Introduction

Actinomycetes from marine sediments collected at various sites in the Atlantic and Pacific Oceans were screened by HPLC-diode array analysis for the production of novel secondary metabolites. Strain NTK 935 was isolated from the same deep-sea sediment core as Streptomyces sp. NTK 937, which we reported recently to produce caboxamycin, a novel antibiotic with a benzoxazole scaffold.2 Extracts of strain NTK 935 contained a metabolite having an unusual UV-vis spectrum, which was not identified by means of our in-house developed HPLC database.3 HPLC–ESI–MS analysis revealed a molecular mass of 380, which was—together with UV-vis data—not in accordance with other natural products available in the DNP database.4 Therefore, strain NTK 935 was selected for fermentation studies, isolation and characterization of the unusual metabolite; this resulted in the identification of the novel 1,4-benzoxazine structure shown in Figure 1.

Figure 1
figure 1

Structure of benzoxacystol (1).

Full size image

This study describes the taxonomy of the producing strain, fermentation and isolation, structural elucidation and biological activity of the new benzoxazine-type metabolite, which was named benzoxacystol.

Results

Taxonomy and phylogenetic analysis of the producing strain

Strain NTK 935 contained LL-diaminopimelic acid in the peptidoglycan, hydrogenated menaquinones with nine isoprene units, and produced straight spore chains of yellowish color on the white aerial mycelium, properties typical of members of the genus Streptomyces.5

The phylogenetic analysis of the almost complete 16S rRNA gene sequence revealed that strain NTK 935 was closely related to the type strains of a group of several Streptomyces species sharing similarities with them of approximately 100% (Figure 2). Members of this group are S. mediolani NBRC 15427T (100% similarity), S. rubiginosohelvus NBRC 12912T (100%), S. griseoplanus AS 4.1868T (99.9%), S. tanashiensis IFO 12919T (99.9%), S. globisporus NRRL B-2872T (99.9%) and S. griseus subsp. griseus NBRC 15744T (99.8%). The phylogenetic data clearly shows that strain NTK 935 is a member of the 16S rRNA S. griseus clade.6 Quite interestingly, a strain of S. globisporus was shown to produce a 1,4-benzoxazine-type compound similar to that of strain NTK 935.7, 8

Figure 2
figure 2

Phylogenetic tree of Streptomyces sp. strain NTK 935, including representatives of the most closely-related type strains and additional members of the genus Streptomyces. Bootstrap values are given in percentage (only numbers above 50% are shown).

Full size image

Screening, fermentation and isolation

Compound 1 was produced by strain NTK 935 in shake-flask cultures in various complex media as monitored by HPLC-diode array analysis. The compound represented a dominant peak in the extracted culture filtrates at a retention time of 7.4 min (Figure 3). HPLC–ESI–MS analysis revealed a molecular mass of 381.1 [M+H]+ in the ESI positive mode for compound 1. Fermentation studies resulted in a maximal biomass at 48 h, whereas production of 1 reached a maximal yield of 14 mg l−1 at 72 h, when mannitol and phosphate were depleted in the medium.

Figure 3
figure 3

HPLC analysis of a culture filtrate extract from Streptomyces sp. NTK 935 at a fermentation time of 72 h, monitored at 280 nm; insert: UV-vis spectrum of 1.

Full size image

Compound 1 was isolated from the culture filtrate of a 10-l fermentation by Amberlite XAD-16 (Rohm and Haas, Frankfurt, Germany) column chromatography, followed by ethyl acetate extraction. The raw product was purified by subsequent chromatography steps on diol-modified silica gel, Sephadex LH-20 and Toyopearl HW-40S columns. Compund 1 was obtained in an amount of 25.2 mg as a yellowish powder after concentration to dryness.

Structure determination

The mass spectrum derived from the HPLC–ESI–MS chromatogram for 1 showed a molecular mass of 381.1 [M+H]+. The exact molecular mass was measured by HPLC-ESI-Orbitrap-FT-MS in negative ion mode as 379.05965 Da, which corresponds to the molecular formula C16H15N2O7S ([M-H]−theor=379.06004 Da, Δm=1.029 p.p.m.). The physico-chemical properties of 1 are given in Table 1.

