Introduction

Prodigiosins are cytotoxic antibiotics characterized by three pyrrole rings in the structure [1]. Roseophilin (2) and dechlororoseophilin (3) are unique prodigiosin-related compounds produced by Streptomyces griseoviridis [2, 3]. These metabolites contain a furan ring in place of the central pyrrole ring and a bicyclic alkyl chain (Fig. 1). The roseophilin producer also produces prodigiosin R1 (4) [4], which is a typical prodigiosin containing a simple cyclic alkyl chain with the same carbon skeleton as 2. It was suggested that most of the biosynthesis pathways of 2 and 4 are common. The rph gene cluster has been found in S. griseoviridis as involved in the biosynthesis of these prodigiosin derivatives [5], although the cluster seems not to contain genes for furan ring formation or chlorination. Then, we searched for biosynthetic intermediates of 2 to add more information for its biosynthesis.

Fig. 1
figure 1

Structure of prodigiosin derivatives from the roseophilin producer

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Results and discussion

S. griseoviridis 2464-S5 was cultured at 27 °C for 60 h with reciprocal shaking in a seed medium (glycerol 1%, Bacto Soytone 1%, molasses 1%, pH 7.2). The seed culture at 2% was transferred to 500-ml Erlenmeyer flasks containing 100 ml of a medium consisting of glycerol 4%, molasses 1%, soybean meal 1.5%, and CaCO3 0.4% (pH 6.8). The fermentation was carried out on a rotary shaker at 27 °C for 6 days. The ethyl acetate extract obtained from the mycelial acetone extract was analyzed by HPLC (PEGASIL ODS SP-100, Senshu Scientific, Tokyo, Japan) with 90% methanol-5 mM disodium hydrogen citrate at 530 nm. The chromatogram revealed peaks for 2, 3, 4, and 11-methyldodecylprodiginine (5) as shown in Fig. 2. No other distinct peak was observed in various culture conditions. The productivity of each prodigiosin in 1-L cultivation was calculated as 82 mg for 2, 36 mg for 3, 22 mg for 4, and 5 mg for 5. In the previous study, only 8.2 mg of 3 was isolated from the 2-L culture under the similar condition [3], while this fraction was expected to contain 72 mg of prodigiosin derivatives. Silica gel TLC analysis and silica gel column fractionation identified a new prodigiosin designated prodigiosin R2 (1) in the HPLC fraction containing 3.

Fig. 2
figure 2

HPLC analysis of prodigiosin derivatives produced by S. griseoviridis 2464-S5

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The fermented broth (2.0 L) was centrifuged and the mycelium was extracted with acetone. After evaporation, the aqueous concentrate was extracted with ethyl acetate. The extract was partitioned between hexane and 90% methanol. The 90% methanol layer was concentrated and applied to a silica gel column, which was washed with hexane-chloroform (2:1) and eluted with hexane-chloroform (1:1). The eluate contained 1 and 4, and the two pigment bands (1: Rf 0.84, 4: Rf 0.93) were separated by preparative silica gel TLC with chloroform-methanol-29% ammonia water (200:20:1). The fraction containing 1 was dissolved in ethyl acetate, and the solution was washed with 0.1 M NaOH and water. After evaporation, the residue was dissolved in methanol and acidified by addition of equivalent hydrogen chloride. The material was concentrated to dryness to give a hydrochloride salt of 1 (5.6 mg).

The molecular formula of 1 was established as C27H35N3O by high-resolution FAB-MS. The 13C and 1H NMR data for 1 are summarized in Table 1. All one-bond 1H–13C connectivities were confirmed by an HMQC experiment [6].

Table 1 13C and 1H NMR data for 1 and 2 in CDCl3
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COSY and HMBC [7] analyses established the structure of 1 as shown in Fig. 3. Three aromatic protons (H-1 to H-3) and an exchangeable proton (1-NH) were coupled with each other. Long-range correlations from H-1 to C-2, C-3, and C-4, from H-2 to C-1 and C-4, and from H-3 to C-1 and C-4 indicated the presence of a 2-pyrrolo group. 1H–13C long-range couplings were observed from H-6 to C-5 and C-8, from an exchangeable proton (5-NH) to C-7, and from a methoxy proton (H-27) to C-7. In addition to these correlations, an NOE from H-27 to H-6 and a high-field chemical shift for C-6 (δ 93.9) constructed a 2,5-disubstituted 3-methoxypyrrole moiety. The third pyrrole ring was identified by 1H–13C long-range correlations from H-12 to C-10, C-11, and C-13, and a 1H–1H long-range correlation between H-12 and an exchangeable proton (10-NH). Proton spin networks from H-14 to a methine (H-22) and from a methine (H-23) to two doublet methyls (H-25 and H-26) revealed the presence of bridged alkyl chains, which were connected by 1H–13C long-range couplings from H-23 to C-21 and C-22. Furthermore, H-23 displayed long-range correlations to C-9, C-10, and C-11, and an NOE to H-12, indicating that C-23 was located on C-11 and a five-membered ring was composed of C-9, C-10, C-11, C-22, and C-23. A long-range correlation between H-14 and C-13 connected the alkyl chain to the C-13 position in the third pyrrole ring. NOEs from H-3 to H-6 and H-27 confirmed the connection of the first and the central pyrrole rings. The remaining bond was formed between C-8 and C-9 to complete the planar structure of 1. The 13C chemical shift similarity (±0.8 p.p.m.) in the alkyl region of 1 and 2 (Table 1) established the structure including the relative stereochemistry.

