Streptomycetes isolated from the rhizosphere of Norway spruce were grown in submerged culture in various complex media, and extracts prepared from culture filtrates and biomass were screened by HPLC-diode array analysis to detect novel secondary metabolites.2 Strain Tü 6384 was found to produce a new anthracycline-type compound that showed a high similarity with aranciamycin, an antibiotic isolated from Streptomyces echinatus, in its UV–visible spectrum.3 The compound was characterized as aranciamycin anhydride (1).

Strain Tü 6384 was isolated from the rhizosphere of Norway spruce collected in Rammert forest near Tübingen, Germany. It was examined for a number of key properties that are known to be of value in streptomycete systematics.4, 5 Whole-cell hydrolysates of strain Tü 6384 contained LL-diaminopimelic acid, and hexa- and octahydrogenated menaquinones with nine isoprene units were the predominant isoprenologs. Partial sequencing of the 16S rRNA gene led to a similarity of 99% with Streptomyces prunicolor.

Batch fermentations of strain Tü 6384 were carried out in a 10-l stirred tank fermentor (Biostat S; B Braun, Melsungen, Germany) in a complex medium that consisted of (per liter tap water) oatmeal (Holo Hafergold, Neuform, Germany) 20 g, and 5 ml of a trace element solution that was composed of (per liter deionized water) CaCl2 × 2H2O 3 g, iron(III) citrate 1 g, MnSO4 × 1H2O 200 mg, ZnCl2 100 mg, CuSO4 × 5H2O 25 mg, Na2B4O7 × 10H2O 20 mg, CoCl2 × 6H2O 4 mg and Na2MoO4 × 2H2O 10 mg; the pH was adjusted to 7.3 (5 M HCl) before sterilization. The fermentor was inoculated with 5% by volume of a shake flask culture grown in a seed medium at 27 °C in 500-ml Erlenmeyer flasks with a single baffle for 72 h on a rotary shaker at 120 r.p.m. The seed medium consisted of glucose 10 g, glycerol 10 g, oatmeal 5 g, soybean meal (Schoenenberger, Magstadt, Germany) 10 g, yeast extract (Ohly Kat, Deutsche Hefewerke, Hamburg, Germany) 5 g, Bacto Casamino acids 5 g and CaCO3 1 g in 1 l tap water. The fermentation was carried out for 4 days with an aeration rate of 0.5 volume air per volume per min and agitation at 250 r.p.m. The production of 1 reached a maximal yield of 50 mg l−1 at 72 h of incubation. The culture filtrate (6 l) was applied to an Amberlite XAD-16 column (60 × 4 cm i.d.; Rohm and Haas, Frankfurt, Germany), washed with each of 3 l H2O and H2O-EtOH (6:4), and 1 was eluted with 3 l EtOH and concentrated in vacuo. The crude product was dissolved in CH2Cl2 and added to a diol-modified silica gel column (45 × 2.6 cm i.d., LiChroprep Diol; Merck, Darmstadt, Germany). The separation was accomplished by a step gradient from CH2Cl2 to 5% EtOH. The combined fractions containing 1 were concentrated in vacuo and yielded 385 mg crude product, which was purified by preparative RP-HPLC (Nucleosil-100 C-18, 10 μm, 25 × 1.6 cm i.d.; Maisch, Ammerbuch, Germany) with CH3CN–0.1% HCOOH using a linear gradient elution from 40 to 80% CH3CN over 20 min at a flow rate of 20 ml min−1. Compound 1, 45 mg, was obtained as a red-orange powder (Table 1).

