Thioviridamide (1) is an N-acylated undecapeptide antibiotic that induces apoptosis selectively in E1A-transformed cells.1 The most unique structural feature of thioviridamide is the presence of five thioamide bonds between the amino acids.2 Recently, the thioviridamide biosynthesis gene cluster of Streptomyces olivoviridis NA05001 was identified, and the heterologous expression of thioviridamide in Streptomyces lividans TK23 was demonstrated.3 Thioviridamide is synthesized by the posttranslational modification of a ribosomal precursor peptide containing a VMAAAASIALHC sequence.3 Heterologous expression of a bacterial artificial chromosome (BAC) clone prepared from S. olivoviridis OM13 containing the entire gene cluster for thioviridamide biosynthesis in S. avermitilis SUKA17 strain,4, 5 which is the suitable host for heterologous expression, resulted in the expression of a novel thioviridamide derivative, JBIR-140 (2), together with thioviridamide. Here, we report the isolation, structure determination and biological activity of 2.

The BAC genomic library of S. olivoviridis OM13 was prepared according to a previously reported method.4 S. olivoviridis OM13 genome was partially digested with BamHI in an agarose gel and then subjected to contour-clamped homogeneous electric field electrophoresis. Fragments (size, 100–130 kb) were then excised from the gel. The purified DNA fragments were ligated to the BamHI fragment of the integrating BAC vector pKU503.5 The ligated DNAs were transformed into Escherichia coli NEB 10-β cells (New England Biolabs, Ipswich, MA, USA) by electroporation.4, 5 Each BAC clone was stored in five 384-well plates containing Plusgrow II (100 μg ml−1 ampicillin and 20% glycerol) at −80 °C. Clones carrying the entire gene cluster for thioviridamide biosynthesis were screened by PCR amplification using two pairs of primers corresponding to the upstream and downstream regions of the gene cluster (upstream primer pair, forward: 5′-CAGATGGACACGTACACGCAGAC-3′ (corresponding to the region from nucleotides 5539 to 5561 of the thioviridamide biosynthesis gene cluster3) and reverse: 5′-CACGTTCGTAGTAGCTGTCGGAGA-3′ (6187–61643); downstream primer pair, forward: 5′-AGTGGAGCGGTGCTACGACATC-3′ (28 124–28 1453)) and reverse: 5′-TCCGGTACAGCTTCTTGTACTCCA-3′ (27 567–27 5443)). The amplification was performed under the following conditions: denaturation at 95 °C for 5 min, 35 cycles of 95 °C for 30 s, 58 °C for 30 s and 72 °C for 60 s, and a final incubation at 72 °C for 5 min, and then soaked at 12 °C. One of the BAC clones, pKU503thvP3-F8 (inset size: 71 900 bp), contained the entire gene cluster for thioviridamide biosynthesis. Unmethylated pKU503thvP3-F8 was prepared by transforming the plasmid into E. coli GM2929 hsdS::Tn10 cells.5 The resultant DNA preparation was introduced into S. avermitilis SUKA17 by polyethylene glycol-assisted protoplast transformation.4, 5 Integration of the plasmids was confirmed by neomycin resistance and PCR analysis using the above-mentioned primer pairs.

SUKA17 carrying pKU503thvP3-F8 was cultivated in 50-ml test tubes, each containing 15 ml of a seed medium consisting of 0.5% glucose (Kanto Chemical, Tokyo, Japan), 1.5% soybean meal (Nisshin Oillio Group, Tokyo, Japan) and 0.5% yeast extract (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) at pH 7.5 (adjusted before sterilization). The test tubes were shaken on a reciprocal shaker (320 r.p.m.) at 28 °C for 2 days. Aliquots (2.5 ml) of the culture were transferred into 500-ml baffled Erlenmeyer flasks filled with 100 ml of a production medium consisting 4.0% β-cyclodextrin (Kanto Chemical, Tokyo, Japan), 0.5% glycerin, 2.0% pharmamedia (Traders Protein, Lubbock, TX, USA), 5 mg l−1 CuSO4, 5 mg l−1 MnCl2 and 5 mg l−1 ZnSO4 at pH 7.2 (adjusted before sterilization), and were cultured on a rotary shaker (180 r.p.m.) at 28 °C for 5 days.

