Enzymatic synthesis of bioactive O-glucuronides using plant

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Enzymatic synthesis of bioactive O-glucuronides using plant glucuronosyltransferases Tian Yue, Ridao Chen, Dawei Chen, Jimei Liu, Kebo Xie, and Jungui Dai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01769 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 14, 2019

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Journal of Agricultural and Food Chemistry

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Enzymatic synthesis of bioactive O-glucuronides using plant

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glucuronosyltransferases

3

Tian Yue a, b, c, Ridao Chen a, b, c, Dawei Chen a, b, c, Jimei Liu a, b, c, Kebo Xie a, b, c *, and

4

Jungui Daia, b, c *

5

a

6

CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs;

7

Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese

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Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan

9

Street, Beijing, 100050, P.R. China

10

*

State Key Laboratory of Bioactive Substance and Function of Natural Medicines; c

b

NHC Key

Corresponding author. Tel: +86 10 63165762; fax: +86 10 63017757. E-mail:

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[email protected] (K. X.), Tel: +86 10 63165195; fax: +86 10 63017757.

12

E-mail: [email protected] (J. D.).

1

ACS Paragon Plus Environment


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Abstract: Many O-glucuronides exhibiting various pharmacological activities have

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been found in nature and in drug metabolism. The glucuronidation of bioactive natural

15

products or drugs to generate glucuronides with better activity and druggability is

16

important in drug discovery and research. In this study, by using two uridine

17

diphosphate (UDP)-dependent glucuronosyltransferases (GATs, UGT88D4 and

18

UGT88D7) from plants, we developed two glucuronidation approaches, pure enzyme

19

catalysis in vitro and recombinant whole-cell catalysis in vivo, to efficiently

20

synthesize bioactive O-glucuronides by the glucuronidation of natural products. In

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total, fourteen O-glucuronides with different structures, including flavonoids,

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anthraquinones, coumarins and lignans, were obtained, 7 of which were new

23

compounds.

24

kaempferol-7-O-β-D-glucuronide

25

phosphatase (PTP) 1B with an IC50 value of 8.02  106 M. Some of the

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biosynthesized O-glucuronides also exhibited significant antioxidant activities.

27

Key words: O-glucuronides; glucuronidation; glucuronosyltransferases; enzyme

28

catalysis; whole-cell catalysis

Furthermore,

one

of

the

(3a),

biosynthesized

potently

inhibited

2


O-glucuronides, protein

tyrosine

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INTRODUCTION

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Glucuronidation occurs during phase II conjugative drug metabolism and the

31

metabolism of the chemical components of foods, and these processes are catalyzed

32

by glucuronic acid transferases/glucuronosyltransferases (GATs)

33

glucuronide metabolites of drugs have higher water solubility and polarity than the

34

parent drug compounds. Thus, glucuronidation serves as an integral step in

35

transforming lipophilic substrates into hydrophilic glucuronides and facilitates the

36

drug transport and elimination, which is generally regarded as a detoxification

37

reaction 4. However, some glucuronide metabolites have more potent pharmacological

38

activities than the corresponding aglycons, and they are the real active drug molecule

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exerting the pharmacological effect. For example, morphine-3-O-glucuronide (M3G)

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and morphine-6-O-glucuronide (M6G) are the two in vivo glucuronide metabolites of

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morphine 5. M3G is the main metabolite but without analgesic activity. In contrast,

42

M6G, the minor metabolite, is the potent opioid receptor agonist involved in the

43

analgesic effect of morphine, and the anesthetic effect of M6G is 100 times more

44

potent than that of morphine 6.

1‒3.

Usually, the

45

Natural glucuronides are widely distributed in plants, and many natural

46

glucuronides with various pharmacological activities have been isolated from plants.

47

For instance, the main component of the clinical injectable drug breviscapine is

48

scutellarin-7-O-glucuronide, which has been used in the clinic for more than ten years

49

to treat cardio-cerebral vascular diseases

50

in plants such as Scutellaria baicalensis and Chrysanthemum morifolium and has a

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variety of pharmacological activities, including anti-atherosclerosis, antioxidant,

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anti-inflammatory, anti-complement, and aldose reductase inhibitory activities

53

Unlike

mammalian

GATs,

which

7, 8.

are

Apigenin-7-O-glucuronide is distributed

generally

3


considered

9‒12.

physiological


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membrane-bound enzymes, plant GATs are soluble and able to recognize extremely

55

diverse sugar acceptors. Chemical routes to the synthesis of glucuronides face

56

challenges associated with regioselectivity, stereoselectivity, protection and

57

deprotection of functional groups. Therefore, enzymatic glucuronidations catalyzed

58

by soluble plant GATs are a potential approach for generating target bioactive

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glucuronides in drug discovery.

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Hence, to construct efficient methods for the synthesis of bioactive glucuronides,

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two plant GATs (UGT88D4 and UGT88D7) were employed and developed as

62

enzyme tools in this study. Two kinds of catalytic systems, pure enzyme catalysis in

63

vitro with high efficiency and whole-cell catalysis in vivo without adding expensive

64

sugar donors, were both successfully established and utilized to synthesize novel and

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structurally diverse bioactive glucuronides.

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MATERIALS AND METHODS

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General. Uridine diphosphate glucuronic acid (UDPGA) was purchased from

68

Sigma-Aldrich (St Louis, USA). Compounds 1‒17 (Figure 5C) were purchased from

69

Nanjing Zelang Biological Technology Co., Ltd. (Nanjing, China). All restriction

70

enzymes were purchased from Takara Biotechnology Co., Ltd (Dalian, China).

