MBJ-0086 and MBJ-0087, new bicyclic depsipeptides, from Sphaerisporangium sp. 33226

We have constructed a library of isolated natural products referred to as the ‘CB library’ for efficient implementation of biological screenings.¹ To expand and variegate this library, we have conducted chemical and biological screenings using the advanced compound-identification system based on accumulated HPLC-MS profiling data combined with strain information designated as ‘MBJ’s special selection’. During screening, we have identified several new natural bioactive compounds such as the cytotoxic eremophilane derivatives MBJ-0009, MBJ-0010, MBJ-0011, MBJ-0012 and MBJ-0013 from Nectria sp. f26111² and Apiognomonia sp. f24023,³ the chaetoglobosin derivatives MBJ-0038, MBJ-0039 and MBJ-0040 from Chaetomium sp. f24230,⁴ a hydroxamate metabolite MBJ-0003 from Micromonospora sp. 29867⁵ and the aziridine containing-peptide MBJ-0035 from Streptosporangium sp. 32552.⁶ Further screening was used to isolate new bicyclic depsipeptides designated as MBJ-0086 (1) and MBJ-0087 (2) from the culture broth of Sphaerisporangium sp. 33226 (Figure 1). Here, we describe the fermentation, isolation, structure elucidation and antibacterial activity of 1 and 2.

Figure 1

Structures of MBJ-0086 (1) and MBJ-0087 (2).

Sphaerisporangium sp. 33226 was isolated from a soil sample collected in Kochi Prefecture, Japan. This producing strain was cultivated in 250-ml Erlenmeyer flasks, each containing 25 ml of a seed medium consisting of 2% potato starch (Tobu Tokachi Nosan Kako Agricultural Cooperative Assoc., Hokkaido, Japan), 2% glucose (Junsei Chemical, Tokyo, Japan), 2% soy bean powder (SoyPro, J-Oil Mills, Tokyo, Japan), 0.5% yeast extract powder (Oriental Yeast, Tokyo, Japan), 0.25% NaCl (Junsei Chemical), 0.32% CaCO₃ (Wako Pure Chemical Industries, Osaka, Japan), 0.0005% CuSO₄·5H₂O (Wako Pure Chemical Industries), 0.0005% ZnSO₄·7H₂O (Wako Pure Chemical Industries) and 0.0005% MnCl₂·4H₂O (Junsei Chemical) for 3 days at 28 °C on a rotary shaker at 220 r.p.m. (pH 7.4). The seed culture (0.5 ml) was transferred into 500-ml Erlenmeyer flasks containing 50 ml of the same medium and cultivated for 4 days at 28 °C on a rotary shaker at 220 r.p.m. After cultivation, an equal volume of n-BuOH was added to the culture broth (2 liters). The n-BuOH extract was evaporated in vacuo and partitioned between water (300 ml) and EtOAc (300 ml, 3 times). The EtOAc-soluble material (1.26 g) was subjected to silica gel medium-pressure liquid chromatography (Si-MPLC, Purif-Pack SI-30, Shoko Scientific, Yokohama, Japan) eluted with a gradient system of n-hexane–EtOAc (0–25% EtOAc) followed by a stepwise solvent system of CHCl₃–MeOH (0, 2, 5, 10, 20, 30 and 100% MeOH). Fractions were monitored using the UPLC-DAD-ELS-MS system. The 5% MeOH eluate (553.9 mg) was subjected to Sephadex LH-20 column chromatography (CHCl₃/MeOH=1:1, GE Healthcare BioSciences AB, Uppsala, Sweden). The target fraction (415.1 mg) was then separated by Si-MPLC (Purif-Pack SI-30) using isocratic elution with 4% MeOH in CHCl₃ to give fractions A (199.5 mg) and B (129.6 mg). Fraction A was subjected to a second round of chromatography using Si-MPLC (Purif-Pack SI-30, 3% MeOH in CHCl₃, isocratically) to obtain crude 2 (59.5 mg). The pure sample of 2 (8.2 mg, retention time: 11.0 min) was obtained by two preparative HPLC runs on an XSelect CSH C₁₈ column (5.0 μm, 19 i.d. × 150 mm; Waters, Milford, MA, USA) with 55% aq. MeCN containing 0.1% formic acid (flow rate: 10 ml min^–1). Fraction B was re-chromatographed using Si-MPLC (Purif-Pack SI-30, isocratic, 3% MeOH in CHCl₃) to give semi-purified 1 (51.5 mg). Final purification of 1 was carried out by reversed-phase HPLC (column: CAPCELL PAK C18 MGII column, 5.0 μm, 20 i.d. × 150 mm, Shiseido, Tokyo, Japan; solvent: 40% aq. MeCN containing 0.1% formic acid; flow rate: 10 ml min^–1; retention time: 5.5 min) to yield 1 (21.6 mg).