The fermentation broth (2 l) was centrifuged, and the pelleted mycelial cake was extracted with acetone (400 ml), two times. The mycelium was removed by filtration, and the acetone extract was evaporated under reduced pressure. The residual aqueous concentrate was partitioned between EtOAc and water (100 ml) three times. The combined EtOAc extract was evaporated to dryness and the dried residue (867 mg) was applied on a normal-phase medium-pressure liquid column (Purif-Pack SI-30; Shoko Scientific, Yokohama, Japan), and successively eluted using a hexane-EtOAc solvent system (0, 5, 10, 20 and 25% EtOAc), followed by a CHCl3-MeOH solvent system (0, 2, 5, 10, 20 and 50% MeOH). The eluate (50% MeOH, 37 mg) was further purified by preparative reversed-phase HPLC using a CAPCELL PAK C18 MGII column (5.0 μm, 20 i.d. × 150 mm; Shiseido, Tokyo, Japan) fitted with a 2996 photodiode array detector (Waters, Taunton, MA, USA) and a 3100 mass detector (Waters), and was developed with 75% aqueous MeOH containing 50 mm ammonium formate for 20 min (flow rate, 10 ml min−1), followed by 90% aqueous MeOH containing 50 mm ammonium formate for 20 min (flow rate, 10 ml min−1) to yield 1 (2.5 mg, retention time=20.7 min) and 2 (15.7 mg, retention time=31.0 min).

Compound 2 was obtained as a colorless amorphous powder ([α]D24−162 (c 0.17, in MeOH)]; UV λmax (ɛ) in MeOH: 247 (28 900) and 272 (31 400) nm). The molecular formula was determined by high-resolution ESI-MS to be C53H88N14O9S7+ (found: 1288.4912 [M+], calculated for C53H88N14O9S7+: 1288.4898), indicating that it was smaller than 1, which had additional C3H4O atoms. The presence of hydroxy and amide moieties in 2 was established from its IR spectrum (νmax (attenuated total reflection): 3450, 1670 and 1520 cm−1).

Direct connectivity between the protons and carbons was established from the heteronuclear single-quantum coherence spectrum. The 13C and 1H NMR spectroscopic data for 2 are listed in Table 1. The 1H and 13C NMR spectra of 2 resembled those of 1, which indicated that 2 was produced by the heterologous expression system. Therefore, 2 was confirmed as biosynthesized by the RiPS (ribosomal peptide synthesis) pathway. Taking into consideration the NMR spectra and the biosynthetic gene cluster, the amino-acid residues and the sequence of 2 except for N-terminal cap moieties were concluded to be identical to that of 1.

Table 1 13C and 1H NMR spectroscopic data for JBIR-140 (2) in CD3OD

Analyses of the double quantum-filtered-COSY and constant time-HMBC6 spectra reconfirmed the presence of a Val, a Met, an Ile, a Leu, a Cys-like, a β-hydroxy-N1, N3-dimethylhistidinium (hdmHis) and five Ala amino-acid residues, in addition to an ethenamine moiety, as shown in Figure 1b. A sulfide linkage between the two partial structures was elucidated by 1H–13C long-range coupling from an olefinic proton (δH 5.47), which was in turn 1H–1H coupled to an olefinic proton (δH 7.26, J=7.6 Hz), to the β-methylene carbon (δC 36.6) in the cysteine-like moiety, no coupling between the olefin proton and the β-methylene protons (δH 3.18, 2.99) and the molecular formula of 2, to generate an S-(2-aminovinyl)cysteine moiety (avCys). An N-terminal lactate moiety (LA) was also established instead of 2-hydroxy-2-methyl-4-oxopentanoic acid in 1 as follows: the 1H–1H spin correlation between a methyl proton (δH 1.46) and an oxymethine proton (δH 4.48), as well as a 1H–13C long-range coupling between the oxymethine proton and an amide carbonyl carbon (δC 173.4), established the presence of LA.