71

Optical rotations were acquired with a Perkin-Elmer Model-343 digital polarimeter

72

(PerkinElmer Inc., USA). UV spectra were measured with a Jasco J-815 circular

73

dichroism (CD) spectrometer (Jasco Corp., Japan). IR spectra were obtained on a

74

Nicolet 5700 FT-IR spectrometer (Thermo Electron Scientific Instrument Corp.,

75

USA). Compounds were characterized by 1D and 2D NMR on Bruker 400/500/600

76

spectrometers (Bruker BioSpin AG, Switzerland). High-resolution electrospray

77

ionization

mass

spectrometry

(HRESIMS)

data

4


were

measured

with

an

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78

ESI-FTICR-MS (LTQ-FT Ultra, ThermoFisher Scientific, USA). The products of

79

enzymatic reactions were analyzed on an Agilent 1260 series HPLC system (Agilent

80

Technologies, Germany) coupled with an LCQ Fleet ion trap mass spectrometer

81

(Thermo Electron Corp., USA) equipped with an ESI source. Samples were analyzed

82

on a Shiseido Capcell pak C18 MG III column (4.6 mm × 250 mm; particle size, 5 μm,

83

Shiseido Co., Ltd., Japan). Purified products were obtained on a Thermo SRD-3200

84

series HPLC with a Shiseido Capcell pak C18 MG II column (10 mm × 250 mm;

85

particle size, 5 μm, Shiseido Co., Ltd., Tokyo, Japan).

86

Gene Cloning and Plasmid Construction. The genes of UGT88D4 (AmUGT10;

87

Gene ID AB362988), UGT88D7 (PfUGT50; Gene ID AB362991), and UDP-glucose

88

dehydrogenase (ugd; Gene ID 946571) were chemically synthesized (Sangon Biotech,

89

Shanghai, China) with optimized codon usage for expression in Escherichia coli.

90

AmUGT10 and PfUGT50 were subcloned into the EcoRI/NotI site of the pCDFDuet-1

91

(Novagen,

92

pCDF-UGT88D4 and pCDF-UGT88D7, respectively.

93

subcloned into the NdeI/XhoI site of pCDF-UGT88D4 and pCDF-UGT88D7,

94

resulting in plasmids named pCDF-UGT88D4-ugd and pCDF-UGT88D7-ugd,

95

respectively. Then, the recombinant plasmids were introduced into E. coli BL21 (DE3)

96

(TransGen Biotech, China) for heterologous expression after verification of the

97

sequences.

98

Expression and Purification of UGT88D4 and UGT88D7. The confirmed E. coli

99

BL21 harboring pCDF-UGT88D4/pCDF-UGT88D7 was cultured in 400 mL of

100

Luria-Bertani (LB) medium containing 40 μg/mL streptomycin sulfate. Cells were

101

grown at 37 °C with shaking at 200 rpm until the OD600 reached 0.6. Then, the

102

expression of UGT88D4 and UGT88D7 was performed by induction with 0.2 mM

Germany)

expression

vector,

resulting

5


in

plasmids

named

Subsequently, ugd was


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103

isopropyl β-D-thiogalactoside (IPTG) for 20 h at 18 °C. The cells were collected by

104

centrifugation at 8,000 g for 10 min at 4 °C and resuspended in 20 mL of binding

105

buffer (20 mM imidazole, 0.5 M NaCl, 20 mM phosphate buffer, pH 7.4) containing 1

106

mM phenylmethylsulfonyl fluoride (PMSF) and 1 mg/mL lysozyme and were

107

disrupted by sonication. The supernatants containing soluble proteins were filtered

108

0.45 μm syringe filter units after centrifuging at 10,000 g for 40 min at 4 °C. The

109

obtained supernatants were immediately loaded onto a column of Ni-NTA resin (GE,

110

USA) which was pre-equilibrated with binding buffer. Subsequently, the resin was

111

washed with binding buffer to remove unbound proteins. The elution was performed

112

with elution buffers containing 50–500 mM imidazole, and the purified protein was

113

desalted with desalting buffer (50 mM NaCl, 1 mM dithiothreitol, 4% glycerol, 50

114

mM Tris-HCl buffer, pH 7.4) by concentration and dilution using an Amicon-Ultra 30

115

centrifugal filter unit (Millipore, USA). The protein purity was verified by sodium

116

dodecyl

117

concentration of pure protein was determined by a Protein Quantitative Kit (TransGen

118

Biotech, China) 13.

119

Effects of Temperature, pH and Divalent Metal Ions on the Catalytic Activities of

120

UGT88D4 and UGT88D7. To optimize the reaction temperature, the reactions were

121

performed at different temperatures ranging from 15–65 °C. To evaluate the effects of

122

pH, reactions were carried out in reaction buffers with pH values of 4.0–6.0 (50 mM

123

citric acid-sodium citrate buffer), 6.0–8.0 (50 mM Na2HPO4-NaH2PO4 buffer),

124

8.0–9.0 (50 mM Tris-HCl buffer) and 9.0–11.0 (50 mM Na2CO3-NaHCO3 buffer). To

125

investigate the dependence of divalent metal ions for UGT88D4 and UGT88D7, 5

126

mM solutions of BaCl2, CaCl2, CoCl2, CuCl2, FeCl2, MgCl2, MnCl2, NiCl2, ZnCl2 and

127

EDTA were added to reactions with the final concentration of 5 mM, respectively.

sulfate-polyacrylamide

gel

electrophoresis

6


(SDS-PAGE)

and

the

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128

Reactions without adding metal ions were used as a control. All assays were carried

129

out with UDPGA as the sugar donor and hesperetin (7) as the acceptor.

