Enzymatic biosynthesis of novel neobavaisoflavone glucosides via Bacillus UDP-glycosyltransferase

Chinese Journal of Natural Medicines 2017, 15(4): 02810287

Chinese Journal of Natural Medicines

Enzymatic biosynthesis of novel neobavaisoflavone glucosides via Bacillus UDP-glycosyltransferase MA Tao 1, DAI Yi-Qun 1∆, LI Nan 1, HUO Qiang 1, LI Hong-Mei 1, ZHANG Yu-Xin 2, PIAO Zheng-Hao 3, WU Cheng-Zhu 1* 1

School of Pharmacy, Bengbu Medical College, Bengbu 233030, China; Department of Biochemistry, Bengbu Medical College, Bengbu 233030, China; 3 Department of Basic Medical Science, School of Medicine, Hangzhou Normal University, Hangzhou 321004, China 2

Available online 20 Apr., 2017

[ABSTRACT] The present study was designed to perform structural modifications of of neobavaisoflavone (NBIF), using an in vitro enzymatic glycosylation reaction, in order to improve its water-solubility. Two novel glucosides of NBIF were obtained from an enzymatic glycosylation by UDP-glycosyltransferase. The glycosylated products were elucidated by LC-MS, HR-ESI-MS, and NMR analysis. The HPLC peaks were integrated and the concentrations in sample solutions were calculated. The MTT assay was used to detect the cytotoxic activity of compounds in cancer cell lines. Based on the spectroscopic analyses, the two novel glucosides were identified as neobavaisoflavone-4′-O-β-D-glucopyranoside (1) and neobavaisoflavone-4′, 7-di-O-β-D-glucopyranoside (2). Additionally, the water-solubilities of compounds 1 and 2 were approximately 175.1- and 4 031.9-fold higher than that of the substrate, respectively. Among the test compounds, only NBIF exhibited weak cytotoxicity against four human cancer cell lines, with IC50 values ranging from 63.47 to 72.81 µmol·L−1. These results suggest that in vitro enzymatic glycosylation is a powerful approach to structural modification, improving water-solubility. [KEY WORDS] Neobavaisoflavone; Glycosylation; UDP-glycosyltransferase; Water-solubility; Cytotoxicity

[CLC Number] R917

[Document code] A

[Article ID] 2095-6975(2017)04-0281-7

Introduction Neobavaisoflavone (NBIF) is a member of the isoflavone subclass of flavonoids isolated from Psoraleae corylifolia and possesses various biological properties such as anticancer, anti-inflammatory, and antioxidant activities [1-4] (Fig. 1). Flavonoids are polyphenolic compounds that are ubiquitous in nature. While the potential biological activities of flavonoids have been recognized, the use of flavonoids as pharmaceutical agents has been limited because of their poor water [Received on] 26- Nov.-2016 [Research funding] This work was financially supported by National Natural Science Foundation of China (No. 81302671), the Education Department of Anhui Natural Science Research Project China (No. KJ2015A273) and Zhejiang Provincial Natural Science Foundation of China (No. LY13H10004). [*Corresponding author] Tel: 86-552-3175232, Fax: 86-552-3171261, E-mail: [email protected]. ∆ Co-first author These authors have no conflicts of interest to declare. Published by Elsevier B.V. All rights reserved

solubility and low bioavailability [5-6]. Commonly, natural products are converted to water-soluble compounds when modified with sugar moieties, displaying improved water-solubility, bioavailability, and pharmacological potency [7-9]. In addition, glycosylation is an important source of natural product diversification. In vitro enzymatic glycosylation is one of the common methods used for natural product glycosylation, which employs glycosyltransferase (GT) to transfer sugar moieties from NDP-sugar to an acceptor [10-11]. UDP-glycosyltransferase (YjiC) is a member of the GT1 family and capable of transferring different types of activated sugars such as UDP-glucose, UDP-galactose, UDP-2-deoxyglucose, and TDP-rhamnose, among others, to acceptor molecules [12-13]. Recently, YjiC has been used for enzymatic glycosylation of phenolic compounds such as apigenin [12], phloretin [14], isobavachalcone [15], 7-hydroxy-8-methoxy-flavone [16], resveratrol [13], geldanamycin analogs [17], anthraquinone [18], and polyketide macrolide [19]. Particularly, YjiC has been shown to have highly flexible non-regiospecific glycosylation ability towards different subclasses of flavonoids acceptors [12, 14-16, 20]. The present study

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was designed to carry out the in vitro glycosylation, structure elucidation, water-solubility determination, and cytotoxicity

testing of two novel NBIF glucosides produced by enzymatic biosynthesis using YjiC.

