Shuai Ji‡
,
Wen-Fei Liang‡,
Zi-Wei Li,
Jin Feng,
Qi Wang,
Xue Qiao and
Min Ye*
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, China. E-mail: yemin@bjmu.edu.cn; Fax: +86 10 82802024; Tel: +86 10 82801516
First published on 15th February 2016
A highly regio- and stereo-specific biocatalytic reaction to produce the glucosides of natural prenylated phenolic compounds by Mucor hiemalis CGMCC 3.14114 is described. M. hiemalis could efficiently catalyze β-O-glucosylation of the isoprenyl-neighboring hydroxyl group, rather than other hydroxyls with low steric hindrance. This reaction was demonstrated by 28 compounds with different basic skeletons. The conversion rate was above 90% for most compounds. Scaled-up biotransformations of 15 substrates yielded 17 products, including 12 new compounds. These products showed enhanced water solubility and improved intestinal absorption in a Caco-2 cell monolayer model. This reaction provides a facile and efficient approach to synthesize the glucosides of prenylated phenolics, which are rare in natural products.
Glycosylation is an effective approach to increase water solubility of natural products, as well as to improve their pharmacological properties.10 Although a number of chemical glycosylation tactics have been established, the glycosylation of vulnerable phenolic compounds with multiple functional groups is still a big challenge.11 Particularly, the reaction yield and selectivity are generally far from ideal.12 Thus far, few reports are available on the glycosylation of prenylated phenolic compounds, possibly due to steric hindrance of the isoprenyl group. Biocatalysis provides an alternative approach for structural modification of natural products. A number of microbial strains have been reported to catalyze stereo- and regio-selective glycosylation reactions under mild conditions.13
In this work, we report highly efficient and selective glucosylation of prenylated phenolic compounds by the filamentous fungus Mucor hiemalis CGMCC 3.14114. M. hiemalis could specifically catalyze β-O-glucosylation of ortho-OH of the isoprenyl group with conversion rates of above 90% to obtain a series of new compounds. Moreover, water solubility, bioavailability, and bioactivities of the biotransformed products were tested.
HPLC-DAD-ESI-MSn analysis was performed on an Agilent 1100 instrument coupled with a Finnigan LCQ advantage ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Samples were separated on an Eclipse XDB-C18 column (4.6 × 250 mm, 5 μm, Agilent, USA) protected with a Zorbax Extend-C18 guard column (4.6 × 12.5 mm, 5 μm). The column temperature was 30 °C. The mobile phase consisted of acetonitrile (A) and water containing 0.1% (v/v) formic acid (B). A linear gradient elution program was used: 0 min, 19% A; 8 min, 28% A; 16 min, 28% A; 17 min, 35% A; 21 min, 50% A; 26 min, 50% A; 38 min, 75% A; 48 min, 95% A; 53 min, 95% A. The flow rate was 1.0 mL min−1. UV spectra were obtained by scanning from 200 to 400 nm. For MS analysis, the effluent was introduced into the ESI source of mass spectrometer at 0.2 mL min−1 via a T-union splitter. The mass spectrometer was operated in the (−)-ESI mode.
Analytical grade reagents were purchased from Beijing Chemical Corporation (Beijing, China). For HPLC and LC/MS analysis, HPLC grade methanol, acetonitrile and formic acid (Mallinkrodt, Phillipsburg, NJ, USA) were used. High-purity nitrogen (99.9%) and helium (99.99%) were from Gas Supple Center of Peking University Health Science Center (Beijing, China). Standard samples of D-glucose, D-galactose, and β-glucosidase were from Sigma-Aldrich (Shanghai, China).