130

Kinetic Parameters of UGT88D4 and UGT88D7. Kinetic analysis of recombinant

131

UGT88D4 and UGT88D7 towards hesperetin (7) was performed in 100 l aliquots

132

with 0.05 mg/mL purified enzyme, 50 mM Na2HPO4-NaH2PO4 buffer (pH 7.0), 5

133

mM UDPGA and varying concentrations of 7 (100–800 M). For UGT88D4, the

134

reactions were incubated at 45 °C for 30 min; for UGT88D7, the reactions were

135

incubated at 40 °C for 30 min and then stopped by adding 200 l of methanol.

136

Subsequently, the reaction mixtures were centrifuged to collect the supernatant, and

137

aliquots were analyzed by HPLC-UV. The kinetic parameters were calculated by

138

nonlinear regression of the Michaelis–Menten equation using Origin 8.0 14.

139

Glucuronidation Reactions Catalyzed by UGT88D4 and UGT88D7 in vitro. The

140

reaction mixtures contained 0.5 mM aglycons (1‒17), 1 mM UDPGA and 1 mg/mL

141

purified enzyme in a final volume of 100 μL. Activity assays, initiated by the addition

142

of enzymes, were performed at 30 °C for 12 h and terminated by addition of 200 μL

143

ice cold methanol. Subsequently, the mixtures were centrifuged at 15,000 g for 30

144

min to collect the supernatant, and aliquots were analyzed by HPLC-UV/ESIMS.

145

Control reactions without adding the enzyme or UDPGA were performed. The elution

146

conditions of HPLC analysis were as follows: solvent A (0.1% formic acid aqueous

147

solution), solvent B (methanol); flow rate (1 mL/min); gradient (15−100% B in 30

148

min followed by 100% B for 5 min for 1−11 and 13−17; 50−100% B in 20 min

149

followed by 100% B for 5 min for 12).

150

Glucuronidation Reactions Catalyzed by Whole-Cell Biocatalyst in vivo. An

151

overnight culture of E. coli BL21 (DE3) harboring pCDF-ugd-UGT88D4 or

152

pCDF-ugd-UGT88D7 was inoculated into 50 mL of fresh LB medium containing an 7



153

appropriate antibiotic (40 μg/mL streptomycin sulfate). Cells were grown at 37 °C

154

with shaking (200 rpm). When the OD600 reached 0.6, genes ugd and UGT in E. coli

155

were induced by adding IPTG to a final concentration of 0.2 mM. Subsequently, the

156

cultures were shaken at 18 °C for 20 h. Cells were harvested by centrifugation and

157

resuspended in fresh M9 medium (Na2HPO4 6.8 g/L, KH2PO4 3.0 g/L, NaCl 0.5 g/L,

158

NH4Cl 1.0 g/L, MgSO4 1.2 g/L, and CaCl2 1.1 g/L) containing 2% glucose and

159

antibiotics. Cell density was maintained at OD600 = 3.0, and the substrates (1–17) for

160

the biotransformation were added into the culture at a final concentration of 0.7 mM,

161

respectively and the total volume of each reaction was 1 mL. Then, the cultures were

162

incubated at 30 °C for 24 h, and the supernatants were analyzed by HPLC-UV/MS. E.

163

coli BL21 (DE3) with pCDFDuet served as the blank control.

164

Preparing the Representative Glucuronides by Biocatalysis. For products prepared

165

by whole cells in vivo, the synthesis reactions were performed as follows. Chrysin (1,

166

10.7 mg) was dissolved in 50 l of dimethyl sulfoxide (DMSO), and this solution was

167

combined with 60 mL of a M9 cell suspension culture of strain B-ugd-UGT88D7 at a

168

final concentration of 0.7 mM with a cell density of OD600 = 3.0. The reaction was

169

performed at 30 °C with shaking at 200 rpm for 24 h, and the reaction mixture was

170

centrifuged at 10,000 g for 30 min to remove precipitated cells. The supernatant

171

containing product 1a was passed through an Amberlite XAD-16 macroporous resin

172

column (50 mL, Rohm & Haas Corp., USA). Aliquots (250 mL) of ultrapure water,

173

50% (v/v) ethanol and 100% (v/v) ethanol were sequentially loaded into the column

174

and then eluted at a flow rate of 1 mL/min. The product 1a was eluted with 50% (v/v)

175

ethanol, and the fraction was concentrated under reduced pressure. The residue

176

containing 1a was further purified by reversed-phase semipreparative HPLC (solvent

177

A: 0.1% formic acid aqueous solution; solvent B: methanol; flow rate: 3 mL/min; 8


Page 8 of 35

Page 9 of 35


178

gradient: 30−90% B in 20 min followed by 100% B for 5 min) to afford 1a.

179

Following the above procedure, the other substrates, including diosmetin (2),

180

kaempferol (3), fisetin (4), morin (5), (2R, 3R)-dihydroquercetin (6), hesperetin (7)

181

and biochanin A (9), were also glucuronidated by whole cells in vivo, and

182

corresponding glucuronides 2a, 3a, 4a, 5a, 6a, 7a and 9a were successfully obtained.