Fig. 1 Structures of compounds 1 and 2 and neobavaisoflavone (NBIF)

Materials and Methods General experimental procedures The mass of the glucosylated products were confirmed by liquid chromatography-mass spectrometry (LC-MS; LTQ-XL linear ion trap mass spectrophotometer) (Thermo Electron, Waltham, MA, USA) equipped with an electrospray ionization (ESI) source. The nuclear magnetic resonance (NMR) spectroscopic data were acquired on a Bruker Avance II 600 NMR spectrometer (Bruker, Billerica, MA, USA). The high-resolution (HR)-ESI-MS spectra were recorded on an Agilent 6538 Accurate Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Semi-preparative reversed-phase HPLC was carried out on 2535Q system (Waters, Milford, MA, USA). The YjiC enzyme expression vector (pET302-YjiC) was obtained from Prof. Jae Kyung Sohng of Sun Moon University (Asan, Chungcheongnam-do, South Korea). UDP-Glucose and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). NBIF was isolated from the fruit of P. corylifolia, and its structure was confirmed by comparing ESI-MS and 1H NMR data with that of the reference substance [1]. ESI-MS m/z [M + H]+ 323.3, [M − H]− 321.3; 1H NMR (600 MHz, methanol-d4): δ 8.05 (1H, s, H-2), 8.04 (1H, d, J = 8.7 Hz, H-5), 7.24 (1H, d, J = 2.1 Hz, H-2′), 7.14 (1H, dd, J = 8.7, 2.4 Hz, H-6′), 6.92 (1H, dd, J = 8.7, 2.4 Hz, H-6), 6.82 (1H, d, J = 2.1 Hz, H-8), 6.80 (1H, d, J = 8.7 Hz, H-5′), 5.34 (1H, m, H-2′′), 3.37 (2H, m, H-1′′), 1.72 (6H, s, H-4′′, 5′′). Enzymatic biosynthesis, purification, and identification of NBIF glucosides The methods for the expression and purification of YjiC in the present study were as described in our previous reports [15, 17]. The in vitro glycosylation reaction using YjiC

was performed in a total volume of 50 mL in reaction buffer (100 mmol·L−1 Tri-HCl, pH 8.0) containing 1 mmol·L−1 of MgCl2, 50 µg·mL−1 of YjiC enzyme, 3 mmol·L−1 of UDPGlucose, 3 mmol·L−1 of NBIF, and 10 mL of MeOH. After incubation at 28 ºC for 10 h, the reaction mixture was quenched by adding an equal volume of EtOAc and then mixed by vortexing. The EtOAc extracts were used for further analyses, including HPLC and LC-MS. Finally, the EtOAc extract was purified using a 2535Q semi-prep HPLC system (Waters) with a SunFireTM C18 column (250 mm × 10 mm, Waters) with 30% acetonitrile (CH3CN−H2O, 4 mL·min−1) to yield compounds 1 (15.2 mg) and 2 (2.1 mg). Determination of water solubility Excess compounds 1 and 2 and NBIF were mixed with 0.5 mL of distilled water in an Eppendorf tube at room temperature. After sonication at room temperature for 45 min and centrifugation at 12 000 r·min−1 for 10 min to remove insoluble material, the solution was subjected to HPLC analysis. The HPLC peaks were integrated and sample solution concentrations were calculated as previously described [9, 17]. MTT assay Human hepatocellular carcinoma (HepG2 and SMMC7721), breast cancer (MDA-MB-231), and colon adenocarcinoma (SW480) cell lines were purchased from the Shanghai Cell Line Bank (Shanghai, China). The cells were seeded into 96-well plates at a density of 8 000 cells/well for 1 day before the start of treatment. We treated the cells with various concentrations of the test compounds for 72 h, with geldanamycin as a positive control. The in vitro assay of cytotoxicity against the aforementioned four human cancer cell lines was evaluated using a standard tetrazolium-based colorimetric assay (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, MTT assay) [21].

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Results and Discussion In vitro enzymatic glycosylation of NBIF NBIF has two phenolic hydroxyl groups at the C-7 and C-4′ positions, indicating that the glycosylation at these two positions is possible. HPLC analysis of the products of the in vitro glycosylation reactions revealed the presence of two new

product peaks with retention times (tR) being 7.8 (1) and 4.7 (2) min, respectively (Fig 2). These two products were further analyzed by LC-MS, which showed that the NBIF glucosides 1 and 2 were the mono-glucoside (observed mass: m/z 507 [M + Na]+, 485 [M + H]+, and 529 [M + HCOOH − H]−) and di-glucoside of NBIF (observed mass: m/z 647 [M + H]+ and 691 [M + HCOOH − H]−), respectively (Fig. 3).

