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 mono-fluorophenols and their glycosides in both medium and N. tabacum cells. On the basis of the spectroscopic analyses such as FABMS, ¹H- and ¹³C-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.

The FABMS spectrum of 3 included a pseudomolecular ion [M+Na]⁺ peak at m/z 459, indicating that the compound was composed of one molecule of the substrate 1 and two hexoses. The ¹H-NMR spectrum of 3 showed two anomeric proton signals at δ 4.36 (1H, d, J=7.6 Hz) and 4.96 (1H, d, J=7.6 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 β-primeverosides 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 4-fluorophenols 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.

The substrate 3-fluorophenol (4), was subjected to biotransformation with the same transformation system as used with 2-fluorophenol (1) (Scheme 2). Product 5 (17%) was detected in the methanol cell extracts. The structure of product 5 was determined to be 3-fluorophenyl β-glucoside by spectroscopic methods [16]. No further glycosylation products such as β-gentiobioside were obtained.

The time-course of the biotransformation of 3-fluorophenol (4) showed that the amount of product 5 increased with time during the reaction with N. tabacum cell cultures (Figure 2).

Incubation of N. tabacum cell cultures with 4-fluorophenol (6) for five days gave the biotransformation products 7 and 8 (Scheme 3). The structures of 7 and 8 were determined as 4-fluorophenyl β-glucoside (32%) [16] and 4-fluorophenyl β-gentiobioside (6%), respectively. The β-gentiobioside 8 was identified for the first time. The time-course in the biotransformation of 6 showed that the amounts of β-glucoside 7 and β-gentiobioside 8 increased with time (Figure 3).

The FABMS spectrum of 8 included an ion attributed to [M+Na]⁺ pseudomolecular ion (m/z 459), suggesting that 8 contained one substrate molecule 6 and two hexoses. Its ¹H- and ¹³C-NMR data were in good agreement with those of β-gentiobioside 3. The HMBC spectrum of 8 included correlations between the anomeric proton signal at δ 4.83 (H-1′) and the carbon signal at δ 155.1 (C-1) and between the anomeric proton signal at δ 4.38 (H-1″) and the carbon signal at δ 69.7 (C-6′), indicating that the inner glucopyranosyl residue was attached to the phenolic hydroxyl group of 4-fluorophenol (6) and that the pair of β-d-glucopyranosyl residues was 1,6-linked. The structure of 8 was determined to be 4-fluorophenyl β-gentiobioside.

The substrates, 2-, 3-, and 4-fluorophenols, were purchased from Sigma-Aldrich Co. The NMR spectra were recorded in CD₃OD using a Varian XL-400 spectrometer (¹H at 400 MHz and ¹³C 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 on a CrestPak C18S column (150 x 4.6 mm) [solvent: dioxane:H₂O (1:9, v/v); detection: UV (270 nm); flow rate: 1.0 mL/min]. Cultured suspension cells of N. tabacum were cultivated in 300 mL conical flasks containing Murashige and Skoog’s medium (100 mL, pH 5.7) and grown with continuous shaking on a rotary shaker (120 rpm) at 25 °C in the dark.

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.

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.