183

For products prepared by purified enzymes in vitro, the reactions were performed

184

as follows. Epicatechin (8, 10.4 mg) was dissolved in 50 l of DMSO, and this

185

solution along with UDPGA (35 mg) were added to 10 mL of desalting buffer

186

containing 1 mg/mL purified UGT88D7 extracted from 4 g (wet weight) of induced E.

187

coli cells containing pCDF-UGT88D7. The reactions were performed at 30 °C with

188

shaking at 90 rpm for 48 h. After centrifugation, the supernatant containing product

189

8a was passed through an Amberlite XAD-16 macroporous resin column. Target

190

product 8a was eluted with 50% (v/v) ethanol, and this fraction was concentrated. The

191

residue containing 8a was further purified by reversed-phase semipreparative HPLC

192

(solvent A: 0.1% formic acid aqueous solution; solvent B: methanol; flow rate: 3

193

mL/min; gradient: 15−58% B in 15 min followed by 100% B for 5 min) to give 8a.

194

Following the above procedure, the other substrates, including phloretin (10),

195

7-hydroxy-4-methylcoumarin (11), emodin (12) and magnolol (13), were also

196

glucuronidated by purified UGT88D4 and UGT88D7 in vitro, and corresponding

197

glucuronides 10a, 10b, 11a, 12a and 13a were obtained.

198

The Spectroscopic Data of Prepared Glucuronides. Chrysin-7-O-β-D-glucuronide

199

(1a, isolated yield 36%). ESI-MS m/z 429.30 [M  H], 859.11 [2M−H]; 1H NMR

200

(500 MHz, DMSO-d6): δH 12.82 (1H, s, 5-OH), 8.19 (2H, d, J = 7.6 Hz, H-2', 6'),

201

7.65-7.58 (3H, m, H-3', 4', 5'), 7.07 (1H, s, H-3), 6.90 (1H, d, J = 1.8 Hz, H-8), 6.48

202

(1H, d, J = 1.8 Hz, H-6), 5.17 (1H, d, J = 7.4 Hz, H-1''), 3.80-3.16 (4H, m, H-2'', 3'', 9



Page 10 of 35

203

4'', 5''); 13C NMR (125 MHz, DMSO-d6): δC 182.2 (C-4), 171.0 (C-6''), 163.7 (C-2),

204

163.1 (C-7), 161.1 (C-5), 157.2 (C-9), 133.3 (C-4'), 130.6 (C-1'), 129.2 (C-3', 5'),

205

126.6 (C-2', C-6'), 105.6 (C-10), 105.5 (C-3), 99.7 (C-6), 99.3 (C-1''), 94.9 (C-8), 76.2

206

(C-5''), 74.5 (C-3''), 72.9 (C-2''), 71.7 (C-4'').

207

Diosmetin-7-O-β-D-glucuronide (2a, isolated yield 67%). ESI-MS m/z 475.48

208

[MH], 951.21 [2MH]; 1H NMR (500 MHz, DMSO-d6): δH 12.95 (1H, s, OH-5),

209

9.56 (1H, s, OH-3'), 7.58 (1H, d, J = 8.4 Hz, H-6'), 7.46 (1H, s, H-2'), 7.10 (1H, d, J =

210

8.4 Hz, H-5'), 6.84 (2H, d, J = 7.1 Hz, H-3, H-8), 6.45 (1H, s, H-6), 5.20 (1H, d, J =

211

7.2 Hz, H-1''), 3.87 (3H, s, -OCH3), 3.53-3.11 (4H, m, H-2'', 3'', 4'', 5'');

212

(100 MHz, DMSO-d6): δC 182.0 (C-4), 171.0 (C-6''), 164.1 (C-2), 163.0 (C-7), 161.5

213

(C-5), 157.0 (C-9), 151.3 (C-4'), 146.9 (C-3'), 122.9 (C-1'), 118.9 (C-6'), 113.1 (C-2'),

214

112.2 (C-5'), 105.4 (C-10), 103.8 (C-3), 99.5 (C-6, 1''), 94.6 (C-8), 76.2 (C-3''), 74.4

215

(C-5''), 72.9 (C-2''), 71.7 (C-4'').

216

Kaempferol-7-O-β-D-glucuronide (3a, novel, isolated yield 57%). Yellow powder;

217

[]25 D −84 (c 0.40, DMSO); UV (MeOH) λmax (log ε) 366 (3.52), 323 (3.25), 267

218

(3.49), 207 (3.61) nm; IR νmax: 3345, 2924, 1601, 1010, 950, 831 cm-1; ESI-MS m/z

219

461.50 [MH], 923.13 [2MH]; HRESIMS m/z 461.0508 [MH] (calcd for

220

C21H17O12, 461.0715); 1H NMR (600 MHz, DMSO-d6): δH 12.50 (1H, s, OH-5), 10.18

221

(1H, br.s, OH-4'), 8.07 (2H, d, J = 8.8 Hz, H-2', 6'), 6.94 (2H, d, J = 8.9 Hz, H-3', 5'),

222

6.82 (1H, d, J = 2.0 Hz, H-8), 6.43 (1H, d, J = 2.0 Hz, H-6), 5.23 (1H, d, J = 7.3 Hz,

223

H-1''), 3.96 (1H, d, J = 9.3 Hz, H-5''), 3.41-3.16 (3H, m, H-2'', 3'', 4''); 13C NMR (150