Table 1 Physico-chemical properties of benzoxacystol (1)
Full size table

The 1H-NMR spectrum of 1 showed six signals in the aromatic/olefinic region and five further signals in the aliphatic region. Integration of the signals revealed 15 protons, with the signals at δH 1.95 p.p.m. and δH 3.78 p.p.m., corresponding to one methyl and one methoxy group, respectively. The 13C-NMR and DEPT spectrum showed one methyl, one methoxy, one aliphatic and one olefinic methylene, two aromatic and one aliphatic methine and nine quaternary carbon atoms. Detailed inspection of the 2D-NMR data (COSY, HSQC and HMBC; Table 2) allowed assignment of the structure of 1 (Figure 1). The 1H-1H-COSY spectrum only showed the connection between H-1′, H-2′ and H-4′, suggesting the presence of a cysteine or serine residue in the structure. The HMBC correlations from H-8 to C-1 and C-2, from H-3 to C-2a, C-4, C-5 and C-6a, from H-5 to C-3, C-4, C-6a and C-10, and from H-9 to C-4 in combination with the chemical shift gave proof for the 1,4-benzoxazine core structure of 1. Further HMBC correlation from H-1′ to C-10, C-2′ and C-3′, from H-2′ to C-1′, C-3′ and C-5′, from H-4′ to C-3′ and C-5′, and from H-6′ to C-5′ gave rise to an N-acetylcysteine thioester attached to C-10 (Figure 4).

Table 2 1H- and 13C-NMR spectroscopic data of benzoxacystol (1) in dimethyl sulfoxide-d6
Full size table
Figure 4
figure 4

1H-1H COSY and HMBC correlations of benzoxacystol (1).

Full size image

The X-ray crystal of 1 obtained from CH3CN gave further proof of the structure (Figure 5). To elucidate the absolute stereochemistry, 1 mg of 1 was subjected to acid hydrolysis and subsequent amino acid analysis by chiral GC–MS. Comparison of the retention time (Rt=12.53 min) to D-cysteine (Rt=11.35 min) and L-cysteine (Rt=12.35 min) showed the R configuration of the stereogenic center in 1.

Figure 5
figure 5

X-ray crystal structure of benzoxacystol (1). A full color version of this figure is available at The Journal of Antibiotics journal online.

Full size image

Biological activity

Compound 1 was tested for antimicrobial and antiproliferative activity by performing a variety of bioassays. It showed a weak antiproliferative activity against the mouse fibroblast cell line NIH-3T3 at a concentration of 50 μM (18% inhibition). On the other hand, it was found to be inactive in the antimicrobial assays. Interestingly, compound 1 showed an inhibitory activity against glycogen synthase kinase 3β (GSK-3β) and inhibited the recombinant enzyme in an in vitro enzyme activity assay with an IC50=1.35 μM±0.15 μM. GSK-3β is a key regulator of numerous signaling pathways and thus has emerged as a prominent target for the treatment of Alzheimer's disease and type 2 diabetes.

Discussion

Strain NTK 935, a member of the Streptomyces griseus 16S rRNA gene clade,6 was isolated from a deep-sea sediment collected from the Canary Basin at the edge of the Saharan debris flow near the Canary Islands at a depth of 3,814 m, using a piston corer. The age of the upper turbidite fraction (0 to 38 cm) of the core, from which the strain was isolated, was deposited approximately 1000 years ago.9 It was shown that the number of actinobacteria—determined by operational taxonomic units (OTUs) based on 16S rRNA gene similiarity—decreased with the depth of the sediment core.10 1406 OTUs were determined in the core section 5 to 12 cm, 308 OTUs in 15 to 18 cm and 212 OTUs in 43 to 46 cm, respectively. However, only 9% of OTUs showed a 100–99% homology in their 16S rDNA sequences with cultured actinobacteria, which demonstrated the huge diversity of unknown species in this unexploited habitat. This first study was corroborated by applying the multistage differential centrifugation technique (DDC) to the isolation of actinobacteria from marine sediments which resulted in an increased isolation of cultivable members of novel taxa.11

Recently we reported on caboxamycin, a new benzoxazole antibiotic from Streptomyces sp. NTK 937, which was isolated from the same sediment sample as strain NTK 935.2 Both strains were screened by HPLC–diode array together with a further 36 streptomycete isolates from the Canary Basin sediment and resulted in the identification of known antibiotics, such as alteramide, antimycins, chromomycin, cyclothiazomycin, elaiophylin, lysolipin and sporaviridin. Besides strains NTK 935 and NTK 937, further strains were identified as producers of unknown metabolites, which are still under investigation and will be reported in forthcoming communications.