Compound 1 was a colorless amorphous powder; [α]²⁶_D +107 (MeOH; c 0.05). The IR spectrum of 1 showed absorption bands for hydroxy (3400 cm⁻¹) and amide (1640 cm⁻¹) functionalities. The UV spectrum showed absorption maxima at 286 (logɛ 4.0) and 303 (logɛ 4.0) nm in MeOH. The molecular formula of 1 was found to be C₆₇H₈₆N₁₀O₁₆ by high-resolution-ESI-MS (m/z 1287.6339 [M+H]⁺, calcd for C₆₇H₈₇N₁₀O₁₆ 1287.6302). The ¹H and ¹³C NMR spectra of 1 suggested the peptidic nature of this molecule, including 4 amide resonances, signals for 9 α-methine protons, 4 N-methyl groups and 10 amide carbonyl carbons. The direct connectivity between protons and carbons was determined based on the heteronuclear single quantum coherence spectrum; the tabulated ¹³C and ¹H NMR spectroscopic data for 1 are shown in Table 1.

Table 1 ¹³C (150 MHz) and ¹H (600 MHz) NMR spectroscopic data for MBJ-0086 (1) and MBJ-0087 (2) in CDCl₃

Detailed analyses of DQF-COSY and constant-time HMBC⁷ spectra revealed the presence of Ala, N-MeVal (MeVal), two 5-MePro (5MePro-1 and 5MePro-2), N,N-diMeThr (Me2Thr), dehydro-N-MeTyr (Δ-MeTyr), β-hydroxy-4-methoxyTyr (4-methoxydroxidopa, mDOPS), 2-phenylglycine (PhGly) and a pipecolic acid (Pip) residues, as shown in Figure 2a.

Figure 2

(a) Structure determination of 1. (b) Structure determination of Ceo moiety of 2.

The structure of the residual amino acid moiety with a 2,2′-bioxirane moiety (abbreviated as Bx), which is considered to be derived from Ile, was determined based on the COSY correlations from an amide proton NH-Bx (δ_H 9.59) to a doublet α-methine proton H-Bx-2 (δ_H 4.77) and from an oxymethine proton H-Bx-5 (δ_H 3.48) to oxymethylene protons H₂-Bx-6 (δ_H 2.85 and 2.83), and HMBCs from the H-Bx-2 to an amide carbonyl carbon C-Bx-1 (δ_C 167.8), an oxygenated quaternary carbon C-Bx-3 (δ_C 57.6), an oxymethylene carbon C-Bx-4 (δ_C 49.5) and an oxymethine carbon C-Bx-5 (δ_C 51.4). High-field shifted ¹³C NMR chemical values of C-Bx-3, C-Bx-4, C-Bx-5 and C-Bx-6 (δ_C 45.5), and the large ¹J_CH values of C-Bx-4 (174 Hz) and C-Bx-6 (180 Hz) also supported this bis-epoxide substructure.

The amino acid sequence of 1 was determined based on ¹H–¹³C long-range couplings from an α-proton H-5MePro-1-2 (δ_H 4.38) to an amide carbonyl carbon C-5MePro-2-1 (δ_C 170.1), from an α-proton H-5MePro-2-2 (δ_H 4.66) to an amide carbonyl carbon C-PhGly-1 (δ_C 167.8), from an α-proton H-PhGly-2 (δ_H 5.75) to an amide carbonyl carbon C-Ala-1 (δ_C 170.6), from an α-proton H-Ala-2 (δ_H 4.58) to an amide carbonyl carbon C-Me2Thr-1 (δ_C 168.6), from a β-proton H-Me2Thr-3 (δ_H 5.08) to an amide carbonyl carbon of Δ-MeTyr (δ_C 163.2), from an N-methyl proton of Δ-MeTyr (δ_H 3.32) to an amide carbonyl carbon C-MeVal-1 (δ_C 170.2), from an N-methyl proton of MeVal (δ_H 3.33) to an amide carbonyl carbon C-mDOPS-1 (δ_C 168.9), from an α-proton H-mDOPS-2 (δ_H 4.68) to an amide carbonyl carbon C-Pip-1 (δ_C 171.1) and from an α-proton H-Pip-2 (δ_H 5.12) to an amide carbonyl carbon C-Bx-1. The presence of an ether bond between C-Δ-MeTyr-7 and C-mDOPS-6 was determined based on the rotating frame NOE between the H-Δ-MeTyr-6 (δ_H 7.11) and H-mDOPS-5 (δ_H 5.32), and index of hydrogen deficiency deduced from the molecular formula. Furthermore, the ¹H and ¹³C NMR chemical shifts of mDOPS moiety in 1 were well matched with those of RA-IV, a cyclic hexapeptide obtained from Rubiae Radix.⁸ Hence, the structure determination of 1 was accomplished as shown in Figure 1. The stereochemistry of the trisubstituted double bond was determined as Z by means of a J-resolved HMBC-2 spectrum.⁹ The ester smaller ¹H–¹³C long-range coupling value of 4.2 Hz between carbonyl carbon signal of Δ-MeTyr and H-Δ-MeTyr-3 indicated that these two nuclei were cis to each other, thus concluding the Z geometry for the double bond.^{10, 11}