Fig. 2 Representative HPLC chromatograms of analysis of the in vitro glycosylation reaction mixture, Compounds 1 and 2, and NBIF. (A) In vitro enzymatic glycosylation with NBIF and UDP-glucose for 10 h of incubation; (B) 1, neobavaisoflavone4′-O-β-D-glucopyranoside; (C) 2, neobavaisoflavone- 4′, 7-di-O-β-D-glucopyranoside; (D) standard NBIF used for the reactions

Structure elucidation of NBIF glucosides Compound 1 was obtained as a yellow amorphous solid. The HR-ESI-MS revealed a pseudo-molecular ion peak m/z 485.181 3 [M + H]+, consistent with the molecular formula, C26H28O9. In the 1H NMR spectrum, the most representative signals were a glucose moiety [δ 4.82 (1H, d, J = 7.2 Hz, H-1′′′), 3.74−3.67 (2H, overlap, H-6′′′), 3.34−3.29 (3H, overlap, H-2′′′, H-3′′′, H-4′′′), and 3.17 (1H, m, H-5′′′) ] and a prenyl group [5.33 (1H, t, J = 7.4 Hz, H-2′′), 3.48 (1H, dd, J = 5.8, 11.8 Hz, H-1′′), 3.41 (1H, m, H-1′′), 1.69 (3H, s, H-5′′), and 1.67 (3H, s, H-4′′)]. The NMR data of 1 was very similar to that of known NBIF, except for the presence of a glucose moiety [1] (Table 1). Furthermore, the relative positions of the glucose moiety and prenyl group were determined based on heteronuclear multiple bond (HMBC) correlations [δ 4.82 (H-1′′′) with 155.4 (C-4′) and δ 3.48 (H-1′′) with 155.4 (C-4′), 130.4 (C-3′), and 123.3

(C-2′′)] (Fig. 4). In addition, the glucose unit in 1 was identified as a β conformation, based on the coupling constant (J = 7.2 Hz) of the anomeric proton. Therefore, the structure of 1 was identified as neobavaisoflavone-4′-O-β-D-glucopyranoside (Fig. 1). Compound 2 was obtained as a white amorphous powder. Its HR-ESI-MS profile exhibited a pseudo-molecular ion peak at m/z 647.234 2 [M + H]+, corresponding to the molecular formula C32H38O14. The 1H and 13C NMR data of 2 were very similar to those of NBIF, except for the presence of two glucose moieties [1] (Table 1). The locations of the two glucose moieties at the C-4′ (δ 155.0) and C-7 (δ 161.4) were confirmed, based on the HMBC correlations (Fig. 4). Additionally, the two anomeric proton signals at δ 4.84 (1H, d, J = 7.5 Hz, H-1′′′) and 5.11 (1H, d, J = 7.4 Hz, H-1′′′′) of 2 were determined as β and β-conformations, respectively. Thus, structure of 2 was identified as neobavaisoflavone-4′, 7-di-O-β-D-glucopyranoside (Fig. 1).

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Fig. 3 Representative LC-MS spectra of in vitro glycosylation reaction mixture. (A) Total ion chromatogram of the in vitro glycosylation reaction mixture of NBIF; (B) MS spectra of selected ion peak at 10.06 min. The ion peak at m/z 507 [M + Na]+, 485 [M + H]+, and 529 [M + HCOOH − H]− corresponds to mono-glucoside form of NBIF as expected; (C) MS spectra of selected ion peak at 8.07 min. The ion peak at m/z 647 [M + H]+ and 691 [M + HCOOH − H]− corresponds to di-glucoside form of NBIF as expected Table 1 NMR data for compounds 1 and 2 in DMSO-d6 δH (J, Hz)

δc

1

2

1

2

2

8.20, s

8.41, s

153.2 (d)

153.7 (d)

3

—

—

123.6 (s)

125.2 (s)

4

—

—

174.9 (s)

174.6 (s)

5

7.87, d (8.7)

8.04, d (8.9)

127.4 (d)

126.9 (d)

6

6.82, d (9.0)

7.15, dd (2.3, 8.9)

117.0 (d)

115. 6 (d)

7

—

—

166.3 (s)

161.4 (s)

8

6.69, s

7.24, d (2.3)

102.4 (d)

103.4 (d)

9

—

—

158.4 (s)

157.0 (s)

10

—

—

115.4 (s)

118.5 (s)

1'

—

—

126.3 (s)

123.6 (s)

2'

7.30, s

7.34, s

130.3 (d)

129.8 (d)

3'

—

—

130.4 (s)

130.0 (d)

4'

—

—

155.4 (s)

155.0 (s)

5'

7.11, d (8.4)

7.13, s

115.2 (d)

114.7 (d)

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MA Tao, et al. / Chin J Nat Med, 2017, 15(4): 281287

Continued δH (J, Hz)

δc

1

2

1

2

6'

7.31–7.28, overlap

7.33, m

127.9 (d)