No. | 1aa | 7a | 9a | 10aa | ||||
---|---|---|---|---|---|---|---|---|
δH (J in Hz) | δC, type | δH (J in Hz) | δC, type | δH (J in Hz) | δC, type | δH (J in Hz) | δC, type | |
a Data were recorded at 600 MHz for 1H NMR and 150 MHz for 13C NMR. | ||||||||
2 | 159.9, C | 8.48, s | 154.7, CH | 8.16, s | 155.2, CH | 8.38, s | 154.5, CH | |
3 | 122.0, C | 122.0, C | 119.6, C | 121.9, C | ||||
4 | 7.87, s | 135.9, CH | 180.9, C | 179.9, C | 180.0, C | |||
5 | 154.7, C | 159.8, C | 161.9, C | 162.0, C | ||||
6 | 120.1, C | 6.62, s | 98.2, CH | 6.23, d (1.8) | 99.0, CH | 6.23, d (2.0) | 99.1, CH | |
7 | 158.3, C | 160.5, C | 164.3, C | 164.4, C | ||||
8 | 6.95, s | 98.4, CH | 108.2, C | 6.40, d (1.8) | 93.8, CH | 6.40, d (2.0) | 93.7, CH | |
9 | 152.8, C | 154.1, C | 157.6, C | 157.5, C | ||||
10 | 108.1, C | 105.9, C | 104.3, C | 104.4, C | ||||
1′ | 113.2, C | 121.1, C | 107.1, C | 122.6, C | ||||
2′ | 156.1, C | 7.40, d (8.4) | 130.2, CH | 151.1, C | 7.01 d (0.8) | 121.8, CH | ||
3′ | 6.38, d (2.4) | 102.7, CH | 6.82, d (8.4) | 115.1, CH | 112.6, C | 145.2, C | ||
4′ | 158.6, C | 157.5, C | 153.1, C | 142.0, C | ||||
5′ | 6.28, dd (8.4, 2.4) | 106.3, CH | 6.82, d (8.4) | 115.1, CH | 6.72, d (8.4) | 111.1, CH | 120.6, C | |
6′ | 7.14, d (8.4) | 131.6, CH | 7.40, d (8.4) | 130.2, CH | 7.05, d (8.4) | 131.3, CH | 7.15 d (0.8) | 121.7, CH |
1′′ | 3.46, m; 3.27, m | 22.5, CH2 | 3.58, m; 3.30, br s | 21.3, CH2 | 6.79, d (9.6) | 116.8, CH | 6.42, d (10.0) | 117.9, CH |
2′′ | 5.21, t (7.2) | 122.6, CH | 5.17, br s | 122.2, CH | 5.70, d (9.6) | 129.6, CH | 5.79, d (10.0) | 131.5, CH |
3′′ | 130.8, C | 131.2, C | 76.0, C | 76.4, C | ||||
4′′ | 1.63, s | 25.5, CH3 | 1.62, s | 25.5, CH3 | 1.30, s | 27.6, CH3 | 1.41, s | 27.6, CH3 |
5′′ | 1.75, s | 17.8, CH3 | 1.78, s | 17.8, CH3 | 1.30, s | 27.6, CH3 | 1.40, s | 27.3, CH3 |
1′′′ | 4.98, d (7.8) | 100.7, CH | 5.00, d (6.4) | 100.5, CH | 4.91, d (7.2) | 100.9, CH | 4.83, d (7.2) | 100.7, CH |
2′′′ | 3.31, d (6.0) | 73.4, CH | 3.30, br s | 73.4, CH | 3.25, m | 73.3, CH | 3.28, d (5.6) | 73.2, CH |
3′′′ | 3.31, d (6.0) | 76.8, CH | 3.30, br s | 76.6, CH | 3.25, m | 76.9, CH | 3.28, d (5.6) | 76.5, CH |
4′′′ | 3.18, br s | 69.8, CH | 3.16, d (4.8) | 69.7, CH | 3.16, br s | 69.8, CH | 3.18, br s | 69.7, CH |
5′′′ | 3.46, m | 77.2, CH | 3.41, m | 77.2, CH | 3.27, m | 77.1, CH | 3.33, m | 77.1, CH |
