Microbial Transformation of Prenylquercetins by Mucor hiemalis

Quercetin, one of the most widely distributed flavonoids, has been found to show various biological activities including antioxidant, anticancer, and anti-inflammatory effects. It has been reported that bioactivity enhancement of flavonoids has often been closely associated with nuclear prenylation, as shown in 8-prenylquercetin and 5′-prenylquercetin. It has also been revealed in many studies that the biological activities of flavonoids could be improved after glucosylation. Three prenylated quercetins were prepared in this study, and microbial transformation was carried out in order to identify derivatives of prenylquercetins with increased water solubility and improved bioavailability. The fungus M. hiemalis was proved to be capable of converting prenylquercetins into more polar metabolites and was selected for preparative fermentation. Six novel glucosylated metabolites were obtained and their chemical structures were elucidated by NMR and mass spectrometric analyses. All the microbial metabolites showed improvement in water solubility.


Introduction
Flavonoids, a large group of unique compounds that are widely distributed throughout the plant kingdom [1], have been reported to perform an essential role in some physiological courses and exhibited a variety of promising pharmaceutical properties for human health [2,3]. The activities of flavonoids are commonly associated with their antioxidant properties and capabilities of modulating the particular enzymes and cell receptors [4]. The antioxidant activity of Alpinia plants (Zingiberaceae family) has been reported to be influenced by the presence of flavonoids [5,6].
Quercetin, one of the active flavonoid aglycones of Alpinia plants, is distributed in various vegetables and fruits [6,7]. It exhibits high and diverse biological activities such as anticancer, antibacterial, anti-inflammatory, and gastroprotective effects [7]. Prenylation increases the lipophilicity of flavonoids, which would increase their uptake through a higher affinity to semi-permeable membranes and thus improve the rate of absorption and bioavailability [8,9]. For example,  showed higher cellular uptake and lower efflux than quercetin in Caco-2 and C2C12 myotube cells and enhanced the bioavailability of quercetin in different tissues in vivo [10]. 5 -Prenylquercetin (uralenol) exhibited higher inhibitory effects against tyrosinase, PTP1B and α-glucosidase enzymes, and the growth of hepatic stellate cells compared with those of quercetin [11][12][13]. The notion that the biological activities of quercetin were enhanced by prenylation led to the synthesis of 8-PQ (1) and other derivatives.
Although the biological properties of flavonoids are enhanced after prenylation, their potential applications are still limited by their poor solubility in aqueous solvents [14][15][16]. It has been demonstrated that glucosylation could significantly increase the water solubility of flavonoids and enhance their stability and bioavailability [17,18]. For instance, the quercetin glucosides were A total of 11 microbial cultures were screened for their ability to metabolize prenylquercetins 1-3, and the fungus M. hiemalis KCTC 26779 was selected for the scale-up fermentation studies (Supplementary Table S1). Substrate and culture control studies were carried out during the screening procedures, confirming that the metabolites were produced as a result of enzymatic activity by the fungus instead of a consequence of chemical or non-metabolic conversion.
High-resolution electrospray ionization mass spectral (HRESIMS) analysis of 4 showed a quasi-molecular ion peak [M + Na] + at m/z 555.1479 (calcd for C 26 H 28 O 12 Na, 555.1478), which established the molecular formula of 4 as C 26 H 28 O 12 , indicating that it was a glycosylated derivative of 1. Presence of the sugar moiety was also confirmed by the additional proton and carbon signals (δ H 5.00 and 3.16-3.71 (6H); δ C 100.9, 77.6, 77.1, 73.8, 70.1, 61.1) observed in the 1 H and 13 C NMR data of 4. The sugar was assigned to be a glucopyranose on the basis of NMR data and by comparison of the TLC R f value with that of the d-glucose reference standard after acidic hydrolysis of 4. The sugar was determined to be in β-configuration by the large coupling constant of 7.5 Hz of the anomeric proton signal at δ H 5.00. The significant downfield shift of the aromatic proton signal at δ H 6.60 (H-6) suggested that this sugar was attached to C-7 through an ether linkage. These spectral data were very similar to those of quercetin 7-O-β-d-glucopyranoside except for the appearance of the 8-prenyl group [32]. Thus, metabolite 4 was assigned as 8-prenylquercetin 7-O-β-d-glucopyranoside.
