G. K. Oleinikova

Bacillomycin D from the marine isolate of Bacillus subtilis KMM 1922

Strain KMM 1922 of Bacillus subtilis, a producer of the antifungal iturin peptide antibiotic bacillomycin D, was isolated from a specimen of the Kuril sponge Stelletta validissima. The structure of the compound was proved using two-dimensional NMR spectroscopy, tandem electrospray mass spectrometry, and literature data. The peptide was shown to exhibit a pH-dependent cytotoxic activity.

Secondary metabolites of the marine fungus Aspergillus ustus KMM 4640

Marine mycelial fungi are known to produce various biologically active compounds with unusual chemical structures [1, 2]. In continuation of research on secondary metabolites of marine fungi-micromycetes, we found that the strain Aspergillus ustus (Bainier) Thom & Church KMM 4640 that was isolated from sediment of the Okhotsk Sea shelf of Sakhalin Island (27 m depth) produced compounds with antimicrobial activity. This strain was cultivated in wort-agar medium with seawater at 22°C for 14 d [3]. Mycelium was extracted with EtOAc. The resulting extracts were concentrated at 10 mm Hg. The dry residue was dissolved in aqueous EtOH (10%) and subsequently extracted with hexane and EtOAc. Repeated column chromatography over silica gel using hexane:EtOAc (20:1) isolated from the EtOAc extract compounds 1 (5.3 mg), 2 (0.5 mg), 3 (5.3 mg), 4 (1.3 mg), 5 (1.3 mg), and 6 (4.4 mg).

Indolic metabolites from the new marine bacterium Roseivirga echinicomitans KMM 6058T

N-Acetyl-(1) and N,N-diacetyl-(2) tryptamines were isolated from the butanol extract of culture medium of the new marine bacterium Roseivirga echinicomitans KMM 6058T. The structures of the compounds were proved using mass spectrometry, UV, PMR, and 13C NMR spectroscopy and by comparing these data with mass and NMR spectra of synthetic samples of 1 and 2. Compound 2 was isolated from a natural source and synthesized for the first time. The cytotoxic activity of the compounds was studied using Erlich carcinoma tumor cells, murine erythrocytes, and sperm and egg cells of the sea urchin Strongylocentrotus intermedius.

Nonpolar compounds and free fatty acids from marine fungi Aspergillus ustus

Marine mycelial fungi are known to produce biologically active compounds with unusual chemical structures [1, 2]. Fungi synthesize such compounds because they must adapt to specific marine habitats. One of the consequences of such adaptation is the unusual composition of cellular and extracellular nonpolar compounds and fatty acids isolated from extracts of fungal marine isolates. It was shown that marine fungi produce such important acids as 16:0, 18:0, 18:1n9, 18:2n6, 18:3n3, and 20:4n6 [3]. An unusually high percentage of branched and unsaturated acids was noted in them [4]. Extracts of fungal marine isolates also contained phthalates, which inhibit the catepsin B proteinase enzyme [5]. In continuation of a study of secondary metabolites from marine fungi-micromycetes, we isolated two strains of Aspergillus ustus from sediment collected on the Okhotsk Sea shelf (27 m depth) of Sakhalin Island. The fungus strains were cultivated in standard wort agar-agar medium in seawater for 14 d [6]. Cultures were extracted with EtOAc. The resulting extracts were concentrated at reduced pressure (10 mm Hg). The dry residue was dissolved in aqueous EtOH (10%) and extracted successively with hexane and EtOAc. The hexane fractions were concentrated at reduced pressure (10 mm Hg) and analyzed by GC–MS by comparing the results with mass-spectrometric fragmentation of standards using the NIST98 database. Table 1 presents the results. The hexane fractions of the studied marine fungi contained linear saturated hydrocarbons, linear hydrocarbons with a terminal double bond, linear hydrocarbons from C16 to C24 with a terminal cyclohexane group for strain 1 and from C14 to C26 for strain 2. Both fractions also contained diene hydrocarbons with C18, C20, and C24. The hexane extract of culture 2 also contained squalene. Both fractions contained di(2-ethylhexyl)phthalate. The EtOAc fractions of each culture were chromatographed over a column of silica gel using a gradient of hexane:EtOAc (100:0 0:100) to isolate fractions of free fatty acids. The resulting total acids were analyzed as methyl esters (methylation by diazomethane in ether) and pyrrolidides [7] using GC–MS. The derivatives were identified by comparing their mass spectra with those of standards using the NIST98 database. The results are given below:

Nonpolar Compounds and Free Fatty Acids from Several Marine Isolates of Fungus Aspergillus ustus and Actinobacterium Nocardiopsis umidischolae