6′′′ | 3.75, d (10.2); 3.47, m | 60.8, CH2 | 3.71, m; 3.44, m | 60.6, CH2 | 3.68, d (10.8); 3.45, m | 60.7, CH2 | 3.69, d (11.6); 3.50, m | 60.7, CH2 |
OCH3 | 3.79, s | 63.0, CH3 |
No. | 4a | 19a | 22a | 24a | ||||
---|---|---|---|---|---|---|---|---|
δH (J in Hz) | δC, type | δH (J in Hz) | δC, type | δH (J in Hz) | δC, type | δH (J in Hz) | δC, type | |
2 | 5.46, s | 64.2, CH2 | 147.4, C | 155.4, C | 5.45, m | 79.2, CH | ||
3 | 107.4, C | 136.0, C | 133.3, C | 3.16, m; 2.64, br d (16.0) | 43.2, CH2 | |||
4 | 144.2, C | 176.1, C | 177.8, C | 190.6, C | ||||
5 | 152.9, C | 159.4, C | 158.5, C | 7.48, s | 126.1, CH | |||
6 | 117.6, C | 111.8, C | 6.30, s | 98.3, CH | 124.1, C | |||
7 | 156.3, C | 160.6, C | 161.7, C | 161.4, C | ||||
8 | 6.57, s | 100.1, CH | 6.87, s | 93.1, CH | 107.0, C | 6.68, s | 102.4, CH | |
9 | 152.7, C | 154.0, C | 153.6, C | 161.2, C | ||||
10 | 104.9, C | 104.4, C | 104.0, C | 114.6, C | ||||
1′ | 116.9, C | 122.2, C | 122.8, C | 129.1, C | ||||
2′ | 155.5, C | 8.07, d (8.8) | 129.6, CH | 8.23, d (9.2) | 130.7, CH | 7.33, d (8.4) | 128.4, CH | |
3′ | 7.00, d (2.0) | 98.0, CH | 6.94, d (8.8) | 115.5, CH | 7.06, d (9.2) | 113.7, CH | 6.79, d (8.4) | 115.1, CH |
4′ | 156.1, C | 156.8, C | 161.2, C | 157.7, C | ||||
5′ | 6.77, dd; (8.4, 2.0) | 112.4, CH | 6.94, d (8.8) | 115.5, CH | 7.06, d (9.2) | 113.7, CH | 6.79, d (8.4) | 115.1, CH |
6′ | 7.34, d (8.4) | 119.2, CH | 8.07, d (8.8) | 129.6, CH | 8.23, d (9.2) | 130.7, CH | 7.33, d (8.4) | 128.4, CH |
1′′ | 3.44, m; 3.25, m | 22.2, CH2 | 3.44, m; 3.20, m | 21.2, CH2 | 2.75, t (8.4) | 17.4, CH2 | 3.27, m | 27.2, CH2 |
2′′ | 5.21, t (6.4) | 123.5, CH | 5.23, t (7.2) | 121.6, CH | 1.56, t (8.4) | 42.9, CH2 | 5.32, m | 122.2, CH |
3′′ | 129.9, C | 130.7, C | 68.8, C | 132.1, C | ||||
4′′ | 1.63, s | 25.5, CH3 | 1.62, s | 25.5, CH3 | 1.17, s | 29.1, CH3 | 1.67, s | 25.6, CH3 |
5′′ | 1.75, s | 17.7, CH3 | 1.75, s | 17.8, CH3 | 1.17, s | 29.1, CH3 | 1.71, s | 17.6, CH3 |
1′′′ | 4.80, d (7.2) | 101.0, CH | 5.04, d (6.8) | 100.4, CH | 5.50, d (7.2) | 101.0, CH | 4.95, m | 100.1, CH |
2′′′ | 3.28, m | 73.4, CH | 3.31, m | 73.4, CH | 3.22, m | 74.2, CH | 3.27, m | 73.3, CH |
3′′′ | 3.27, m | 76.7, CH | 3.30, m | 76.8, CH | 3.22, m | 76.4, CH | 3.27, m | 76.5, CH |
4′′′ | 3.14, m | 69.7, CH | 3.17, m | 69.7, CH | 3.10, br s | 69.9, CH | 3.12, br s | 69.6, CH |
5′′′ | 3.37, m | 77.1, CH | 3.47, m | 77.2, CH | 3.10, br s | 77.5, CH | 3.36, m | 77.0, CH |