Fig. 3
figure 3

NMR analysis of prodigiosin R2 (1)

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The cytotoxic activities of the prodigiosin derivatives were evaluated using the MTT method against HeLa human cervical carcinoma cells and HT1080 human fibrosarcoma cells. The IC50 values against HeLa and HT1080 cells were 0.41 and 0.82 μM for 1, 4.2 and 5.7 μM for 2, and 1.7 and 3.3 μM for 4, respectively. Prodigiosin derivatives isolated from the roseophilin producer exhibited potent cytotoxicity against both the cell lines. Prodigiosin R2 (1) was more cytotoxic than the other prodigiosin derivatives.

Prodigiosins are known to possess antimicrobial, antifungal, antitumor, antimalarial, and immunosuppressive activities [8], and are reported to be H+/Cl− symporters, which uncouple lysosomal vacuolar-type ATPase [9]. The activity of prodigiosin derivatives as H+/Cl− symporters can be evaluated by cytotoxicity. The similar cytotoxic profiles of 2 and 3 have been reported [3]. The higher cytotoxicity of 1 indicated that the bicyclic alkyl chain and the central pyrrole ring increase the activity of prodigiosins. These structure-activity relationships will be helpful for structural optimization.

Prodigiosin R2 (1) contains a tripyrrole moiety with a bicyclic alkyl chain. Among known prodigiosin derivatives, such an alkyl chain has been found in only 1, 2, and 3. In the rph gene cluster, four genes rphG, rphG2, rphG3, and rphG4 exhibited similarity to prodigiosin cyclization genes. Among them, rphG has been established to cyclize undecylprodiginine [10] and is considered to be involved in the formation of prodigiosin R1 (4) from 11-methyldodecylprodiginine (5). Since only the roseophilin producer has four genes for prodigiosin cyclization, rphG2, rphG3, and rphG4 might construct a system for cyclization to form the bicyclic alkyl chain. In this study, no chlorinated derivative of 1 has been found in the culture extract (data not shown). This result suggests that the substrate of chlorination is only 3 or chlorinated 1 is immediately converted into 2. Further biosynthetic studies are in progress.

Experimental section

General experimental procedures

UV and visible spectra were measured on a UV-1700 spectrometer (Shimadzu, Kyoto, Japan). IR spectra were obtained on a Spectrum 100 FT-IR spectrometer (PerkinElmer, Waltham, MA, USA) in the ATR (attenuated total reflection) mode. Mass spectra were measured on a JMS-SX102A spectrometer (JEOL, Akishima, Tokyo, Japan) in the FAB mode using m-nitrobenzyl alcohol as matrix and polyethylene glycol as internal standard. NMR spectra were obtained on a JNM-LA400 spectrometer (JEOL) with 1H NMR at 400 MHz and with 13C NMR at 100 MHz. Chemical shifts were referenced relative to CDCl3 (δH 7.24 and δC 77.0).

Physico-chemical properties of prodigiosin R2 (1)

Red amorphous powder; m.p. 65–68 °C; high-resolution FAB-MS m/z 418.2856 ([M + H]+, calcd for C27H36N3O, 418.2858); UV λmax (ε) 271 nm (4000), 293 nm (4700), 342 nm (4100), 513 nm (80300) in MeOH, 293 nm (4800), 342 nm (4400), 513 nm (82800) in 0.01 M HCl–MeOH, 289 nm (6800), 459 nm (29100) in 0.01 M NaOH–MeOH; IR (ATR) νmax 3140, 2930, 1580, 1320 cm−1.

Cell culture and cell viability assay

HeLa and HT1080 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum and 0.1% glucose. The cells were plated and incubated for 48 h with various concentrations of samples. After the cells were treated with 0.5 mg ml−1 of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 4 h at 37 °C, the relative cell number was measured as absorbance at 540 nm. IC50 values were calculated by linear interpolation between the two drug concentrations above and below the 50% inhibition line.