Compound 5 13 C NMR data of 5. The sugar was assigned to be a glucopyranose on the basis of NMR data and by comparison of the TLC R f value with that of the d-glucose reference standard after acidic hydrolysis of 5. The sugar was determined to be in β-configuration by the large coupling constant of 7.2 Hz of the anomeric proton signal at δ H 5.48. The upfield shift of the aromatic proton signal at δ H 7.64 (H-2 ) suggested that this sugar should be attached to C-3 through an ether linkage. This was confirmed by the downfield chemical shift of C-2 at δ C 156.5 compared to 1 and the HMBC correlation between H-1 and C-3 (Scheme 2). These spectral data were very similar to those of quercetin 3-O-β-d-glucopyranoside except for the appearance of the 8-prenyl group [32]. Thus, metabolite 5 was assigned as 8-prenylquercetin 3-O-β-d-glucopyranoside. Presence of the sugar moiety was confirmed by the additional proton and carbon signals (δ H 5.02 and 3.19-3.68 (6H); δ C 100.3, 77.5, 76.8, 73.5, 69.9, 61.0) observed in the 1 H and 13 C NMR of 6. The sugar was assigned to be a glucopyranose on the basis of NMR data and by comparison of the TLC R f value with that of the d-glucose reference standard after acidic hydrolysis of 6. The sugar was determined to be in β-configuration by the large coupling constant of 7.3 Hz of the anomeric proton signal at δ H 5.02. The downfield shift of the aromatic proton signal at δ H 6.65 (H-8) and 6.43 (H-6) suggested that this sugar should be attached to C-7 through an ether linkage. These spectral data were very similar to those of quercetin 7-O-β-d-glucopyranoside except for the appearance of the 6 -prenyl group [32]. Thus, metabolite 6 was assigned as 6 -prenylquercetin 7-O-β-d-glucopyranoside.
Compound 7 showed the [M + Na] + peak at m/z 555.1477 (calcd for C 26 H 28 O 12 Na, 555.1478), which established a molecular formula of C 26 H 28 O 12 , indicating that it was also a glycosylated derivative of 2. Presence of the sugar moiety was confirmed by the additional proton and carbon signals (δ H 4.72 and 3.19-3.66 (6H); δ C 102.2, 77.7, 76.5, 73.7, 70.1, 61.0) observed in the 1 H and 13 C NMR of 7. The sugar was assigned to be glucopyranose on the basis of NMR data and by comparison of the TLC R f value with that of the d-glucose reference standard after acidic hydrolysis of 7. The sugar was determined to be in β-configuration by the large coupling constant of 7.3 Hz of the anomeric proton signal at δ H 4.72. The downfield shift of the aromatic proton signal at δ H 6.99 (H-5 ) suggested that this sugar should be attached to C-4 through an ether linkage. It was further confirmed by HMBC correlation between H-1 and C-4 (Scheme 3). These spectral data were very similar to those of quercetin 4 -O-β-d-glucopyranoside except for the appearance of the 6 -prenyl group [32]. Thus, metabolite 7 was assigned as 6 -prenylquercetin 4 -O-β-d-glucopyranoside.  Compound 9 showed the [M + Na] + peak at m/z 623.2108 (calcd for C 31 H 36 O 12 Na 623.2104), which established a molecular formula of C 31 H 36 O 12 , indicating that it was a glycosylated derivative of 3. The presence of the sugar moiety was also confirmed by the additional proton and carbon signals (δ H 5.00 and 3.19-3.75 (6H); δ C 101.1, 77.6, 77.0, 73.8, 70.1, 61.1) observed in the 1 H and 13 C NMR data of 9. The sugar was assigned to be glucopyranose on the basis of NMR data and by comparison of the TLC R f value with that of the d-glucose reference standard after acidic hydrolysis of 9. The sugar was identified as β-d-glucose by the large coupling constant of 7.3 Hz of the anomeric proton signal at δ H 5.00. The downfield shift of the aromatic proton signal at δ H 6.63 (H-6) suggested that this sugar should be attached to C-7 through an ether linkage. It was further confirmed by the HMBC correlation between H-1 and C-7 (Scheme 4). Thus, the structure of metabolite 9 was assigned as 8,6 -diprenylquercetin 7-O-β-d-glucopyranoside.