6′′′ | 3.71, d (9.6); 3.45, m | 61.7, CH2 | 3.73, m; 3.47, d (7.6) | 60.7, CH2 | 3.57, m; 3.35, br s | 60.8, CH2 | 3.67, m; 3.43, m | 60.6, CH2 |
OCH3 | 3.76, s | 58.1, CH3 | 3.79, s | 59.7, CH3 | 3.85, s | 55.4, CH3 |
No. | 8a | 16a | 16b | 16c | ||||
---|---|---|---|---|---|---|---|---|
δH (J in Hz) | δC, type | δH (J in Hz) | δC, type | δH (J in Hz) | δC, type | δH (J in Hz) | δC, type | |
2 | 8.23, s | 156.2, CH | 4.10, br d (10.0); 3.92, t (10.0) | 69.1, CH2 | 4.11, br d (10.4); 3.88, t (10.4) | 69.4, CH2 | 4.23, br d (10.4); 3.73, t (10.4) | 69.6, CH2 |
3 | 121.2, C | 3.32, br s | 30.5, CH | 3.35, br s | 30.4, CH | 3.63, br s | 29.7, CH | |
4 | 181.4, C | 2.79, dd (15.6, 4.4); 2.63, m | 26.2, CH2 | 2.80, m; 2.67, m | 26.1, CH2 | 2.60, m | 27.4, CH2 | |
5 | 157.9, C | 156.8, C | 156.5, C | 156.8, C | ||||
6 | 112.6, C | 113.1, C | 115.4, C | 112.9, C | ||||
7 | 160.6, C | 154.4, C | 154.9, C | 154.9, C | ||||
8 | 6.79, s | 93.2, CH | 6.10, s | 98.7, CH | 6.39, s | 99.3, CH | 6.09, s | 98.7, CH |
9 | 155.5, C | 152.5, C | 152.9, C | 152.8, C | ||||
10 | 106.0, C | 107.0, C | 109.8, C | 107.0, C | ||||
1′ | 109.4, C | 124.2, C | 119.5, C | 121.4, C | ||||
2′ | 154.0, C | 154.9, C | 154.5, C | 154.3, C | ||||
3′ | 115.3, C | 118.7, C | 116.0, C | 126.5, C | ||||
4′ | 156.4, C | 152.9, C | 153.1, C | 153.0, C | ||||
5′ | 6.38, d (8.4) | 106.6, CH | 6.62, d (8.4) | 106.7, CH | 6.34, d (8.4) | 107.2, CH | 6.62, d (8.4) | 111.7, CH |
6′ | 6.75, d (8.4) | 128.7, CH | 6.87, d (8.4) | 123.4, CH | 6.74, d (8.4) | 124.0, CH | 6.87, d (8.4) | 124.6, CH |
1′′ | 3.46, m; 3.24 m | 21.3, CH2 | 3.14, m | 22.3, CH2 | 3.18, d (6.4) | 22.4, CH2 | 3.15, m | 22.2, CH2 |
2′′ | 5.20, m | 122.1, CH | 5.13, t (6.8) | 122.9, CH | 5.15, m | 123.6, CH | 5.13, t (7.2) | 124.0, CH |
3′′ | 130.8, C | 129.2, C | 129.3, C | 129.1, C | ||||
4′′ | 1.61, s | 25.6, CH3 | 1.61, s | 25.5, CH3 | 1.61, s | 25.5, CH3 | 1.62, s | 25.4, CH3 |
5′′ | 1.71, s | 18.7, CH3 | 1.69, s | 17.7, CH3 | 1.71, s | 17.7, CH3 | 1.69, s | 17.6, CH3 |
1′′′ | 3.24, m | 22.4, CH2 | 3.52, m; 3.30, br s | 22.6, CH2 | 3.27, d (6.4) | 22.5, CH2 | 3.52, m; 3.30, br s | 23.0, CH2 |
2′′′ | 5.20, m | 123.5, CH | 5.18, t (6.8) | 124.2, CH | 5.19, m | 124.2, CH | 5.18, t (7.2) | 124.3, CH |
3′′′ | 129.5, C | 129.7, C | 129.6, C | 129.5, C | ||||
4′′′ | 1.61, s | 25.6, CH3 | 1.61, s | 25.6, CH3 | 1.61, s | 25.5, CH3 | 1.62, s | 25.5, CH3 |