224

MHz, DMSO-d6): δC 176.1 (C-4), 170.5 (C-6''), 162.3 (C-7), 160.4 (C-5), 159.4 (C-4'),

225

155.8 (C-9), 147.6 (C-2), 136.1 (C-3), 129.6 (C-2', 6'), 121.5 (C-1'), 115.5 (C-3', 5'),

226

104.8 (C-10), 99.2 (C-6), 98.7 (C-1''), 94.2 (C-8), 75.8 (C-5''), 75.1 (C-3''), 72.8

227

(C-2''), 71.4 (C-4'') 10


13C

NMR

Page 11 of 35


228

Fisetin-7-O-β-D-glucuronide (4a, novel, isolated yield 68%). Yellow powder; []25

229

D −63 (c 1.46, DMSO); UV (MeOH) λmax (log ε) 363 (3.52), 316 (3.23), 251 (3.45),

230

207 (3.78) nm; IR νmax: 3296, 2925, 1611, 1257, 1019, 952, 770 cm-1; ESI-MS m/z

231

461.63 [MH], 923.27 [2MH]; HRESIMS m/z 463.0876 [M+H]+ (calcd for

232

C21H19O12, 463.0871); 1H NMR (400 MHz, DMSO-d6): δH 9.30 (2H, br.s, OH-3', 4'),

233

8.00 (1H, d, J = 8.9 Hz, H-5), 7.72 (1H, d, J = 1.7 Hz, H-2'), 7.56 (1H, dd, J = 8.4 Hz,

234

1.5 Hz, H-6'), 7.31 (1H, d, J = 1.6 Hz, H-8), 7.09 (1H, dd, J =8.9 Hz, 1.7 Hz, H-6),

235

6.90 (1H, d, J = 8.5 Hz, H-5'), 5.24 (1H, d, J = 6.8 Hz, H-1''), 3.85 (1H, d, J = 8.8 Hz,

236

H-5''), 3.66-3.30 (3H, m, H-2'', 3'', 4'');

237

(C-4), 170.2 (C-6''), 161.0 (C-7), 155.8 (C-9), 147.5 (C-4'), 145.8 (C-2), 145.1 (C-3'),

238

137.5 (C-3), 126.2 (C-5), 122.3 (C-1'), 119.7 (C-6'), 116.1 (C-5'), 115.6 (C-2'), 115.2

239

(C-6), 115.1 (C-10), 102.9 (C-8), 99.5 (C-1''), 76.0 (C-3''), 74.7 (C-5''), 74.3 (C-2''),

240

72.9 (C-4'').

241

Morin-7-O-β-D-glucuronide (5a, novel, isolated yield 78%). Yellow powder; []25 D

242

−113 (c 0.48, DMSO); UV (MeOH) λmax (log ε) 371 (3.14), 293 (3.06), 256 (3.34),

243

210 (3.57) nm; IR νmax: 3356, 2920, 1601, 1292, 952, 710 cm-1; ESI-MS m/z 477.49

244

[MH], 955.29 [2MH]; HRESIMS m/z 479.0814 [M+H]+ (calcd for C21H19O13,

245

479.0820); 1H NMR (400 MHz, DMSO-d6): δH 7.71 (1H, d, J = 1.9 Hz, H-3'), 7.56

246

(1H, dd, J = 8.4 Hz, 1.9 Hz, H-5'), 6.90 (1H, d, J = 8.4 Hz, H-6'), 6.77 (1H, d, J = 1.8

247

Hz, H-8), 6.41 (1H, d, J = 1.8 Hz, H-6), 5.13 (1H, d, J = 7.3 Hz, H-1''), 3.74 (1H, d, J

248

= 9.5 Hz, H-5''), 3.51-3.18 (3H, m, H-2'', 3'', 4''); 13C NMR (150 MHz, DMSO-d6): δC

249

176.0 (C-4), 171.4 (C-6''), 162.6 (C-7), 160.3 (C-5), 155.7 (C-9), 148.0 (C-4'), 147.6

250

(C-2'), 145.1 (C-2), 136.1 (C-3), 121.8 (C-5'), 120.1 (C-1'), 115.6 (C-3'), 115.3 (C-6'),

251

104.6 (C-10), 99.5 (C-1''), 98.8 (C-6), 94.1 (C-8), 76.2 (C-4''), 74.4 (C-5''), 72.9

252

(C-2''), 71.7 (C-3'').

13C

NMR (100 MHz, DMSO-d6): δC 171.9

11



253

(2R, 3R)-Dihydroquercetin-7-O-β-D-glucuronide (6a, novel, isolated yield 50%).

254

Yellow powder; []25 D −88 (c 1.16, DMSO); UV (MeOH) λmax (log ε) 375 (3.25),

255

285 (3.76), 209 (3.95) nm; IR νmax: 3385, 3309, 2916, 1643, 1024, 827, 709 cm-1;

256

ESI-MS m/z 479.37 [M  H]; HRESIMS m/z 481.0977 [M + H]+ (calcd for

257

C21H21O13, 481.0977); 1H NMR (500 MHz, DMSO-d6): δH 11.81 (1H, s, OH-5), 9.07

258

(2H, s, OH-3', 4'), 6.90 (1H, s, H-2'), 6.78-6.73 (2H, overlapped, H-5', 6'), 6.17 (2H, s,

259

H-6, 8), 5.84 (1H, s, OH-3), 5.14 (1H, d, J = 7.6 Hz, H-1''), 5.04 (1H, d, J =11.7 Hz,

260

H-2), 4.59 (1H, d, J = 11.0 Hz, H-3), 3.91 (1H, d, J = 8.7 Hz, H-5''), 3.37-3.27 ( 3H,

261

m, H-2'', 3'', 4'');

262

165.0 (C-7), 162.7 (C-9), 162.4 (C-5), 145.9 (C-4'), 145.0 (C-3'), 127.8 (C-1'), 119.5

263

(C-6'), 115.1 (C-2', 5'), 102.1 (C-10), 99.0 (C-1''), 96.7 (C-6), 95.3 (C-8), 83.3 (C-2),

264

75.8 (C-5''), 74.8 (C-3''), 72.7 (C-2''), 71.7 (C-3), 71.4 (C-4'').

