-H, H-3′ and H-5′, H-5′and H-6′ in compound 1. ROESY correlations between H-8 and H-3′′, H-3′′ and H-6′ were not observed (Figure 3). However, ROESY correlation peaks for herbicidin F (2) between H-3′′ and H-6′ were observed, suggesting that the (E)-2-methyl-2-butenoic acid group connected to C-8′ is oriented differently in compounds 1 and 2. The configuration of 1 was confirmed to be 8'-epi-herbicidin F. Another 8′-epi-herbicidin was reported recently in Streptomyces.⁸ To identify its absolute configurations of compound 1, ECD spectrum was used in combination with DFT-ECD calculations.

Table 1 ¹H (500 MHz, CD₃OD) and ¹³C NMR (125 MHz, CD₃OD) data of compounds 1 and 2

Figure 2

Key HMBC and¹H-¹H COSY correlations for compound 1.

Figure 3

ROESY correlations of compounds 1 (left) and 2 (right).

The experimental ECD spectrum of herbicidin F (2) contains a broad negative signal centered at 268 nm and a strong positive signal centered at 220 nm with a shoulder at 203 nm. The experimental ECD spectrum of 8-epi-herbicidin F (1) exhibits a broad negative signal centered at 260 nm, a weakly positive signal centered at 223 nm, and a weakly negative signal centered at 213 nm. The change in ECD spectra was caused by the 8'-(E)-2-methyl-2-butenoic acid moiety located at the equatorial bond in compound 1. The calculated spectrum of 8'-epi-herbicidin F (1) and herbicidin F (2) (Figure 4) was well matched with the experimental spectra. This comparison allowed us to identify compound 1 as 8'-epi-herbicidin F.

Figure 4

Experimental ECD (left) and calculated ECD (right) spectra of 8'-epi-herbicidin F (1) and herbicidin F (2). σ is 0.2 eV. A full color version of this figure is available at The Journal of Antibiotics journal online.

Compounds 2, 3 and 5 showed no marked cytotoxicity on human leukemia cells (HL-60), human lung cancer cells (A549), hepatocellular carcinoma cells (SMMC-7721), human breast adenocarcinoma (MCF-7) or human colorectal carcinoma cells (SW480) at 40 μm. These compounds were tested for antimicrobial activities against five strains of microorganisms (B. subtilis, Staphylococcus aureus, Escherichia coli, Candida albicans and X. oryzae) at 512 μg mL⁻¹. Compounds did not show significant antibacterial activity except for 4 and 5 showed MICs of 32 μg mL⁻¹ against B. subtilis (Table 2). Much more potent inhibitory effect of herbicidins occurred against germination of the plant seeds, such as cabbage. The MICs of 8'-epi-herbicidin F (1), herbicidin F (2), herbicidin A (3), herbicidin B (4) and 2′-O-demethylherbicidin F (5) for germination of the seeds of cabbage were 12.5–50 , <3.1 , 3.1–12.5 , 12.5–50 and 12.5–50 μg mL⁻¹.

Table 2 MICs (μg mL⁻¹) of 1, 2, 3, 4 and 5 in screening of antimicrobial activity