In continuation of research on secondary metabolites of marine fungi and actinobacteria [1, 2], hexane fractions and fractions of free fatty acids and the sterol fraction of actinobacterium were obtained and analyzed from cultures of marine isolates of the fungus Aspergillus ustus KMM 4642 and KMM 4664 isolated from sediment (Okhotsk Shelf, Sakhalin Island, 26.5 m depth) and the actinobacterium Nocardiopsis umidischolae KMM 7036 isolated from Mycale sp. of sponge (Deryugin Basin, Okhotsk Sea). Strain KMM 4642 was cultivated in malt-agar medium prepared with sea water for 14 d [3] and on rice medium prepared with seawater for 21 d [4]. Strain KMM 4664 was cultivated only on rice medium prepared with seawater. Actinobacterium was cultivated in a special medium containing peptone (5.0 g/L), meat extract (3.0), starch (20.0), agar (16.0) and seawater at pH 7.5 and 23°C for 23 d. Cultures were extracted with EtOAc. The extracts were evaporated to dryness. The resulting 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. The data were compared with the mass spectrometric fragmentation of standards using the NIST98 database. The hexane fraction of A. ustus KMM 4642 consisted of 96.88% diethylhexylphthalate. The remainder consisted of fatty acid ethyl esters and pentadecane. The hexane fraction of A. ustus KMM 4664 contained fatty acid ethyl esters, squalene, octadecane, and sterols such as 24-cholest-4-en-3-one, 24-ethylcholest-7-en-3 -ol, 24-ethylcholesta-3,5-diene, and 24-ethylcholest-5-en-3 -ol at concentrations of 1–2% of the studied mixture. These results differed from those obtained earlier for marine isolates of A. ustus [1]. The hexane fraction of actinobacterium N. umidischolae contained hydrocarbons at concentrations of 1.5–3% that included linear C15, C16, C17, C18, C20, and C22; C18, C19, C20, C22, and C24 with an iso-carbon chain; C18, C19, and C22 with a terminal double bond; and dibutylphthalate (5%) and diethylhexylphthalate (45%). The EtOAc fraction of each culture was chromatographed over a column of silica gel using a hexane–EtOAc gradient (100:0 90:10) to afford fractions of free fatty acids. The obtained total acids were analyzed as the methyl esters (methylated by diazomethane in Et2O) and pyrrolides [5] using GC-MS. Derivatives were identified by comparing their mass spectra with those of standards using the NIST98 database. The composition and content of A. ustus acid esters are given below (mass%):

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.

Non-polar compounds and free fatty acids from marine isolates of mycelial fungi

Marine mycelia fungi are known to be producers of biologically active compounds with broad spectra of activity and unusual chemical structures [1, 2]. Such compounds are biosynthesized by marine organisms in order to adapt to specific marine habitats. One of the consequences of such adaptation may be the qualitative and quantitative composition of non-polar compounds and extracellular fatty acids produced by isolates of marine fungi. Previous studies showed that several isolates of marine fungi produced important acids such as 16:0, 18:0, 18:1n9, 18:2n6, 18:3n3, and 20:4n6 and those with normal and branched chains [3]. Extracts of several isolates of marine fungi contained phthalates, which inhibit the protein-catabolism enzyme catepsin B [4]. In continuation of research on metabolites of marine fungi, we studied extracts of marine isolates of 10 strains of the mycelial fungi Penicillium implicatum KMM 4648 (1), P. citrinum KMM 4649 (2) and KMM 4650 (3), P. glabrum KMM 4651 (4), P. restrictum KMM 4652 (5), P. expansum KMM 4653 (6), Isaria felina KMM 4659 (7), Curvularia inaequalis KMM 4660 (8), Scopulariopsis brumptii KMM 4661 (9), and Acremonium roseum KMM 4662 (10). Cultures of facultative marine fungi were isolated from various sources, i.e., 1, 4, 9, from seaweed Zostera sp.; 3, from soft coral Zoantharia sp.; 2, 5, 6, 7, 8, 10, from marine soil. Strains of fungi were cultivated on malt-extract agar [5] (strains 3, 4, 7, 8, 10) and rice [6] (strains 1, 2, 4, 5, 6, 7, 8, 9) using stationary seawater at 25°C for 14 and 21 d, respectively. Fungal cultures were extracted with EtOAc. The extracts were concentrated at reduced pressure. The resulting 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 of the hexane fraction and derivatives of the EtOAc eluate were identified by comparison of their mass spectra with those of standard compounds using the NIST98 database. Strain P. implicatum (1) produced an insignificant amount of linear hydrocarbon C16 (0.2%); strain P. expansum (6), hydrocarbons with a terminal double bond C18 (3%), C20 (4%), C22 (2.4%), and C24 (0.8%) in addition to saturated C20 (43%) and fatty acids proportional to the content in the fatty-acid fraction (Table 1). The hexane fraction of I. felina (7), which was cultivated on marine malt agar, contained linear hydrocarbons C16 (5.3%), C18 (12.2%), C20 (9.4%), and C22 (5.0%); linear hydrocarbons with a terminal double bond C16 (9.2%), C18 (19.7%), C20 (17.7%), C22 (8.6%), and C24 (1.4%); and hydrocarbons with a terminal cyclohexane group C16 (3.4%), C18 (4.4%), and C20 (3.6%). Non-polar compounds were not detected in extracts of the other cultures. The EtOAc fractions of each culture were chromatographed over a column of silica gel using a gradient of hexane:EtOAc (100:0 90:10) to afford fractions of fatty acids. The resulting total acids were analyzed as methyl esters (methylation by diazomethane in Et2O) and pyrrolidides using GC-MS [7]. All cultures produced 16:0, 18:2, 18:1, and 18:0 fatty acids. Strain P. expansum (6) and I. felina (7) produced trace amounts of 14:0 acid; strain P. citrinum (3), which was isolated from soft coral, 17:0 acid. Table 1 presents the results. All culture samples contained phthalates in small (1–3%) quantities. However, their content in extracts of P. glabrum (4) and P. restrictum (5) exceeded 50% of the fraction contents. The qualitative composition of fatty acid fractions of the studied strains agreed in general with previously published data [8]. The results supplemented our previous investigations of non-polar compounds and fatty acids from marine isolates of fungi [9, 10].