Benzoxacystol (1) represents a new 1,4-benzoxazine-type metabolite with an N-acetylcysteine thioester. Only a limited number of 1,4-benzoxazine-type natural products are described in the literature. Benzoxazolinone was isolated from crushed rye plants and shown to be enzymatically transformed in the rye tissue from the 1,4-benzoxazin-3-one glycoside through its aglycone.12 Hydroxamic acids of 1,4-benzoxazin-3-one metabolites were found to exhibit an important role in defense mechanisms in cereal crops.13 The 1,4-benzoxazine chromophore was described as a building block of the antitumor enediyne antibiotic C-1027 produced by Streptomyces globisporus.7, 8 Nevertheless, the majority of benzoxazine compounds described in the literature are of a synthetic nature, showing manifold biological activities, for example, histidine kinase inhibitors as potential antimicrobial agents.14 Inhibition of GSK-3β, a key regulator of numerous signaling pathways, has not been reported from other benzoxazine compounds and benzoxacystol (1) is the first reported to exert this activity.

Methods

Producing organism and its classification

Strain NTK 935 was isolated from an Atlantic Ocean deep-sea sediment core collected in 2001 at the southern edge of the Saharan debris flow near the Canary Islands (27°02′392N, 18°29′022W), at a depth of 3814 m. Isolations were made from the turbidite fraction of the core (0–38 cm below the surface of the sediment), which was deposited approximately 1000 years ago.10

Strain NTK 935 was examined using a combination of genotypic and phenotypic procedures known to produce data of value in the delineation of Streptomyces species.15

Extraction of the DNA, amplification of the 16S rRNA gene sequence, as well as the sequencing procedure, were performed according to Wiese et al.16 The 16S rRNA sequence of strain NTK 935 was edited using BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The Ribosomal Database Project database (http://rdp.cme.msu.edu) was used to select the most clearly related type strains.17 Sequence similarity values were determined with the ‘bl2seq’ tool from the NCBI database (http://www.ncbi.nlm.nih.gov/Blast.cgi).18 The alignment of the 16S rRNA gene sequences of strain NTK 935, closest type strains, representatives of the genus Streptomyces and Micrococcus lylae as outgroup was perfomed with CLUSTAL X software and refined manually.19 All sequences were cut to the same length of 1417 bp. The phylogenetic tree was calculated with PhyML (http://atgc.lirmm.fr/phyml) by maximum likelihood analysis assuming the general time reversal model and applying 500 bootstrap replicates.20, 21 NJplot (http://pbil.univ-lyon1.fr/software/njplot.html) was used to display the phylogenetic tree.22

Biological activity

Antimicrobial activity was tested against Bacillus subtilis DSM 347, Erwinia amylovora DSM 50901, Escherichia coli DSM 498, Pseudomonas fluorescens NCIMB 10586, Pseudomonas syringae DSM 50252, Ralstonia solanacearum DSM 9244, Staphylococcus lentus DSM 6672, Xanthomonas campestris DSM 2405 and Candida glabrata DSM 6425. The antimicrobial assays were performed as described by Helaly et al.23

To determine the cytotoxicity of compound 1, the sensitivity of the cell line NIH-3T3 was evaluated by monitoring the metabolic activity using the CellTiter-Blue−Cell Viability Assay (Promega, Mannheim, Germany). The cultivation of the cell line and the bioassay were performed as described by Schneemann et al.24

GSK-3β inhibition with compound 1 was determined in an in vitro assay adapted from a luminescent assay described by Baki et al.25

Fermentation and isolation

Batch fermentations of strain NTK 935 were carried out in a 10-l stirred-tank fermentor (Biostat E; B.Braun, Melsungen, Germany) in a complex medium consisting of mannitol 20 g and soybean meal 20 g in 1 l tap water; the pH was adjusted to 7.5 before sterilization. The fermentor was inoculated with 5% by volume of a shake flask culture grown in the same medium at 27 °C in 500 ml-Erlenmeyer flasks with a single baffle for 48 h on a rotary shaker at 120 r.p.m. The fermentation was carried out for 3 days with an aeration rate of 0.5 volume air per volume per minute and agitation at 250 r.p.m.