Figure 1
figure 1

(a) Structures of thioviridamide (1) and JBIR-140 (2). (b) Correlations in the double quantum-filtered-COSY (DQF-COSY; bold lines) and constant time-HMBC spectroscopy (CT-HMBC; arrows) spectra of 2.

Three-bond correlations from α-methine protons to thioamide carbons established five thioamide linkages between Ala 4 and Ala′ 3, Ala′ 3 and Ala′ 2, Ala′ 2 and Al ′1, Ala′ 1 and Met′, and Met′ and Val′. To finalize the structure of 2, five amide bonds between Val′ and LA, Ile and avCys, Ala5 and Ile, Leu and Ala5 and hdmHis and Leu were identified by three-bond couplings from α-methine protons to amide carbonyl carbons. A 1H–13C long-range coupling from an olefin proton (δH 7.26) to a carbonyl carbon (δC 167.6) indicated the presence of an additional amide linkage between the carbonyl group of hdmHis and the ɛ-amino group of avCys. The two partial structures should be directly joined from the molecular formula of 2. Finally, an amide linkage between avCys and Ala4 established the planar structure of 2, an N-terminal lactate derivative of thioviridamide (1), as shown in Figure 1a. The N-terminal 5-dimethyl-2-oxohexanoate of polytheonamides A and B and the N-terminal lactate of epilancin 15X have been reported to be derived from threonine and serine, respectively.7, 8 In the case of thioviridamide, although heterologous expression of the thioviridamide biosynthesis gene cluster resulted in the production of both 1 and 2, S. olivoviridis OM13 expressed only 1. Posttranslational modification genes related to N-terminal modification have not been identified in the thioviridamide biosynthesis gene cluster. Further biosynthetic studies of 1 and 2 are in progress.

The absolute configurations of the standard amino-acid residues in 2 were determined using the Marfey's method.9 Compound 2 was subjected to complete acid hydrolysis (6 n HCl, 110 °C for 16 h), followed by reaction with FDAA (Nα-(5-fluoro-2,4-dinitrophenyl)-l-alaninamide) to obtain the respective FDAA derivatives. The retention times of these FDAA amino-acid derivatives were established by monitoring the UV absorption at 340 nm using ultra-performance liquid chromatography and negative-ion mode ESI-MS. All derivatives were identified based on a comparison of their retention times, molecular formulae and UV spectra with those of standard amino acids derived from FDAA-conjugated compounds. The absolute configurations of the standard amino acids in 2 were identified as L-Val, L-Met, L-Ile and L-Leu. As two L-Ala and three D-Ala units were obtained, their locations could not be established conclusively.

The cytotoxic activities of 1 and 2 against human ovarian adenocarcinoma SKOV-3, malignant pleural mesothelioma Meso-1 and Jurkat cells were examined. All cell lines were seeded in 384-well plates at a density of 1000 cells per 20 μl per well and incubated at 37 °C in a humidified incubator with 5% CO2. After 4 h, cells were treated with various concentrations of compounds for 72 h. The vehicle solvent (dimethylsulfoxide) was used as a control. Cell viabilities were measured using WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) by a colorimetric assay (Cell Counting Kit-8; Dojindo; Kumamoto, Japan). Compound 1 exhibited cytotoxic activities against SKOV-3, Meso-1 and Jurkat cells with the IC50 values of 38.0, 27.8 and 12.5 μm, respectively. Compound 2 exhibited two to three times higher activities against SKOV-3, Meso-1 and Jurkat cells with IC50 values of 10.8, 14.3 and 5.4 μm, respectively. Further studies on the absolute configuration and biological activities of 1 and 2 are under way.