265

Hesperetin-7-O-β-D-glucuronide (7a, isolated yield 50%). ESI-MS m/z 477.34 [M 

266

H]; 1H NMR (500 MHz, DMSO-d6): δH 12.04 (1H, s, OH-5), 9.16 (1H, s, OH-3'),

267

6.93-6.94 (2H, m, H-2', 5'), 6.89 (1H, dd, J = 8.1 Hz, 2.2 Hz, H-6'), 6.18 (1H, d, J =

268

1.8 Hz, H-8), 6.14 (1H, s, H-6), 5.51 (1H, dd, J = 12.4 Hz, 2.2 Hz, H-2), 5.08 (1H, d,

269

J = 7.9 Hz, H-1''), 3.81-3.79 (1H, m, H-5''), 3.77 (3H, s, -OCH3), 3.32-3.21 (4H, m,

270

H-3axial, 2'',3'',4''), 2.75 (1H, dd, J =17.1 Hz, 2.0 Hz, H-3equatorial); 13C NMR (125 MHz,

271

DMSO-d6): δC 197.1 (C-4), 171.0 (C-6''), 165.1 (C-7), 162.9 (C-5), 162.6 (C-9), 148.0

272

(C-4'), 146.5 (C-3'), 130.9 (C-1'), 117.8 (C-6'), 114.4 (C-2'), 112.0 (C-5'), 103.3

273

(C-10), 99.1 (C-1''), 96.5 (C-6), 95.4 (C-8), 78.5 (C-2), 76.0 (C-4''), 74.6 (C-5''), 72.8

274

(C-2''), 71.5 (C-3''), 55.7 (-OCH3), 42.2 (C-3).

275

Epicatechin-7-O-β-D-glucuronide (8a, isolated yield 61%). ESI-MS m/z 465.46 [M 

276

H]; 1H NMR (400 MHz, Acetone-d6): δH 7.07 (1H, s, H-2'), 6.86 (1H, d, J = 8.7 Hz,

277

H-6'), 6.80 (1H, d, J = 8.2 Hz, H-5'), 6.23 (1H, s, H-6), 6.17 (1H, d, J = 1.8 Hz, H-8),

13C

NMR (100 MHz, DMSO-d6): δC 198.7 (C-4), 170.5 (C-6''),

12


Page 12 of 35

Page 13 of 35


278

5.03 (1H, d, J = 7.7 Hz, H-1''), 4.92 (1H, s, H-2), 4.24 (1H, s, H-3), 4.08 (1H, d, J =

279

9.6 Hz, H-5''), 3.72 (1H, t, J = 9.0 Hz, H-4''), 3.62-3.48 (2H, m, H-2'',3''), 2.91 (1H,

280

dd, J = 16.8 Hz, 4.4 Hz, H-4), 2.78 (1H, dd, J = 16.7 Hz, 2.8 Hz, H-4); 13C NMR (100

281

MHz, Acetone-d6): δC 170.2 (C-6''), 157.9 (C-7), 157.5 (C-5), 157.1 (C-9), 145.3

282

(C-3', 4'), 132.1 (C-1'), 119.5 (C-6'), 115.5 (C-2'), 115.3 (C-5'), 102.8 (C-10), 102.0

283

(C-1''), 97.4 (C-6), 96.8 (C-8), 79.6 (C-2), 77.1 (C-3''), 76.1 (C-5''), 74.3 (C-2''), 72.7

284

(C-4''), 66.7 (C-3), 29.1 (C-4).

285

Biochanin A-7-O-β-D-glucuronide (9a, novel, isolated yield 35%). White powder;

286


287

(3.73) nm; IR νmax: 3359, 2916, 1657, 1026, 828, 703, 566 cm-1; ESI-MS m/z 459.41

288

[M  H], 919.33 [2M  H]; HRESIMS m/z 461.1067 [M + H]+ (calcd for C22H21O11,

289

461.1078); 1H NMR (400 MHz, DMSO-d6): δH 12.91 (1H, s, OH-5), 8.47 (1H, s, H-2),

290

7.52 (2H, dd, J = 8.7 Hz, 2.1 Hz, H-2', 6'), 7.01 (2H, dd, J = 8.7 Hz, 2.1 Hz, H-3', 5'),

291

6.75 (1H, s, H-8), 6.50 (1H, s, H-6), 5.24 (1H, d, J = 7.3 Hz, H-1''), 3.96 (1H, d, J

292

=9.2 Hz, H-5''), 3.79 (3H, s, -OCH3), 3.38-3.27 (3H, m, H-2'', 3'', 4''); 13C NMR (150

293

MHz, DMSO-d6): δC 180.4 (C-4), 170.4 (C-6''), 162.7 (C-7), 161.7 (C-5), 159.2 (C-4'),

294

157.2 (C-9), 155.0 (C-2), 130.2 (C-2', 6'), 122.7 (C-1'), 122.2 (C-3), 113.8 (C-3', 5'),

295

106.2 (C-10), 99.5 (C-6), 99.2 (C-1''), 94.5 (C-8), 75.8 (C-5''), 75.0 (C-2''), 72.8

296

(C-4''), 71.4 (C-3''), 55.2 (-OCH3).