MBJ-0087 (2) was a colorless amorphous powder with the following properties: [α]²⁶_D +113 (MeOH; c 0.1); UV λ_max (logɛ) in MeOH: 286 (4.0) and 303 (4.0) nm; high-resolution-ESI-MS: m/z 1321.5912 [M–H]^–, calcd for C₆₇H₈₆³⁵ClN₁₀O₁₆ m/z 1321.5912; and IR absorption (ν_max) 3400 and 1640 cm^–1. Analyses of 1D and 2D NMR spectra of 2 revealed that the partial structure of 2 was the same as that of 1 with the exception of the 2,2′-bioxirane moiety, as described below.

The presence of a 2-amino-2-(2-(2-chloro-1-hydroxyethyl)oxiran-2-yl)acetic acid (Ceo) moiety was determined based on COSY correlations from an amide proton NH-Ceo (δ_H 8.70) to an α-methine proton H-Ceo-2 (δ_H 4.88), from an oxymethine proton H-Ceo-5 (δ_H 4.20) to chlorinated methylene protons H₂-Ceo-6 (δ_H 3.84, 3.60), and ¹H–¹³C long-range couplings from the H-Coe-2 to an amide carbonyl carbon C-Coe-1 (δ_C 168.0), an oxygenated quaternary carbon C-Coe-3 (δ_C 58.7) and an oxymethine carbon C-Coe-5 (δ_C 69.0), from oxymethylene protons H₂-Ceo-4 (δ_H 3.26, 3.02) to the C-Coe-3 and from the H₂-Ceo-6 to the C-Coe-3 and C-Coe-5, and low-field shifted chemical shifts of C-Ceo-3 and C-Ceo-4 (δ_C 48.6; Figure 2b). HMBCs from the H-Ceo-2 to an amide carbonyl carbon C-5MePro-1-1 (δ_C 172.0) and from an α-methine proton H-Pip-2 (δ_H 5.18) to C-Ceo-1 revealed the gross structure of 2 as shown in Figure 1.

Antimicrobial activity against Bacillus subtilis was tested. First, 20 μl of a log-phase B. subtilis culture (2 × 10⁶ CFU ml⁻¹) was dispensed into each well in a 384-well plate and compounds were added at various concentrations. Dimethyl sulfoxide (1%) was used as a vehicle control. After incubation for 18 h at 37 °C, the OD₆₂₀ was measured using a 2103 Envision Multilabel Reader (PerkinElmer, Waltham, MA, USA). Compounds 1 and 2 were active against B. subtilis with IC₅₀ values of 1.1 and 24 μM, respectively. In contrast, neither compound showed cytotoxicity against human ovarian adenocarcinoma SKOV-3 cells (IC₅₀>50 μM) or antimicrobial activity against Micrococcus luteus (IC₅₀>50 μM).

The structures of 1 and 2 are extremely unique and include bicyclic depsipeptides, diphenyl ether and rare amino acid residues such as 5-MePro, Δ-MeTyr, mDOPS and Bx (1) or Ceo (2). Compounds 1 and 2 are the first natural peptide compounds possessing Bx (1) or a Ceo (2) units, respectively. Furthermore, there have been no reports describing peptide compounds possessing a diphenyl ether moiety except for dityromycin¹² isolated from Streptomyces sp. and the bicyclic hexapeptides isolated from Rubiaceae.^{13, 14}