127.5 (d)

1''

3.48, dd (11.8, 5.8); 3.41, m

3.44,m; 3.28, m

28.6 (t)

28.2 (t)

2''

5.33, t (7.4)

5.34, m

123.3 (d)

122.7 (d)

3''

—

—

131.8 (s)

131.3 (s)

4''

1.67, s

1.68, s

18.2 (q)

17.7 (q)

5''

1.69, s

1.70, s

26.1 (q)

25.6 (q)

1'''

4.82, d (7.2)

4.84, d, (7.5)

101.8 (d)

101.2 (d)

2'''

3.34–3.29, overlap

3.39–3.55, overlap

74.0–77.6 (d)

73.4–76.8 (d)

3'''

3.34–3.29, overlap

3.39–3.55, overlap

74.0–77.6 (d)

73.4–76.8 (d)

4'''

3.34–3.29, overlap

3.39–3.55, overlap

74.0–77.6 (d)

73.4–76.8 (d)

5'''

3.17, m

3.18, m

70.3 (d)

69.8 (d)

6'''

3.74–3.67, overlap

3.72, m

61.3 (t)

60.8 (t)

1''''

—

5.11, d (7.4)

—

99.9 (d)

2''''

—

3.39–3.55, overlap

—

73.4–76.8 (d)

3''''

—

3.39–3.55, overlap

—

73.4–76.8 (d)

4''''

—

3.39–3.55, overlap

—

73.4–76.8 (d)

5''''

—

3.18, m

—

69.6 (d)

6''''

—

3.72, m

—

60.6 (t)

7-OH

8.30, s

—

—

—

1

13

H and C NMR spectra data (δ) were obtained at 600 and 150 MHz, respectively

gested that enzymatic biosynthesis of novel glucosides of NBIF greatly enhanced their water solubility. Cytotoxicity The anti-proliferative activities of compounds 1 and 2 and NBIF against various cancer cell lines were determined using the MTT assay. Following a 72-h treatment, only NBIF showed weak cytotoxicity in the four human cancer (HepG2, SMMC7721, MDA-MB-231, SW480) cell lines, with IC50 values ranging from 63.47 to 72.81 µmol·L−1 (Table 3). Table 2 Water solubility of compounds 1 and 2 and NBIF Solubility in water (μmol·L−1)

Compounds

Fig. 4 Key HMBC correlations of compounds 1 and 2

787.9

175.1

2

18 143.5

4 031.9

4.5

1.0

NBIF

Water solubility To determine water solubility, excess compounds 1 and 2, and NBIF were mixed with water and centrifuged, and the water solution was then analyzed by HPLC. The water - solubility of novel NBIF glucosides 1 and 2 was evaluated by comparison with the substrate. The values of solubility of compounds 1 and 2 in water were 787.9 and 18 143.5 µmol·L−1, respectively, which were approximately 175.1- and 4031.9fold higher than that of the substrate (Table 2). Our data sug-

Relative solubility

1

Table 3 Cytotoxicity (IC50, μmol·L−1) of compounds 1 and 2 and NBIF

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Compounds

HepG2 SMMC7721

SW480

1

> 200

> 200

> 200

> 200

2

> 200

> 200

> 200

> 200

64.93

63.74

59.91

72.81

1.74

0.11

0.62

0.51

NBIF Geldanamycin a

MDA-MB-231

a

Geldanamycin was used as a positive control

MA Tao, et al. / Chin J Nat Med, 2017, 15(4): 281287

NBIF consists of phenolic hydroxyl groups at the C-7 and C-4′ positions, indicating that the glycosylation at these two positions is possible. The 7-OH and 4′-OH form important networks at the active position for biological activities, including antioxidant, antibacterial, and anticancer activities [22]. Thus, the cytotoxic activity of compounds 1 and 2 decreased dramatically, which may have been a consequence of the bulkiness of glycosylation at the C-7 hydroxyl group (A ring) and the C-4′ hydroxyl group (B ring) [15, 17]. NBIF glucosides 1 and 2 were more hydrophilic and may be difficultly to absorb by cells via passive diffusion [23]. In some cases, such cytotoxic compounds can be inactivated by glycosylation and reactivated for cytotoxic activity by glycosidase [24]. Therefore, we expect that further development of novel NBIF glucosides with higher water solubility will improve the pharmacokinetic properties of these compounds over NBIF.

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Cite this article as: MA Tao, DAI Yi-Qun, LI Nan, HUO Qiang, LI Hong-Mei, ZHANG Yu-Xin, PIAO Zheng-Hao, WU Cheng-Zhu. Enzymatic biosynthesis of novel neobavaisoflavone glucosides via Bacillus UDP-glycosyltransferase [J]. Chin J Nat Med, 2017, 15(4): 281-287.

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