297

Phloretin-4'-O-β-D-glucuronide (10a, novel, isolated yield 58%). White powder;

298

[]25 D −91 (c 0.45, DMSO); UV (MeOH) λmax (log ε) 280 (3.41), 225 (3.44) nm; IR

299

νmax: 3282, 2921, 1630, 1261, 1083, 946, 740, 583 cm-1; ESI-MS m/z 449.41 [M  H],

300

899.23 [2M  H]; HRESIMS m/z 433.1128 [M + H  H2O]+ (calcd for C21H21O10,

301

433.1129); 1H NMR (600 MHz, DMSO-d6): δH 12.37 (2H, s, OH-2', 6'), 9.15 (1H, s,

302

OH-4), 7.02 (2H, d, J = 8.4 Hz, H-2, 6), 6.66 (2H, d, J = 8.4 Hz, H-3, 5), 6.05 (2H, s, 13



Page 14 of 35

303

H-3', 5'), 5.01 (1H, d, J =7.7 Hz, H-1''), 3.86 (1H, d, J = 9.5 Hz, H-5''), 3.41-3.18 (5H,

304

m, H-2'', 3'', 4'', α-CH2), 2.78 (2H, t, J = 7.8 Hz, β-CH2);

305

DMSO-d6): δC 205.1 (C=O), 170.3(C-6''), 163.8 (C-2', 6'), 163.0 (C-4'), 155.4 (C-4),

306

131.5 (C-1), 129.2 (C-2, 6), 115.1 (C-3, 5), 105.4 (C-1'), 99.1 (C-1''), 95.0 (C-3', 5'),

307

75.7 (C-5''), 75.3 (C-3'') , 72.8 (C-2''), 71.4 (C-4''), 45.8 (C-α), 29.3 (C-β).

308

Phloretin-2'-O-β-D-glucuronide (10b, isolated yield 9%). ESI-MS m/z 449.42 [M 

309

H]; 1H NMR (400 MHz, DMSO-d6): δH 13.52 (1H, s, OH-6'), 9.10 (1H, s, OH-4),

310

7.04 (2H, d, J = 8.4 Hz, H-2, 6), 6.64 (2H, d, J = 8.4 Hz, H-3, 5), 6.14 (1H, d, J = 1.6

311

Hz, H-3'), 5.91 (1H, d, J = 2.0 Hz, H-5'), 4.98 (1H, d, J = 7.0 Hz, H-1''), 3.70-3.29 (6

312

H, m, H-2'', 3'', 4'', 5'', α-CH2), 2.78 (2H, t, J = 7.3 Hz, β-CH2); 13C NMR (150 MHz,

313

DMSO-d6): δC 204.6 (C=O), 171.0 (C-6''), 165.4 (C-6'), 164.6 (C-4'), 160.6 (C-2'),

314

155.2 (C-4), 131.5 (C-1), 129.2 (C-2, 6), 115.0 (C-3, 5), 105.0 (C-1'), 100.4 (C-1''),

315

96.8 (C-5'), 94.3 (C-3'), 76.3 (C-5''), 74.8 (C-3'') , 72.9 (C-2''), 71.6 (C-4''), 44.9 (C-α),

316

29.0 (C-β).

317

4-Methylcoumarin-7-O-β-D-glucuronide (11a, isolated yield 75%). ESI-MS m/z

318

351.47 [M  H], 703.36 [2M  H]; 1H NMR (400 MHz, DMSO-d6): δH 7.70 (1H, d,

319

J = 8.7 Hz, H-5), 7.06 (2H, m, H-6, 8), 6.24 (1H, s, H-3), 5.21 (1H, d, J = 6.9 Hz,

320

H-1'), 3.98 (1H, d, J = 9.1 Hz, H-5'), 3.40-3.28 (3H, m, H-2', 3', 4'), 2.40 (3H, s,

321

-CH3);

322

154.4 (C-8a), 153.3 (C-4), 126.5 (C-5), 114.2 (C-6), 113.3 (C-4a), 111.8 (C-3), 103.1

323

(C-8), 99.4 (C-1'), 75.8 (C-5'), 75.2 (C-2'), 72.9 (C-3'), 71.4 (C-4'), 18.1 (-CH3).