5′′′ | 1.74, s | 17.3, CH3 | 1.73, s | 17.9, CH3 | 1.71, s | 17.8, CH3 | 1.71, s | 18.0, CH3 |
1′′′′ | 5.03, d (6.4) | 100.4, CH | 4.71, d (7.6) | 101.3, CH | 4.71, d (6.8) | 101.3, CH | 4.46, d (7.6) | 104.7, CH |
2′′′′ | 3.26, m | 73.4, CH | 3.24, m | 73.5, CH | 3.17, br s | 73.4, CH | 3.22, m | 73.8, CH |
3′′′′ | 3.26, m | 76.7, CH | 3.24, m | 77.0, CH | 3.17, br s | 76.9, CH | 3.13, m | 76.3, CH |
4′′′′ | 3.17, m | 69.7, CH | 3.14, m | 69.8, CH | 3.14 m | 69.8, CH | 2.99, m | 70.1, CH |
5′′′′ | 3.46, m | 77.3, CH | 3.32, br s | 77.0, CH | 3.26, m | 77.1, CH | 2.97, m | 77.4, CH |
6′′′′ | 3.72, m; 3.44, m | 60.7, CH2 | 3.69, br d (11.2); 3.46, m | 60.0, CH2 | 3.70, d (10.0); 3.43, m | 60.1, CH2 | 3.72, m; 3.35, br s | 61.4, CH2 |
OCH3 | 3.60, s | 60.8, CH3 | 3.63, s | 60.8, CH3 | 3.57, s | 59.9, CH3 |
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Fig. 1 Glucosylation of glycycoumarin (1) by Mucor hiemalis. (A) The biocatalytic reaction. (B) HPLC chromatograms of 1 and its biotransformed product 1a. (C) (−)-ESI-MS and MS/MS spectra for 1a. |
A scaled-up biotransformation of 1 in M. hiemalis for three days was conducted, and 1a was purified by column chromatography and semi-preparative liquid chromatography. The HRESIMS spectrum established the molecular formula of 1a as C27H30O11 ([M + H]+ m/z 531.1856, calcd for C27H31O11, 531.1861). The 1H and 13C NMR spectra showed additional resonances corresponding to a glucosyl residue (δH 4.98, d, J = 7.8 Hz; δC 100.7, 77.2, 76.8, 73.4, 69.8 and 60.8). The δH 4.98 and δC 100.7 signals could be assigned to the anomeric H-1′′′ and C-1′′′, respectively, according to the HSQC spectrum. H-1′′′ showed a long-range correlation with δC 158.3 (C-7), indicating the glucosyl residue was linked to 7-OH (instead of 2′- or 4′-OH) of 1. In accordance, the resonance for C-7 shifted upfield by 1.8 ppm, whereas C-6 and C-10 shifted downfield by 1.7 and 1.8 ppm, respectively, when compared to 1 (Table 1). The coupling constant of H-1′′′ (7.8 Hz) suggested β-configuration of the glycosidic bond. To further confirm the glucosyl residue, 1a was subjected to acid hydrolysis, and then analyzed by ion chromatography coupled with pulsed amperometric detection (IC-PAD).21 The hydrolysis product showed a peak at the same retention time as D-glucose (Fig. S2†). On the basis of the above deductions, 1a was identified as glycycoumarin 7-O-β-D-glucoside, which is a new compound.
