M. P. Sobolevskaya

NEW 3-(2(R)-HYDROXYBUTYL)-7-HYDROXYPHTHALIDE FROM MARINE ISOLATE OF THE FUNGUS Penicillium claviforme

The new phthalide derivative 3-[2′(R)-hydroxybutyl]-7-hydroxyphthalide (2) and the five known compounds (−)-3-butyl-7-hydroxyphthalide (1), isopatulin (3), m-hydroxybenzyl alcohol (4), cyclopenin (5), and cyclopeptine (6) were isolated from the marine isolate of the fungus Penicillium claviforme associated with the seagrass Zostera marina. The structures of the compounds were established using NMR spectroscopy and mass spectrometry. The absolute configuration of the C-2′ asymmetric center was determined using a modified Mosher method.

Metabolites from the marine actinobacterium Streptomyces sp. KMM 7210

In continuation of the search for biologically active metabolites from marine actinobacteria, we studied the strain Streptomyces sp. KMM 7210 that was isolated from sediment samples of Troits Bay (Poset Gulf, Sea of Japan). Cultivation of this strain in medium consisting of edible potato starch (10 g/L), peptone (2 g/L), yeast extract (2 g/L), CaCO3 (1 g/L), pH 7.7, and distilled water:seawater (1:1) for 6 d at ~20°C produced compounds with cytotoxic activity against sea urchin Strongylocentrotus intermedius embryos. The cultivation liquid (20 L) was centrifuged for 30 min at 500 g. The resulting cells were suspended in distilled H2O (100 mL) and destroyed with cooling by ultrasound for 2 min at 20-second intervals. The suspension of destroyed cells was extracted (3 ) successively with EtOH and acetone. The supernatant was extracted with EtOAc (3 ). The resulting extracts were combined and evaporated to dryness. The dry solid (600 mg) was chromatographed over a column of SiO2 using a gradient of hexane:EtOAc (10:1, 5:1, 2:1, 3:2, 1:1), EtOAc, and EtOAc:EtOH (20:1, 10:1). This produced pure 1 (3 mg), 2 (2 mg), 3 (2.2 mg), and 4 (2.5 mg).

Metabolites of the sea isolate of bacteria Streptomyces SP 6167

Sea isolate of Streptomyces sp. 6167 was found to produce four macrolide antibiotics feigrisolides A, B, and D, and dinactin. The chemical structures of the compounds were determined using 1D and 2D NMR spectrometry and electrospray mass spectrometry (ESMS). It was shown that the feigrisolides are cytotoxic to Ehrlich carcinoma tumor cells and to egg-cells of the sea urchin Strongylocentrotus intermedius and antimicrobial to the bacteria Bacillus cereus and Escherichia coli.

Spiroketals from Marine Isolates of the Fungi Penicillium thomii KMM 4645 and P. lividum KMM 4663

Recent research showed that micromycete fungi isolated from marine sources are interesting as producers of biologically active compounds [1–3]. In continuation of work on the discovery of biologically active compounds in extracts of marine isolates of micromycete fungi, we isolated the two strains Penicillium lividum and P. thomii from the surface of Sargassum miyabei (collected in Lazurnaya Bay, Peter the Great Gulf, Sea of Japan). Work with the fungi was carried out in parallel. Each fungus was cultivated on standard solid medium. The biomass was worked up with EtOAc followed by separation of the dry residue over silica gel using eluent of increasing polarity. The resulting fractions were separated by reversed-phase HPLC to afford from P. lividum compounds 1 (5 mg) and 2 (38 mg); from P. thomii, 1 (2 mg) and 2 (30 mg).

Non-polar compounds and free fatty acids from several marine isolates of fungi of the genus Aspergillus

