Actinobacteria have been extensively studied thus far, and they are known to produce pharmaceutically important compounds.1, 2 Soil serves as the primary source of actinobacteria and, therefore, we attempted to obtain novel metabolites from actinobacteria isolated from soil samples. During our screening program for novel metabolites, a new metabolite possessing a thioester named JBIR-69 (1, Figure 1a), together with a known compound, 3-isopropylmalate methyl ester, was isolated from the culture broth of Streptomyces sp. OG05. This paper describes the fermentation, isolation, structural elucidation, and, in brief, the biological activity of 1.

Figure 1
figure 1

(a) Structure of 1. (b) Key correlations in the DQF-COSY (bold line) and HMBC (arrow) spectra of 1.

Full size image

Streptomyces sp. OG05 was isolated from a soil sample collected in Okinawa Prefecture, Japan, and was cultured on a rotary shaker (180 r.p.m.) at 27 °C for 5 days in 500-ml Erlenmeyer flasks containing 100 ml of 2% glycerol (Nacalai Tesque, Kyoto, Japan), 1% molasses (Dai-Nippon Meiji Sugar, Tokyo, Japan), 0.5% casein (Kanto Chemical, Tokyo, Japan), 0.1% polypeptone (Nihon Pharmaceutical, Tokyo, Japan), and 0.4% CaCO3 (Kozaki Pharmaceutical, Tokyo, Japan; pH 7.2 before sterilization).

The mycelium collected from the culture broth (1 l) by centrifugation was extracted with Me2CO (200 ml). After concentration in vacuo, the residue aqueous concentrate with pH adjusted to 2–3 using 2 N HCl was extracted twice with EtOAc. The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The dried residue (180 mg) was subjected to normal-phase medium-pressure liquid chromatography (Purif-Pack SI-60, size: 20, Moritex, Tokyo, Japan) with a Hexane-EtOAc (0–30% EtOAc) linear gradient system, followed by elution with a CHCl3–MeOH (0–90% MeOH) linear gradient system; then, peak detection was carried out with UV absorption at 254 nm. The 10–20% MeOH elute fractions (30 mg) were further purified by reversed-phase high-pressure liquid chromatography (HPLC) using a PEGASIL ODS column (Senshu Pak, 20 i.d. × 150 mm, Senshu Scientific, Tokyo, Japan) with a H2O–MeOH (0–100% MeOH) linear gradient system containing 0.1% formic acid to yield JBIR-69 (1, 15 mg) together with 3-isopropylmalate methyl ester (6 mg).3

Compound 1 was obtained as a colorless oil ([α]D −11.8, 29 °C, c 0.28 (CHCl3), UV (MeOH) λmax (ɛ) 233 (15 800)). The infrared (IR) spectrum (KBr) of 1 revealed the characteristic absorptions of esters (vmax 1730 cm−1), amide (vmax 1670 cm−1), hydroxyl, and/or amide NH (vmax 3400 cm−1) groups. Its molecular formula was established as C13H21NO7S (m/z [M-H]− 334.0960, +0.2 mmu) by high-resolution electrospray ionization-mass spectrum.

The structure of 1 was mainly elucidated by the analyses of NMR spectra, including heteronuclear single-quantum coherence, double-quantum-filtered correlation (DQF-COSY), and constant-time heteronuclear multiple-bond correlation (HMBC).4 The 1H and 13C NMR spectral data revealed by the heteronuclear multiple quantum coherence spectrum for 1 are listed in Table 1. The DQF-COSY and HMBC analyses revealed the two partial structures (Figure 1b). The sequence from an amide proton 2-NH (δH 6.68) to methylene protons 3-H (δH 3.67 and 3.22) through an α-methine proton 2-H (δH 4.91, δC 52.2) was observed in the DQF-COSY of 1. In the HMBC spectrum, 1H–13C long-range couplings from the α-methine proton 2-H and the methylene protons 3-H to a carbonyl carbon C-1 (δC 171.6), and from the amide proton 2-NH and a singlet methyl proton 10-H (δH 2.12) to an amide carbonyl carbon C-9 (δC 172.4) were observed. Thus, the carbonyl and acetyl moieties were elucidated to be substituted at the C-2 and 2-N positions, respectively. In a similar manner, the sequence from an oxymethine proton 6-H (δH 4.46) to a methyl proton 12-H (δH 0.96) through methine protons 5-H (δH 2.64) and 11-H (δH 2.30), which in turn spin coupled to a methyl proton 13-H (δH 1.06), was established. 1H–13C long-range couplings from 5-H and 11-H to a carbonyl carbon C-4 (δC 200.3) and from 5-H and 6-H to an ester carbonyl carbon C-7 (δC 174.7) were recognized in the HMBC spectrum of 1. A methoxyl proton 8-H (δH 3.81) was long-range coupled to the ester carbonyl carbon C-7, which established the substitution position of the methoxyl functional group. By taking into consideration the molecular formula of 1 and the long-range coupling from 3-H to C-4, together with the 13C chemical shifts of C-3 (δC 29.7) and C-4, these partial structures should be connected through a sulfur atom. Finally, the structure of 1 was determined to be 2-acetamido-3-(3-hydroxy-2-isopropyl-4-methoxy-4-oxobutanoylthio)propanoic acid, as shown in Figure 1. This structure was also supported by alkaline hydrolysis (2 N NaOH, 40 °C, 1 h), which yielded an N-acetyl cystein and 2-hydroxy-3-isopropylsuccinic acid residues.

Table 1 1H and 13C NMR spectral data for 1 and 3-isopropylmalate methyl ester
Full size table

The absolute stereochemistry of 1 was established as follows. The N-acetyl cystein obtained from 1 by alkaline hydrolysis was determined as R by comparing the optical rotations ([α]D 6.25, c 0.24 (MeOH), 25 °C; authentic sample: [α]D 6.08, c 0.25 (MeOH), 25 °C). The absolute stereochemistry at C-5 and C-6 was determined by the modified-Mosher method5 and the J-based method.6, 7, 8, 9 Compound 1 was treated with trimethylsilyldiazomethane to afford a methyl ester of 1. This methyl ester compound was then reacted with (R)- and (S)-MTPA chloride in pyridine. The differences in chemical shift values obtained by subtracting the (R)-MTPA ester values from (S)-MTPA ester values (δΔ = δ(S)-MTPA−δ(R)-MTPA) are summarized in Figure 2a. From these values, the absolute configuration at C-6 was concluded to be 6R. A small coupling constant (<2 Hz) between 5-H and C-6 revealed that the oxygen atom and 5-H were in an anti relationship. A large coupling constant between 6-H and C-4 (3JH6−C4 = 8.8 Hz) and a small one between 6-H and C-11 (3JH6−C11<2 Hz) indicated that they were in anti and gauche locations, respectively, as shown in Figure 2b. These results revealed the absolute configuration at C-5 to be 5S. Thus, the absolute structure of 1 was established, as shown in Figure 1a.

Figure 2
figure 2

(a) Absolute configuration at C-6 revealed by modified Mosher's method. (b) Absolute configuration at C-5 established by J-based analysis.

Full size image

The cytotoxic activity of 1 against human acute myelogenous leukemia HL-60 cells was tested by the WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] colorimetric assay (Cell Counting Kit, Dojindo, Kumamoto, Japan). It was found that 1 exhibited a weak cytotoxic effect against HL-60 cells for 48 h with an IC50 value of 210 μM. We also attempted to investigate the antimicrobial activitiy of 1. However, 1 did not exhibit antimicrobial activity against Micrococcus luteus, Escherichia coli, and Schizosaccharomyces pombe.