Natural Phthalate Derivatives from the Bacterium Burkholderia cepacia K 87

In continuation of our screening of antifungal active compounds from the fermentation extracts of soil borne bacteria Burkholderia cepacia K87 afforded three phthalate derivatives, dibutyl phthalate (1), dioctyl phthalate (2), and phthalate ester of alkylated 9hydroxynonanoic acid (3). Compound 3 was reported first time. These phthalate derivatives were found to be marginally active or inactive against a number of bacteria and fungi.


General methods
High resolution TOF mass spectra (positive ESI mode) were measured on a Waters LCT Premier mass spectrometer coupled with a Waters Alliance HPLC system.Optical rotations were measured on a Perkin-Elmer 341-LC Polarimeter. 1 H-and 13 C-NMR spectra were recorded on a Varian Mercury 400 Spectrometer operating at 400 MHz for 1 H NMR and 100 MHz for 13 C spectra.The chemical shifts were given in ppm (δ) and were referenced relative to CDCl 3 (δ 7.26 and 77.24 ppm for 1 H and 13 C NMR, respectively).2D NMR spectra (COSY, HSQC, HMBC and NOESY) were recorded using the manufacturer's software VNMR 6.1C.Flash column chromatography was carried out on a column packed with reversed phase C-18 silica gel (40-63 μm, 90 × 100 mm, Merck).Medium-pressure liquid chromatography (MPLC) was carried out on a Yamazen instrument using a column packed with reversed phase C-18 silica gel (cosmosil 40 C18 Prep, 40 X 320 mm) at 5 mL/min.Preparative HPLC was performed on a Waters 600 model system (Microsorb 100-5 C-18, 21.4 x 250 mm) at 7 mL/min at 210 nm.

Extraction and isolation
Solid culture media were harvested with MeOH.The methanolic extract (180 g) was suspended in water (100 mL) and subjected to reversed phase C-18 flash column chromatography with a stepwise gradient elution of a mixture of water and MeOH to give eleven fractions (1 L/each fraction).The 90-100% aqueous MeOH eluate (brownish residue, 650 mg) was further chromatographed on a reversed phase C-18 MPLC with a gradient elution of 60% aqueous MeOH to 100% MeOH for 160 min followed by reversed phase HPLC (75%-100% aqueous MeOH for 60 min) to yield compounds 1 (20 mg, R t = 29.4min), 2 (7.6 mg, R t = 33.8min), and 3 (3.8mg, R t = 81.3min).

Antimicrobial and cytotoxicity assays
Antimicrobial and cytotoxicity assays were carried out according to the methods described in the literatures [3,17,18].

Results and Discussion
The broth of B. cepacia K87 (10 L) was extracted with MeOH [3].The methanolic extract was subjected to a series of chromatography (reversed-phase C-18 flash column chromatography followed by C-18 MPLC and HPLC).Compounds, 1, 2, and 3 were obtained in yield of 2.8, 20, and 7.6 mg, respectively.Compounds 1 and 2 were isolated as colorless oil and established as dibutyl phthalate, and dioctyl phthalate, respectively [4,5].Compound 3 was obtained as colorless oil with specific rotation of - where the methine groups might be repeated due to severe overlapping of the signals.
From interpretation of HSQC and HMBC (Fig. 2) spectral data, the substructures were connected to provide two alkylated 9-hydroxynonanoate moiety which were ester-linked with the phthalate moiety as evidenced from the correlations between the carbonyl groups at δ 167.0 (2C) and the hydroxylated methine protons at δ 4.23 and 4.20.The methoxy group at δ H 3.66 was correlated with one of the carbonyl groups of the nonanate at δ C 174.0.The numbers of severely overlapped methylene and methine groups were counted from the molecular formula deduced from the MS data.Due to severe overlapping the propyl and butyl groups could be interchangeable between the chains.Thus, we suggest the structure of 3 could be either [2-ethyl-4-  Compounds 1-3 were tested against the bacterial strains like Streptococcus mutans, Bacillus subtilis, Shigella sonnei, Pseudomonas aeruginosa, and the fungal strains like Candida kruisii, Candida glabrata, Candida albicans, and Rhizoctonia solani.Compounds 1 and 2 were found to be inactive against all the microbial strains tested at 100 μg.Compound 3 possessed marginal antifungal activity only against C. kruisii (zone of inhibition 9 mm at 200 μg) but inactive against other bacteria.
Synthetic phthalate esters are used commonly in paints and polymer products as plasticizers [6,7], and widely detected in the environments, such as sediments, natural waters, soils, plants, and aquatic organisms [8].Although phthalate derivatives are useful chemicals, they are regarded as environmental health hazards due to their toxicity, carcinogenicity, teratogenicity, and mutagenicity [7,[9][10][11].It was reported that prenatal exposures of animal to some phthalates, particularly di(2-ethylhexyl) phthalate, dibutyl phthalate, and benzyl butyl phthalate caused male reproductive anomalies including decreased anogenital distance, testicular dysgenesis, hypospadias, cryptorchidism, malformations of the male reproductive organs, somniferous tubule degeneration [12][13][14].Phthalate exposure in childhood has also caused an increased occurrence of atopic disease including allergic rhinitis, wheezing, and eczema [9,15].However, there are reports that phthalate esters are naturally produced extracellularly by microorganisms such as bacteria, fungi and yeasts [4,6,16].Compounds 1-3 were isolated from the extract of the culture broth of plant-growth promoting rhizobacteria B. cepacia K87.It is unclear whether they were true metabolites of the bacterium or artifacts formed during isolation.