Glycosylation of Fluorophenols by Plant Cell Cultures

Fluoroaromatic compounds are used as agrochemicals and released into environment as pollutants. Glycosylation of 2-, 3-, and 4-fluorophenols using plant cell cultures of Nicotiana tabacum was investigated to elucidate their potential to metabolize these compounds. Cultured N. tabacum cells converted 2-fluorophenol into its β-glucoside (60%) and β-gentiobioside (10%). 4-Fluorophenol was also glycosylated to its β-glucoside (32%) and β-gentiobioside (6%) by N. tabacum cells. On the other hand, N. tabacum glycosylated 3-fluorophenol to β-glucoside (17%).


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halogen bonds in their structures have been described to be much more resistant to microbial degradation than otherwise substituted analogs [1].
Plant cell cultures are ideal systems for studying biotransformations specific to plants such as glycosylation reactions [10]. Plant cells can convert exogenous toxic compounds into glycosides [11][12][13][14][15], so glycosylation of halophenols by plant cell cultures is of importance from the viewpoint of pollution control. This study focused on the metabolism of monofluorophenols by plant cell cultures of Nicotiana tabacum. (1) After plant cell cultures of N. tabacum were incubated with 2-fluorophenol (1) for five days, the biotransformation products 2 and 3 (Scheme 1) were isolated from the cells by homogenization and extraction with methanol. On the other hand, no products were detected in the medium by HPLC analysis. No additional glycosylation products were detected in the methanol extracts of the cells despite careful HPLC analyses. HPLC analyses of the control cell cultures of N. tabacum, which had not been treated with 2-, 3-, and 4-fluorophenols (1, 4, and 6), showed that there were no monofluorophenols and their glycosides in both medium and N. tabacum cells. On the basis of the spectroscopic analyses such as FABMS, 1 H-and 13 C-NMR, the chemical structures of the products were determined to be 2-fluorophenyl β-glucoside (2, 60%) [16] and 2-fluorophenyl β-gentiobioside (3, 10%) of which 3 has not been identified before.

Glycosylation of 2-fluorophenol
The Hz). From the coupling pattern of the proton signals and the chemical shifts of the carbon signals due to the sugar moiety of 3, it was indicated to be β-gentiobioside [17]. Additionally, HMBC correlations were observed between the anomeric proton signal at δ 4.96 (H-1') and the carbon signal at δ 146.3 (C-1) and between the anomeric proton signal at δ 4.36 (H-1'') and the carbon signal at δ 69.6 (C-6') which confirms that the inner glucopyranosyl residue was attached to the phenolic hydroxyl group of 2-fluorophenol (1) and that the pair of β-D-glucopyranosyl residues was 1,6-linked. Thus, the structure of 3 was determined to be 2-fluorophenyl β-gentiobioside. A time-course experiment was carried out to investigate the scope of the ability of N. tabacum cell cultures to glycosylate 2-fluorophenol (1) (Figure 1). The amount of products 2 and 3 increased with incubation time. In our recent papers, we reported that the amount of disaccharides such as βprimeverosides and β-vicianosides increased with a corresponding decrease of that of β-glucosides in the biotransformation of phenolic compounds, i.e., capsaicinoids, by plant cell cultures and that conversion of the substrates to their β-glucosides occurred first, followed by further glycosylation to the corresponding disaccharides [18]. However, it is not clear that stepwise glycosylations occurred in the present case.
Recently, we reported that cultured Eucalyptus perriniana cells converted 2-, 3-, and 4fluorophenols into the corresponding β-glucosides [16]. On the other hand, it has been reported that exogenously added α-tocopherol was glycosylated to its β-gentiobioside by N. tabacum cell cultures [17]. The glycosylation of exogenous phenolic compounds to their gentiobiosides was a characteristic reaction for N. tabacum cell cultures.
Plant cells can conjugate sugar residues, not only to endogenous metabolic intermediates but also to xenobiotics [19]. Glycosylation reactions serve diverse functions in plants, i.e., increasing the solubility and stability of the aglycones, decreasing the toxicity of xenobiotics, and activating biosynthetic intermediates. The present biotransformation system using N. tabacum cell cultures would be useful for glycosylation of fluoroaromatic compounds. Furthermore, we have reported that immobilized plant cells of N. tabacum in sodium alginate gel glycosylated bisphenol A and benzophenone in higher yields than normal N. tabacum cell cultures [20], so immobilized N. tabacum cells could be applicable for phytoremediation of fluorophenols to be removed from contaminated water. The glycosylation of these compounds by immobilized N. tabacum cells will be reported in the near future.

General
The substrates, 2-, 3-, and 4-fluorophenols, were purchased from Sigma-Aldrich Co. The NMR spectra were recorded in CD 3 OD using a Varian XL-400 spectrometer ( 1 H at 400 MHz and 13 C at 100 MHz, respectively). The chemical shifts were expressed in δ (ppm) referring to tetramethylsilane. The FABMS spectra were measured using a JEOL MStation JMS-700 spectrometer. HPLC was carried out Biotransformation experiments were performed by addition of substrate (0.1 mmol/flask) into ten 300 mL conical flasks containing the suspension of cultured cells of N. tabacum and the cultures were incubated at 25°C for five days on a rotary shaker (120 rpm) in the dark. After incubation, the cells and medium were separated by filtration with suction. The filtered medium was extracted with ethylacetate. The medium was further extracted with n-butanol. The cells were homogenized and extracted with methanol. The glycoside products were detected in methanol fractions by HPLC analyses. The methanol fraction was concentrated and partitioned between water and ethylacetate. The ethylacetate fractions were combined and analyzed by the HPLC. The water fraction was applied to a Diaion HP-20 column and the column was washed with water followed by elution with methanol. The methanol eluate was subjected to preparative HPLC to give glycoside products.
Spectral data of new compounds are as follows.

Time-course experiment
Suspension cells of N. tabacum (50 g, fr. wt) was partitioned into each of eight 300 mL conical flasks containing Murashige and Skoog's medium (100 mL). Substrate (0.1 mmol) dissolved in ethanol was administered to each of conical flasks and the mixtures were incubated on a rotary shaker at 25 °C. At 12 h intervals, one of the flasks was taken out from the rotary shaker, and the cells and medium were separated by filtration. The extraction and analysis procedures were same as described in Section 3.2. The yield of the products was determined on the basis of the peak area from HPLC and expressed as a relative percentage to the total amount of the whole reaction products extracted.