Water Solubility of Prenylquercetins and Their Metabolites
The water solubility of prenylquercetins and their metabolites was examined and summarized in Supplementary Table S2. It was observed that 6 -prenylation improved its water solubility (quercetin: 0.035 mg/mL; 2: 0.063 mg/mL). It was established that glucosylation could significantly enhance the water solubility of prenylquercetins by comparing the solubility of compounds 1-3 with their glucosides. Solubility was affected not only by the position that glucose was attached to, but also by the number of glucose units. In fact, metabolite 8 containing two glucose units showed higher solubility than metabolite 9, which contains one glucose unit.

General Experimental Procedures
The 1 H and 13 C NMR spectra were obtained in DMSO-d 6 on a Bruker Avance III HD 400 spectrometer at 400 and 100 MHz, respectively, using TMS as internal standard. The chemical shift values (δ) are reported in ppm units, and the coupling constants (J) are in Hertz (Hz). UV spectra were recorded on a Jasco V-530 spectrophotometer (Jasco, Tokyo, Japan). HRESIMS was performed on a Waters Synapt G2 QTOF mass spectrometer (Waters, Milford, MA, USA). TLC analyses were carried out on precoated silica gel 60 F 254 plates (Merck, Darmstadt, Germany). The developing system used was CHCl 3 :MeOH solution, and visualization of TLC plates was performed under UV light (254 and 365 nm) or performed with anisaldehyde-H 2 SO 4 spray reagent. The adsorbent used for open column chromatography was silica gel 60-230 mesh. HPLC was performed on a Waters 1525 Binary HPLC pump (Waters Corp., Milford, MA, USA) connected to a Waters 996 Photodiode Array detector using Zorbax RX-C18 (21.2 × 250 mm) and Waters Symmetry C18 (4.6 × 150 mm) columns with HPLC-grade methanol and water.

Chemicals and Ingredients
Quercetin was purchased from Alfa Aesar (Haverhill, MA, USA). All of the ingredients used for microbial culture media, including dextrose, peptone, malt extract, and potato dextrose broth, were purchased from Becton, Dickinson and Co (Detroit, MI, USA). Prenylquercetins 1-3 were semi-synthesized by nuclear prenylation of quercetin, which was similar to the method used for the preparation of prenylapigenins [29]. Briefly, to a stirred solution of quercetin (3.99 g, 13.2 mmol) in dry dioxane, BF 3 -etherate (3.52 mL, 27.8 mmol, 2.1 eq.) was gradually added at room temperature for 30 min. Then a solution of 2-methyl-3-buten-2-ol (2.77 mL, 26.5 mmol, 2.0 eq.) in dry dioxane was added, and the resulting solution was stirred for another 8 h. After keeping at room temperature overnight, diethyl ether was added and then washed with water (three times). The remaining ethereal layer was evaporated to dryness to give an orange-red gum containing PQ. The reaction mixture was subjected to silica gel chromatography eluting with n-hexane-EtOAc gradient mixture (10:1→1:1) to obtain crude prenylquercetins. Then the crude material was purified by HPLC eluting with MeOH:H 2 O at a flow rate of 4.0 mL/min to obtain 8-PQ and related derivatives.