Marine mycelial fungi of the genus Aspergillus are known producers of biologically active compounds with broad spectra of action and unusual chemical structures [1, 2]. The biosynthesis of such compounds may be due to the need to adapt to specific conditions of the marine habitat. One of the consequences of such adaptation can be an unusual composition of non-polar compounds in addition to extracellular fatty acids (FAs) produced by fungal marine isolates. It was shown that they are producers of such important acids as 16:0, 18:0, 18:1n9, 18:2n6, 18:3n3, and 20:4n6 [3]. An unusually high percent content of branched and unsaturated FAs was noted in them [4]. Extracts of fungal marine isolates contained also phthalates, which inhibited the protein catabolism enzyme catepsin B [5]. According to our data, several types of marine isolates of A. ustus produced significant quantities of various hydrocarbons [6]. In continuation of research on metabolites of marine fungi, we investigated fractions of marine isolates of the fungi A. sulfureus KMM 4640, A. versicolor KMM 4644, A. carneus KMM 4646, and A. versicolor KMM 4647 that were isolated from sediment on the Sea of Okhotsk shelf, Sakhalin Island, at a depth of 26-28 m in addition to the fungus A. carneus KMM4638 that was isolated from the marine alga Laminaria sachalinensis (Miyabe). Fungal strains were cultivated in modified rice medium at a constant 25°C for 21 d [7]. Cultures were extracted with EtOH. The extracts were concentrated at reduced pressure. The resulting residuals were dissolved in EtOH (10%) and extracted successively with hexane, EtOAc, and BuOH. The hexane fractions were evaporated at reduced pressure and analyzed by GC-MS. Compounds were identified by comparing their mass spectra with those of standards using the NIST98 database. The hexane extract of A. versicolor KMM 4644 contained hydrocarbons with linear carbon chains from C20 to C32 at concentrations from 2 to 18%. Non-polar compounds were not observed in hexane extracts of the other cultures. The EtOAc fractions from each culture were chromatographed over a column of silica gel with gradient elution by hexane:EtOAc (100:0 90:10) to afford fractions of free FAs. The resulting total acids were analyzed as methyl esters (methylated by Et2O solution of diazomethane) and pyrrolidides [8] using GC-MS. Derivatives were identified by comparing their mass spectra with those of standards using the NIST98 database. All cultures produced significant amounts of heptadecanoic, octadecadienoic, octadecaenoic, and octadecanoic acids. The strain A. carneus that was isolated from laminaria produced insignificant amounts of linear acids with C22–C24 C atoms whereas that isolated from sediment had a broader set of FAs. All fractions contained insignificant amounts of phthalates. The isolate of A. carneus KMM4646 produced small amounts of squalene and 1,4,6-trimethylnaphthalene. The qualitative compositions of the FA fractions agreed in general with those published earlier [9]. Table 1 presents the results.

Nonpolar Compounds and Free Fatty Acids from Several Fungal Marine Isolates of Penicillium thomii

In continuation of research on biologically active compounds in extracts of marine isolates of micromycete fungi [1, 2], we analyzed nonpolar compounds and fatty acids in hexane and EtOAc fractions of marine isolates of the fungus Penicillium thomii that was obtained from the surface of brown seaweed and seagrass. Marine isolates of P. thomii were obtained from the seagrass Zostera marina; strains 1 and 2, from the rhizoplane (rhizome surface); 3 and 4, from the phylloplane (leaf surface); 5 and 6, from the rhizosphere (soil next to root); 7, from the brown seaweed Sargassum miyabei (thallus); and 8, from S. pallidum (thallus) that were collected in the Sea of Japan (2011). Strains 1–6 were from Rifovaya Bay; 7, Troitsa Bay; 8, Vostok Bay (2012). Fungal strains were cultivated for 14 d in malt-extract agar prepared with seawater [3]. The obtained cultures were extracted with EtOAc. The extracts were concentrated at reduced pressure. The resulting dry residues were dissolved in EtOH (10%) and extracted successively with hexane, EtOAc, and BuOH. The hexane fractions were evaporated at reduced pressure and analyzed by GC-MS. Compounds were identified by comparing their mass spectra with those of standards by using the NIST98 database. Table 1 presents the results. The obtained fungal isolates produced hydrocarbons from C16 to C25 that could contain one or two double bonds and were linear or branched at one end of the chain. The qualitative and quantitative contents of the compounds were different in all fractions. The EtOAc fractions of each culture were chromatographed over a column of silica gel using a hexane–EtOAc gradient (100:0 90:10) to isolate fractions of free fatty acids. The obtained total acids were analyzed as methyl esters (methylated by diazomethane in Et2O) and pyrrolidides [4] using GC-MS. The derivatives were identified by comparing their mass spectra with those of standards by using the NIST98 database. Table 2 presents the results. All fractions also contained dibutyland diethylhexylphthalates, which was characteristic for fungal marine isolates [5]. The broader spectrum of hydrocarbons that were identified in the studied cultures was notable. The results agreed in principle with those published previously [6, 7] and supplemented our data on nonpolar compounds and free fatty acids from fungal marine isolates.

Metabolites from the Marine Isolate of the Fungus Aspergillus versicolor KMM 4644

In continuation of the search for biologically active compounds in extracts of marine isolates of mycelial fungi, we isolated the strain Aspergillus versicolor KMM 4644 from sediment of Sakhalin Bay, Sea of Okhotsk. The fungus was cultivated on solid medium for 21 d at room temperature in 20 Ehrlenmeyer flasks (500 cm3), each of which contained medium of the following composition: rice, 15.0 g; yeast extract, 0.02 g; KH2PO4, 0.01 g; sodium tartrate, 0.01 g; seawater, 20 mL. Mycelium with medium was extracted with EtOAc. The extract was evaporated in vacuo. The dry residue (3.15 g) was chromatographed over a column of silica gel using hexane:EtOAc mixtures of increasing polarity. A fraction (hexane:EtOAc, 70:30) was separated by HPLC over a Diaspher-110-C18 column using H2O:MeOH (50:50) to isolate 1 (12 mg), 2 (2.5 mg), and 3 (7 mg). Separation of another fraction (hexane:EtOAc, 95:5) using the same column and H2O:MeOH (85:15) afforded 4 (2 mg) and 5 (6.5 mg).