Table 1 Physico-chemical properties of aranciamycin anhydride1
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The molecular mass of 1 was determined by high-resolution electrospray ionization-FT-ion cyclotron resonance mass spectrometry, which gave the mass of 710.17728, suggesting a molecular formula of C35H34O16 (theoretical: 709.17741, Δ=0.13 p.p.m.). The chemical structure of 1 as shown in Figure 1 was determined by 1H-, 13C- and 2D-NMR experiments (Table 2) and GC–MS. The complete NMR assignments were unambiguously carried out on the basis of COSY and heteronuclear multiple bond correlation (HMBC) experiments. The 1H-NMR and heteronuclear single quantum coherence (HSQC) data showed a total of 30 carbon-attached protons, among which five methyl, two methylene and eleven methine carbons could be assigned. Inspection of the 2D-NMR data (COSY, HSQC and HMBC) allowed assignment of the structure of 1, a new derivative of the aranciamycin family, aranciamycin anhydride (Figure 2). The 1H–1H–COSY spectra of 1 revealed protons attached to two ethyl, one phenyl and one sugar (hexose) moiety, as shown by the bold lines in Figure 2. All expected HMBC correlations of the aranciamycin moiety could be seen and couplings were in good accordance with the data given in literature.6 The connection between the sugar moiety and the aranciamycin chromophore was established by the 1H–13C–long-range coupling from C-10 (δH 5.19, δC 72.4) to H-1″ (δH 5.65, δC 100.5) and vice versa as shown in Figure 2. The constitution of the sugar was determined from COSY and HMBC spectra, which revealed a 6-desoxyhexose moiety. The coupling constants (Table 3) indicated that the sugar was a mannopyranose. A 1H–13C long-range coupling from the methyl group at δH 3.56 to C-2″ (δC 80.3) of the sugar established the position of the methoxy group. Additional examination of the NMR data and chiral GC–MS analysis of the derivatized hydrolysate allowed us to identify the moiety as β-2-O-methyl-L-rhamnose in accordance with the literature.7 In comparison with the molecular formula of aranciamycin, derivative 1 has an additional C8H6O4 moiety. In addition, the NMR spectra showed five quaternary olefinic, two methylene and one methyl moiety compared with the aranciamycin core structure. The 1H–13C long-range couplings from H-9″ (δH 2.77) to C-8″ (δC 171.8), C-10″ (δC 19.9) and C-11″ (δC 142.0), from H-10″ (δH 2.78) to C-8″, C-9″ (δC 31.3) and C-14″ (δC 165.8), and from H-15″ (δH 2.10) to C-11″, C-12″ (δC 142.6) and C-13″ (δC 166.1) gave rise to an anhydride structure in excellent accordance with literature values.8 The connection between the sugar and the anhydride moiety was established by the 1H–13C long-range coupling observed from H-4″ (δH 4.87) to the carbonyl of C-8″, resulting in the structure of 1 (Figure 1).

Figure 1
figure 1

Structure of aranciamycin anhydride (1).

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Table 2 1H- and 13C-NMR assignment of 1 in CDCl3-d (25 °C)
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Figure 2
figure 2

1H–1H–COSY and HMBC correlations observed in 1.

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Table 3 Growth-inhibitory activity of 1 and aranciamycin (μg ml−1) against selected human tumor cell lines
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The antimicrobial activity spectrum of 1 was tested in an agar plate diffusion assay against Bacillus subtilis DSM 10, Escherichia coli K12, Saccharomyces cerevisiae ATCC 9010 and Botrytis cinerea Tü 157 in a concentration of 0.1–1 mg ml−1. Similar to aranciamycin, 1 showed weak antibacterial activity only against Bacillus subtilis. The inhibitory action of 1 on the growth of tumor cells was compared with aranciamycin and tested according to NCI guidelines9 with the human tumor cell lines HM02 (gastric adenocarcinoma), MCF 7 (breast carcinoma) and HepG2 (hepatocellular carcinoma). Cells were grown in 96-well microtiter plates in RPMI 1640 with 10% fetal calf serum in a humidified atmosphere of 5% CO2 in air. Aranciamycin and 1 (0.1–10 μg ml−1) were added to the cells after incubation for 24 h. Stock solutions were prepared in DMSO; the final DMSO concentration of the cultures was 0.1%. The cells were fixed and cell protein analyzed with sulforhodamine B after incubation for 48 h. The cytostatic activity of 1 was somewhat less than that of aranciamycin (Table 3).