PJS, a novel isocoumarin with hexahydropyrimidine ring from Bacillus subtilis PJS

Abstract

Amicoumacin group of antibiotics, such as baciphelacin, 1 amicoumacins, 2 AI-77s, 3 xenocoumacins, 4 Y-05460M-A, 5 PM-94128, 6 Sg17-1-4, 7 bacilosarcins A, B 8 and lipoamicoumacins A-D, 9 is a small family of isocoumarin, which possesses the common chromophore, 3, 4-dihydro-8-hydroxyisocoumarin. Most members of amicoumacin group of antibiotics are produced by the genus Bacillus and exhibit various important bioactivities. The chromophore shows specific UV absorbance at 247 and 314 nm in methanol. Thus, a project based on HPLC-diode array screening and HPLC-MS dereplication was carried out to find new amicoumacin analogues from the secondary metabolites of Bacillus spp. As a result, PJS (1), a new isocoumarin antibiotic was discovered from the fermentation broth of Bacillus subtilis PJS. In this study, we wish to report the fermentation, isolation, physico Àchemical properties, structural elucidation and biological activities of (1) ( Figure 1).The producing strain Bacillus subtilis PJS was isolated from the leaf of an unidentified plant collected at Luopu county, Hetian area, Xinjiang Province, People's Republic of China. On the basis of analysis of 16S rRNA, it was identified as Bacillus subtilis subsp. inaquosorum. A stock culture of the strain Bacillus subtilis PJS was maintained on modified Gause's no. 1 agar slant consisting of soluble starch (Beijing Qi Te Xin Chemical Co. Ltd. China) 20.0 g, NaCl 50.0 g, K 2 HPO 4 0.5 g, KNO 3 1.0 g, MgSO 4 1.0 g, FeSO 4 0.02 g, glucose 1.0 g, peptone 0.5 g, tryptone 0.3 g and agar 20.0 g in 1.0 l distilled water (pH 8.0) at 4 1C. The stock culture was inoculated into 250 ml Erlenmeyer flasks containing 50 ml of seed medium, which was the same modified Gause's no.1 liquid medium as above, but no agar. The culture was incubated on a rotary shaker (180 r.p.m.) at 28 1C for 24 h. Fifty millilitres of the seed culture was transferred to a 5000 ml Erlenmeyer flask containing 1000 ml of the producing medium, which was the same as the seed medium. The fermentation was carried out at 28 1C for 48 h on a rotary shaker (180 r.p.m.).Eighty liters of the fermentation broth was centrifuged at 4500 r.p.m. for 20 min, then, the supernatant was obtained and was extracted twice with 40 l of ethyl acetate each time. The organic layer was pooled and concentrated under reduced pressure at 37 1C to give yellow syrup (2.3 g). It was then separated by preparative thin layer chromatography on 10 Â 10 cm plates (silica gel 60F 254 , Merck KGaA, Darmstadt, Germany) using CHCl 3 -MeOH, 65:35 (v/v) as the developing solvent. Under UV 365 nm, bands with light blue fluorescence at R f ¼ 0.35 were scraped and then eluted with methanol to yield a semipurified sample (260 mg). After dissolved in 1 ml methanol, the sample was filtered through a 0.22 mm membrane and was further purified by HPLC on a shim-Pack PRC-ODS column (250 Â 20 mm, Shimadzu Corp., Tokyo, Japan) with MeOH-H 2 O, 55:45 (v/v) at 2 ml min À1 . Peak at R t ¼ 26 min, showed UV absorbance at 247 and 314 nm detected by prominence diode array ...

Dichloroisocoumarins with Potential Anti-Inflammatory Activity from the Mangrove Endophytic Fungus Ascomycota sp. CYSK-4

“…The structure-activity relationships of these dichloroisocoumarins indicated that a hydroxyl group was more significance than a methoxy group on anti-inflammatory activity. Isocoumarins were previously reported to have radical scavenging and antioxidant [ 14 ], anti-HIV [ 15 ], antimicrobial [ 16 ], anti- γ -secretase [ 17 ], antitumor [ 18 ], immunomodulatory [ 19 ], antifungal [ 20 ], toxicity to zebrafish embryos [ 13 ], and α -glucosidase inhibitory activities [ 21 ]. This is the first report of anti-inflammatory activity of dichloroisocoumarins.…”

Hetiamacin E and F, New Amicoumacin Antibiotics from Bacillus subtilis PJS Using MS/MS-Based Molecular Networking

“…The preparation of the agar slant and cultural broth of strain PJS were the same as described previously [ 14 ]. The strain was inoculated into three 500 mL Erlenmeyer flasks containing 100 mL of modified Gause’s No.1 cultural broth (soluble starch 20.0 g, NaCl 50.0 g, K 2 HPO 4 0.5 g, KNO 3 1.0 g, MgSO 4 1.0 g, FeSO 4 0.02 g, glucose 1.0 g, peptone 0.5 g, tryptone 0.3 g, and N -acyl-asparagine 0.5 g in 1.0 L distilled water, pH 8.0), and cultured on a rotary shaker at 180 rpm and 28 °C.…”

“…The structural variations are ascribed to the differences in an aliphatic, carbohydrate-like chain connected with the dihydroisocoumarin nucleus by an amido bond. To date, more than 40 amicoumacin analogs have been reported and they exhibit a wide range of biological activities, such as antibacterial [ 12 , 13 , 14 , 15 ], antifungal [ 16 ], anticancer [ 17 , 18 , 19 ], anti-inflammatory [ 20 ], antiulcer and herbicidal effects [ 21 , 22 ]. Many amicoumacins showed excellent inhibitory activities against Gram-positive bacteria, especially the methicillin- or oxacillin-resistant strains of Staphylococcus aureus (MRSA or ORSA).…”

“…Bacillus subtilis PJS is an endophytic bacterium isolated from a plant collected in the Hetian area in the Taklamakan desert, Xinjiang Uygur Autonomous Region, China [ 14 ]. In the previous study [ 12 , 14 , 25 ], a series of novel bioactive amicoumacins, hetiamacin A–D, have been obtained from this strain by means of traditional bioassay-guided separation. Recent advances in cheminformatics and analytical techniques spur us to deeply dig metabolites in the fermentation broth of strain PJS by using molecular networking-based dereplication strategy.…”

Damxungmacin A and B, Two New Amicoumacins with Rare Heterocyclic Cores Isolated from Bacillus subtilis XZ-7

“…In our ongoing research on novel amicoumacin products from Bacillus spp. [13,14], the strain Bacillus subtilis XZ-7, isolated from a soil sample collected from Damxung county in Tibet, China, was chosen through the chemical screening approaches and dereplication strategies [15]. Previous investigations of the strain XZ-7 have resulted in the identification and isolation of a series of amicoumacin compounds [15,16].…”