Because all the above substrates contain more than one hydroxyl group, it is essential to determine the glycosylation position. Thus, we conducted scaled-up biotransformation of 15 substrates, and obtained 17 mono-O-glucosylated products. Twelve of them are new compounds, and their structures were fully identified by HRESIMS and NMR spectroscopic analysis. The glucosyl residue exhibited characteristic NMR resonances (the anomeric proton signal at δH 4.46–5.50; the anomeric carbon signal at δC 100.1–104.7; one CH2 signal at δC 60.0–61.7; and four CH signals at δC 69.6–77.5). The coupling constants of the anomeric protons (J = 6.8–8.4 Hz) suggested β-glycosidic linkage for all the products. The glucosyl residue for 16a and 22a was further confirmed by IC-PAD analysis after acid hydrolysis (Fig. S38†). The linkage site for glucosyl residue was determined by HBMC correlations. 13C and 1H NMR spectral data for the new compounds are given in Tables 1–3.
Here we describe structural identification of 16a–16c as typical examples. The scaled-up biotransformation of 16 by M. hiemalis yielded three glycosylated products, namely 16a–16c. They had the same molecular formula C32H42O10, according to their HRESIMS spectra. In the MS/MS spectra, all the [M − H]− ions (m/z 585) could lose 162 Da to yield m/z 423 (Fig. S18†), suggesting they were mono-O-glucosides. In the HMBC spectra, the anomeric proton H-1′′′ showed a long-range correlation with δC 154.9 (C-2′, 16a), δC 154.9 (C-7, 16b), and δC 153.0 (C-4′, 16c), respectively, indicating the glucosyl residue was linked to 2′-OH of 16a, 7-OH of 16b, and 4′-OH of 16c. In accordance, the ortho-carbons resonated at remarkably low fields when compared to 16. For instance, C-1′ (δC 124.2) and C-3′ (δC 118.7) of 16a shifted down-field by 4.5 and 2.8 ppm, respectively (Table S4†). Based on the above deductions, the structures for 16a–16c were identified as licoricidin 2′-O-β-D-glucoside, licoricidin 7-O-β-D-glucoside, and licoricidin 4′-O-β-D-glucoside, respectively. All of them are new compounds.
Through the above structural elucidations, we found that all the prenylated phenolic compounds could be efficiently glucosylated at the isoprenyl-neighboring hydroxyl group (Fig. 2). This rule applied when the isoprenyl group was located at C-6 (1, 4–6, 18, 19, 24) or C-8 (7, 20, 26) (please be noted that carbon numbering for chalcones is different from the other subclasses of flavonoids). For both groups of compounds (C-6 or C-8), 7-O-glucoside was the only or predominant product, though other free hydroxyl groups are also present in the substrate structures. It was important to note that compounds 5, 6, 18 and 19 contained two isoprenyl-neighboring hydroxyl groups (5-OH and 7-OH) in their structures, and all of them were only glucosylated at 7-OH. This was probably due to the intramolecular hydrogen bonding between 5-OH and the carbonyl group at C-4. Compound 16 contains two isoprenyl groups at C-6 and C-3′, and yielded three glucosides at 7-OH, 2′-OH, and 4′-OH. Compound 8 also contains two isoprenyl groups, and yielded 7-O-glucoside as the predominant product. The three minor products could only be observed by LC-MS analysis (Fig. S10†). The above evidences indicated that glucosylation occurred more readily at 7-OH than at 2′- or 4′-OH. Aside from the normal isoprenyl (γ,γ-dimethylallyl) chain, a cyclized isoprenyl group (such as a 2,2-dimethylbenzopyran ring) could also facilitate the glucosylation reaction, as exemplified by allolicoisoflavone B (9) and semilicoisoflavone (10). Licoarylcoumarin (2), licochalcone A (27) and rhodomyrtone (28) contain varied isoprenyl groups (α,α-dimethylallyl- or 3-methyl-1-oxo-butyl-). All of them could be efficiently glucosylated with conversion rates of above 95%. The two isoprenyl chains of 6,8-diprenylgenistein (13) did not hinder the glucosylation reaction. In fact, it was completely metabolized into one single product, which was expected to be the 7-O-glucoside. Puerarol (3) contains a C10 monoterpene group (two isoprenyl units). It could also be metabolized into O-glucosides, though the conversion rate decreased to 30%. Compound 22 contains a hydroxylated isoprenyl group (3-hydroxy-3-methyl-butyl-). Although it could also be efficiently glycosylated, the product was identified as 3-O-glucoside. This special isoprenyl group may suppress 7-O-glucosylation.