Fatty-acid compositions of marine isolates of the actinobacteria Nocardiopsis umidischolae KMM 7036 and Streptomyces sp. KMM 7210

Actinobacteria are the leading producers of antibiotic, cytotoxic, and other biologically active compounds [1–3]. The marine world has been for several decades a rich source of these producers [4–6]. In continuation of the search for biologically active compounds in actinobacteria of the Okhotsk Sea, we determined the composition of free fatty acids from Streptomyces sp. KMM 7210 [7] that were studied previously by us, and Nocardiopsis umidischolae KMM 7036. Actinobacterium Streptomyces sp. KMM 7210 was isolated from sediment collected at a depth of 3 m (Troits Bay, Poseta Gulf, Sea of Japan). The strain was cultivated with rocking for 5 d at 22°C in Ehrlenmeyer flasks (300-mL), each of which contained potato starch (1.0 g), peptone (0.2 g), yeast extract (0.2 g), CaCO3 (0.1 g), and distilled water and seawater (50 mL each) at pH 7.7. The culture medium was worked up with EtOAc and evaporated in vacuo. Actinobacterium Nocardiopsis umidischolae KMM 7036 was isolated from a sponge Mycale sp. collected at a depth of 606 m (Deryugin Bank, Sea of Okhotsk). The strain was cultivated for 28 d at 28°C in 40 Petri dishes, each of which contained agar (0.4 g), FeSO4 (0.00125 g), MgSO4 (0.0025 g), filtered millet broth (15 mL), and seawater (10 mL) at pH 7.5. The millet broth was prepared from millet (100 g) and distilled H2O (600 mL). The biomass was worked up with EtOAc and evaporated in vacuo. Total fractions of fatty acids were obtained by chromatography of EtOAc extracts over silica gel using hexane:EtOAc (0 10%). Fatty acids as methyl esters were analyzed by GC [HP-5SM column (0.2 mm 30 m); Agilent 6850, Germany]. The temperature program was injector 270°C, 100°C (1 min), 10°C/min, 270°C (20 min), detector 300°C (He carrier gas). GC–MC of fatty-acid methyl esters was carried out in the same mode on an HP GC 6890 spectrometer with an HP-5 ms column (30 m 250 mm 0.25 m) with 5% phenylmethylsiloxane (MSD5973, Hewlett–Packard, USA) (He carrier gas). Compounds were identified by comparison of experimental mass spectra with those of known compounds using the NIST98 database. Table 1 presents data for the fatty-acid compositions of the bacteria. According to the results (Table 1), both strains produced saturated, unsaturated, and branched acids. The fraction of unsaturated acids was greater than that of saturated. N. umidischolae contained a high percent of palmitic acid (18.9%) whereas its content in the Streptomyces sp. was 4.0% with the fraction of stearic acid in it reaching 13.9%. High contents of palmitic (16:0) and stearic (18:0) acids are characteristic of actinobacteria [8-10], in particular actinobacteria of the genera Streptomyces [10] and Nocardiopsis [9, 11]. N. umidischolae produced tuberculostearic acid (10-methyl-C18:0), the presence of which is characteristic of all representative of this genus [9, 11–13]. Actinobacteria are known to produce fatty acids (saturated, unsaturated, branched) with chain lengths in the range C10-C18 [11–14]. Our results agreed with those in the literature. The fatty-acid composition of N. umidischolae isolated from the marine world was studied for the first time.

New Thomimarine E from Marine Isolate of the Fungus Penicillium thomii

The new eudesmane-type sesquiterpene thomimarine E (1) and the known compounds guaiadiol A (2) and 4,10,11-trihydroxyguaiane (3) were isolated from the marine isolate of the fungus Penicillium thomii. The structures of the compounds were established using NMR spectroscopy and high-resolution mass spectrometry. The ability of all compounds to inhibit production of NO in lipopolysaccharide-stimulated murine macrophages (RAW 264.7 line) at nontoxic concentrations was studied.

Fatty-acid composition of several Lake Baikal streptomycetes

The fatty-acid composition of seven strains of aquatic actinobacteria of the genus Streptomyces was studied. GC and GC–MS showed that the microorganisms produced mixtures of saturated, unsaturated, and branched fatty acids. A producer of dehydroabietic acid was found.