Procedure for Microbial Transformation
Microbial transformation studies were carried out according to the standard two-stage procedure [33]. Briefly, the actively growing microbial cultures were incubated in 250 mL flasks containing 50 mL of media and incubated with gentle agitation (200 rpm) at 25 • C in a temperature-controlled shaking incubator. The dimethyl sulfoxide solution (10 mg/mL) of each substrate was added to each flask at a concentration of 16 µg/mL 24 h after inoculation and incubated further under the same condition for 7 days. General sampling and TLC monitoring were performed on precoated TLC plates (CHCl 3 :MeOH) at an interval of 24 h. UV (254 and 365 nm) and anisaldehyde-H 2 SO 4 reagent were used for the identification of metabolites. Culture controls consisted of fermentation cultures in which the microbes were grown without addition of substrates. Substrate controls consisted of substrates added to sterile culture media with no microbes added.

Scale-up Fermentation and Isolation of Metabolites
Preparative-scale fermentation was carried out under the same condition with five 1 L flasks each containing 200 mL medium and 15 mg 8-PQ, 14 mg 6 -PQ, and 11 mg 8,6 -DPQ for 5 days, respectively. The cultures were extracted with EtOAc three times, and the organic layers were combined and concentrated under reduced pressure. The EtOAc extract (270 mg) of 8-PQ (1) culture broth was subjected to reversed-phase HPLC using a gradient solvent system of MeOH:H 2 O (40:60 → 60:40) eluting at a flow rate of 4 mL/min to obtain compounds 4 (6.95 mg, t R 47.4 min) and 5 (45.37 mg, t R 67.9 min). The EtOAc extract (396 mg) of 6 -PQ (2) culture broth was subjected to reversed-phase HPLC using the same elution condition as above to obtain compounds 6 (5.89 mg, t R 42.9 min) and 7 (15.72 mg, t R 56.2 min). Likewise, the EtOAc extract (360 mg) of 8,6 -DPQ (3) culture broth was subjected to reversed-phase HPLC to obtain compounds 8 (6.42 mg, t R 40.3 min) and 9 (19.16 mg, t R 58.6 min).

Acid Hydrolysis
Solutions of compounds 4-9 (1 mg each) in 10% HCl were heated at 80 • C for 2 h. After cooling, each mixture was neutralized and partitioned between EtOAc and H 2 O. The water layer was concentrated to dryness and developed by silica gel TLC (CHCl 3 :MeOH:H 2 O = 13:7:2) in comparison with authentic d-glucose [34].

Water Solubility Test
The solubility of prenylquercetins and their glucosides were determined based on the previously reported method with some modifications [35,36]. Briefly, each compound (4 mg) was dissolved in 1 mL of distilled water at room temperature followed by sonication for 10 min and centrifugation at 3600 rpm for 5 min at room temperature. Then the concentrations of the compounds in the supernatant were measured based on their peak areas utilizing calibration curves determined by the HPLC of authentic samples.

Conclusions
8-PQ (1), 6 -PQ (2), and 8,6 -DPQ (3) were synthesized by nuclear prenylation method using BF 3 ·Et 2 O as catalyst. Six new glucosylated metabolites were furnished during the biotransformation studies with Mucor hiemalis. This and our previous studies [29,30] have shown that glucosylation of the flavonoids biocatalyzed by M. hiemalis took place at the hydroxyl group of the molecules. Glucosylation, however, strongly depended on the position of the hydroxyl group in prenylated flavonoids. For instance, existence of a hydroxyl group at C-3 position, if not sterically hindered, could significantly increase the transformation efficiency. And it was also revealed that M. hiemalis selectively catalyzed glucosylation of the hydroxyl group on the prenyl-substituted aromatic ring. To the best of our knowledge, few investigations have been reported on the glucosylation of prenylflavonoids by M. hiemalis, and this result may provide a new and valuable pathway to acquire prenylflavonoid glucosides. All the glucosylated metabolites increased the solubility in water compared with their aglycones, and an increase in the number of glucose units enhanced the aqueous solubility. Thus, our results suggest that prenylquercetin glucosides appear to be useful, and it may be necessary to conduct further studies for the evaluation of their biological activities.