Hyphlo Super-cel (3%) was added to the fermentation broth, which was separated by multiple sheet filtration into culture filtrate and mycelium. The culture filtrate was applied to an Amberlite XAD-16 column (Rohm and Haas; resin volume 800 ml) and the resin washed with H2O and H2O-MeOH (1:1). Compound 1 was eluted with H2O-MeOH (25:75) and concentrated in vacuo to an aqueous residue. The concentrate was adjusted to pH 4.0, extracted three times with EtOAc and the organic extracts combined and concentrated in vacuo to dryness. The crude product (1 g) was dissolved in CH2Cl2 and applied to a diol-modified silica gel column (30 × 2.5 cm i.d.; LiChroprep Diol, E. Merck, Darmstadt, Germany). Compound 1 was separated using a step gradient of CH2Cl2-MeOH and was eluted with 5% MeOH at a flow rate 250 ml h−1. Further purification was achieved by subsequent chromatography on Sephadex LH-20 (Amersham, Freiburg, Germany) and Toyopearl HW-40S (Tosoh Biosep, Stuttgart, Germany) with MeOH (each column 90 × 2.5 cm i.d.) at a flow rate of 30 ml h−1.

HPLC–diode array analysis

The chromatographic system consisted of an HP 1090M liquid chromatograph equipped with a diode-array detector and an HP Kayak XM 600 ChemStation (Agilent Technologies, Waldbronn, Germany). Multiple wavelength monitoring was performed at 210, 230, 260, 280, 310, 360, 435 and 500 nm, and UV-vis spectra measured from 200 to 600 nm. A 10-ml aliquot of the fermentation broth was centrifuged and the supernatant adjusted to pH 4, and the preparation extracted with the same volume of EtOAc. After centrifugation, the organic layer was concentrated to dryness in vacuo and resuspended in 1 ml MeOH. Aliquots of 5 μl of the samples were injected onto an HPLC column (125 × 3 mm i.d.), fitted with a guard-column (20 × 3 mm i.d.) filled with 5-μm Nucleosil-100 C-18 (Maisch, Ammerbuch, Germany). The samples were analysed by linear gradient elution, using 0.1% ortho-phosphoric acid as solvent A and CH3CN as solvent B at a flow rate of 0.85 ml min−1. The gradient was from 4.5 to 100% for solvent B in 15 min with a 3-min hold at 100% for solvent B.

HPLC–ESI–MS analysis

HPLC–ESI–MS analysis was done with an Agilent 1200 HPLC series equipped with a binary HPLC pump, autosampler, diode array detector, and a LC/MSD Ultra Trap System XCT 6330 (Agilent Technologies). Samples of 2.5 μl were injected onto an HPLC column (Nucleosil-100 C-18, 3 μm, 100 × 2 mm i.d.) and separated by 0.1% aqueous HCOOH as solvent A and 0.06% HCOOH in CH3CN as solvent B by a linear gradient from 10% B to 100% B over 15 min at a flow rate of 400 μl min−1.

Structure determination

ESI–MS spectra were obtained on a QTRAP 2000 LC-MS/MS spectrometer (Applied Biosystems, Darmstadt, Germany). High-resolution HPLC-ESI-Orbitrap-FT mass spectra were recorded on an Orbitrap XL mass spectrometer (Thermo Scientific, Bremen, Germany) coupled to an Agilent 1200 HPLC system (Agilent Technologies), and NMR spectra were recorded on a DRX 500 spectrometer (Bruker, Karlsruhe, Germany) at 500 and 125 MHz for 1H and 13C, respectively. The chemical shifts are given in p.p.m. referred to dimethyl sulfoxide-d6 as 2.50 p.p.m. (1H) and 39.51 (13C).

To analyze the absolute configuration of the amino acid moiety, 1 was first hydrolyzed with 6 M HCl at 110 °C for 24 h. The dry hydrolysate was subjected to ethanolic HCl at 110 °C for 30 min, followed by subsequent evaporation to dryness using a stream of nitrogen and then trifluoroacylated with trifluoroacetic anhydride in CH2Cl2 at 110 °C for 10 min. The reaction mixture was again evaporated to dryness, dissolved in toluene and analyzed by chiral GC–MS on Lipodex E/PS255 (30:70) capillary column (20 m × 0.25 mm i.d.).

X-ray diffraction data were collected on an Oxford Diffraction Xcalibur S diffractometer; the diffractometer was equipped with a Sapphire CCD detector and an enhanced monochromated MoKα source on a four-circle κ-platform (Agilent Technologies, Palo Alto, CA, USA). The diffraction frames were integrated by using the CrysAlisRed program, the set of data were corrected for empirical absorption with SCALE3 ABSPACK.26 The structure was solved by direct methods and refined using the program SHELX97.27 CCDC 807516 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.