We further tested seven other phenolic compounds with no isoprenyl groups (29–35). Only the two flavonols, kaempferol (29) and quercetin (30), could be glycosylated (Table S6, Fig. S31–S37†). Their products were obtained by scaled-up biotransformation. Interestingly, both products (29a and 30a) were 3-O-glucosides. This result indicated that M. hiemalis could catalyze the glucosylation of 3-OH of flavonols, when no isoprenyl group was present. In a previous study, M. hiemalis had also been reported to catalyze the glucosylation of normal phenolic compounds.25
Although prenylated phenolics are common in natural products, only a few of them are present as glycosides.26 Glycyrrhiza uralensis contains tens of prenylated flavonoids and flavonoid glycosides. However, no compounds reported from this plant, thus far, contain both an isoprenyl group and a sugar residue. The present work provides an efficient approach to prepare the glycosides of prenylated phenolic compounds, and the reaction took place specifically at the isoprenyl-neighboring hydroxyl group. Our further experiments demonstrated the glucosylation of 1 could also be catalyzed by extracted crude enzymes from M. hiemalis in Tris–HCl buffer, when uridine diphosphate glucose (UDP-G) was added. After optimization of incubating time, donor concentration, pH value, incubating temperature, activating factor, protease inhibitor, and incubating solvents, the conversion rate could be as high as 81.5% (Fig. S39†). A number of glycosyl transferases have been cloned from plants and microorganism, so far.27 However, few reports are available on glycosyl transferases from filamentous fungi. Molecular cloning of this type of enzymes from M. hiemalis is still under way in our laboratory.
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Fig. 3 The time-transport rate curves of 1 and 1a from the apical (AP) to basolateral (BL) side in the Caco-2 monolayer cell model. |
A lot of prenylated phenolic compounds show significant cytotoxic activities.1 Thus, we tested the cytotoxic activities of several substrates (5, 6, 8–10, 16, 18, 19, 24, 26), together with their glycosides, against HepG2 human hepatocellular carcinoma cell line at 10 μM using the MTS assay. For majority of the tested compounds, the cytotoxic activities decreased remarkably after being glycosylated (Fig. S40†). Another test indicated the glycosides exhibited comparable Nrf2 activation activities to the substrates (Fig. S41†).23 More bioactivity tests, including the hepatoprotective activities of 1a (so as to compare its activities with glycycoumarin), will be conducted in the near future.7
Footnotes |
† Electronic supplementary information (ESI) available: LC-MS analysis of the biotransformed products; cytotoxic and Nrf2 activation activities; spectroscopic data (NMR, MS, UV and IR) for new compounds. See DOI: 10.1039/c6ra00072j |
‡ These two authors contributed equally to this paper. |
This journal is © The Royal Society of Chemistry 2016 |