Diketopiperazines from Marine Isolate of Actinobacterium Nocardiopsis umidischolae KMM 7036

In continuation of research on secondary metabolites of marine isolates of microorganisms [1, 2], we investigated actinobacterium Nocardiopsis umidischolae KMM 7036, which was isolated from Mycale sp. fungi, collected in Deryugin Basin, Sea of Okhotsk. The actinobacterium was cultivated and the culture was processed as described earlier [3]. Multiple column chromatography over silica gel using a gradient of hexane EtOAc (100% hexane 100% EtOAc) of the culture extract isolated 1 (11.4 mg), 2 (0.9 mg), and 3 (2.2 mg).

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.

Nonpolar Compounds and Free Fatty Acids from Several Isolates of Marine Fungus Penicillium antarcticum

In continuation of research on biologically active compounds from extracts of marine isolates of fungi and micromycetes [1, 2], extracts of nine Far-Eastern strains of the marine fungus Penicillium antarcticum were investigated (Table 1). Strains of the fungus were cultivated for 14 d on malt-agar [3] and rice medium [4] that were prepared with seawater. Mycelia together with the medium were extracted with EtOAc. The culture extracts were concentrated at reduced pressure. The resulting dry solids were dissolved in EtOH (10%) and extracted sequentially with hexane, EtOAc, and BuOH. The hexane fractions were concentrated at reduced pressure and analyzed using GC-MS. Compounds were identified by comparing their mass spectra with those of standard compounds using the NIST98 database. Table 2 presents the results. Hexane fractions of all cultures contained linear paraffinic hydrocarbons, linear hydrocarbons with a terminal double bond, and their isomers. Strain 1 on malt-agar produced mainly olefinic hydrocarbons with a terminal double bond such as C16, C18, C20, C22, C24, C25, and C26 in addition to C22-diene-1,4; on rice medium, also linear paraffins C16 (6.79%), C17 (7.92%), and C18. Strain 2 produced olefinic hydrocarbons with a terminal double bond such as i-C18 (2.86%), C24, and C27 (9.48%) in addition to linear paraffins C23 (8.6%), C24, C26, and C27 (25–21%). The hexane fraction of the culture of 3 contained 11 paraffins (C19–C29) and eicosene (8.76%). Culture of 7 gave linear alkanes C20–C29 and i-heptacosane (5.24%). Strain 4 on malt-agar medium produced mainly linear paraffins C12 (19.7%) and C19–C29; on rice medium, C16, C18, C22, C25 with a terminal double bond and C22-diene-1,4. The hexane fraction of culture 5 on malt-agar medium contained only linear hydrocarbons C20–C30 and C32; on rice medium, hydrocarbons were absent. The other cultures produced linear paraffins and olefinic hydrocarbons with a terminal double bond for C12–C30. Strains 8 and 9 produced triacontane (11.15 and 5.03%, respectively). Strains 1, 2, 5, and 6 produced squalene as follows. 1 (on both media), 8.55 and 14.52%, respectively; 2 and 6 (on rice medium), 34.17 and 48.63%, respectively; 5 (on malt-agar medium), 4.03% (% of total mass of hexane fraction). EtOAc fractions of each culture were chromatographed over a column of silica gel using a hexane–EtOAc gradient (100:0 90:10). Free fatty acids and phthalates eluted with the 95:5 fraction. The obtained sums were analyzed by GC-MS as methyl esters (methylated by diazomethane in Et2O) and pyrrolidides [5]. Derivatives were identified by comparing their mass spectra with those of standard compounds using the NIST98 database. Table 3 presents the results. Most cultures produced fatty acids 16:0, 18:2, 18:1, and 18:0. Cultures of 5, 6, 7, and 9 (malt-agar medium) and 4 (rice medium) did not produce 18:2(9,12) acid. Several strains produced small quantities of other acids, e.g., 1, 19:2(9,12) (1.9%, rice medium); 5 and 6, 14:0 (2.15 and 1.57%, respectively, malt-agar medium); 6 and 1, 15:0 (1.15 and 0.86%, respectively, malt-agar medium). Extracts of cultures contained phthalates (5–28% of the fatty-acid fraction weight). The total phthalate contents in extracts of cultures grown on rice medium were less than in those grown on malt-agar medium. Phthalates were not detected in extracts of cultures of 3 and 4 grown on rice medium.