324

Emodin-3-O-β-D-glucuronide (12a, novel, isolated yield 24%). Yellow powder;

325


326

(3.49), 225 (3.62) nm; IR νmax: 3387, 2921, 1629, 1086, 761 cm-1; ESI-MS m/z 445.25

327

[M  H], 891.08 [2M  H]; HRESIMS m/z 445.0562 [M  H] (calcd for C21H17O11,

13C

13C

NMR (150 MHz,

NMR (100 MHz, DMSO-d6): δC 170.5 (C-6'), 160.1 (C-2), 159.7 (C-7),

14


Page 15 of 35


328

445.0765); 1H NMR (400 MHz, DMSO-d6): δH 7.50 (1H, s, H-5), 7.24 (1H, s, H-4),

329

7.18 (1H, s, H-7), 6.98 (1H, s, H-2), 5.27 (1H, d, J = 5.8 Hz, H-1'), 3.95 (1H, d, J =

330

6.5 Hz, H-5'), 3.48-3.25 (3H, m, H-2', 3', 4'), 2.42 (3H, s, -CH3); 13C NMR (100 MHz,

331

DMSO-d6): δC 190.1 (C-9), 181.1 (C-10), 170.4 (C-6'), 164.0 (C-3), 163.6 (C-8),

332

161.5 (C-1), 148.6 (C-6), 134.9 (C-4a), 132.8 (C-10a), 124.2 (C-7), 120.6 (C-5),

333

113.5 (C-8a), 110.9 (C-9a), 108.9 (C-2), 108.7 (C-4), 99.4 (C-1'), 75.7 (C-5'), 75.0

334

(C-4'), 72.8 (C-2'), 71.4 (C-3'), 21.6 (-CH3).

335

Magnolol-O-β-D-glucuronide (13a, isolated yield 79%). ESI-MS m/z 441.36 [M 

336

H], 883.00 [2M  H]; 1H NMR (400 MHz, DMSO-d6): δH 7.09- 6.80 (6H, m, H-3,

337

3', 4, 4' 6, 6'), 5.99-5.89 (2H, m, H-8, 8'), 5.10 (1H, d, J = 8.0 Hz, H-1''), 5.06- 4.98

338

(4H, m, H-9, 9'), 3.75 (1H, d, J = 8.2 Hz, H-5''), 3.33- 3.26 (6H, m, H-7, 7', 3'', 4''),

339

3.08 (1H, m, H-2''); 13C NMR (100 MHz, DMSO-d6): δC 170.7 (C-6''), 152.5 (C-2'),

340

152.3 (C-2), 138.4 (C-8), 138.0 (C-8'), 132.6 (C-5'), 131.8 (C-5), 131.6 (C-6'), 130.0

341

(C-6), 128.2 (C-4'), 128.1 (C-1, 4), 125.4 (C-1'), 116.2 (C-3'), 115.6 (C-9'), 115.2

342

(C-9), 114.7 (C-3), 100.4 (C-1''), 75.9 (C-5''), 75.0 (C-3''), 73.1 (C-4''), 71.5 (C-2''),

343

40.15 (C-7'), 38.89 (C-7).

344

Protein Tyrosine Phosphatase Inhibitory Activity Assay. Para-nitrophenyl

345

phosphate (pNPP) was used as the substrate of recombinant human protein tyrosine

346

phosphatase (PTP1B). The compounds were preincubated with the enzyme PTP1B at

347

room temperature for 5 min. The hydrolysis of pNPP catalyzed by PTP1B was

348

measured in a 100 l reaction system containing 50 mM HEPES, 5 mM DTT, 150

349

mM NaCl, 2 mM EDTA and 2 mM pNPP at pH 7.0. After incubation at 30 °C for 10

350

min, the PTP1B enzyme reaction was stopped by adding 50 l of NaOH (3 M). The

351

hydrolysate of pNPP has a strong absorption at 405 nm, and the OD values were used

352

to calculate the inhibition rates against PTP1B. A similar system without PTP1B 15



Page 16 of 35

353

protein was used as a blank, and compound CC06240, which is a benzofuran, was

354

used as a positive control 15.

355

Antioxidative activity assay. The antioxidative activities of these compounds were

356

evaluated by means of a 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay

357

Briefly, 10 µl of the sample (200 M in DMSO) was added to 96-well plates, and 190

358

µl of DPPH (200 M in anhydrous ethanol) was added. The reactions were incubated

359

at 37 °C without light for 30 min. Then, the absorbance values (A values) were

360

determined at 517 nm. The scavenging capacity for the free radical was Sc (%) =

361

(1−Asample/Acontrol)100%. The absorbance value of the reaction without the test

362

compounds was used as a control.

363

RESULTS

364

Effects of Temperature, pH and Divalent Metal Ions on the Activities of

365

UGT88D4 and UGT88D7. The genes for UGT88D4 and UGT88D7 were

366

successfully heterologously expressed in E. coli BL21 (DE3) as N-terminal

367

His6-tagged proteins. The two recombinant GATs were purified to homogeneity

368

through Ni-NTA metal affinity chromatography and used for the catalytic reactions

369

(Figure S1, Supporting Information, SI). To obtain the highest catalytic efficiency of

370

UGT88D4 and UGT88D7, the catalytic conditions, including the temperature, pH and

371

divalent metal ions, were optimized. As shown in Figure 1A, the catalytic activity of

372

UGT88D4 increased with increasing temperature in the 15‒45 °C interval but

373

decreased with increasing temperature in the 45‒65 °C interval, which is likely due to

374

denaturation of the enzyme. Thus, the optimum temperature was 45 °C. Similarly, for

375

UGT88D7, the optimum temperature was 40 °C (Figure 1B). The analysis of the

376

enzyme activity over the pH range 4.0 to 11.0 revealed that for both UGT88D4 and

377

UGT88D7, the maximum activity was observed at pH 7.0, while no activity was 16


16.

Page 17 of 35


378

observed at pH 9.0 (Figure 2). The assay of divalent cations showed that

379

both UGT88D4 and UGT88D7 were independent of the presence of metal ions, while

380

the catalytic activities of these two GATs were enhanced by the presence of Mg2+,

381

Ca2+, Fe2+ and Ba2+ (Figure 3).

382

Kinetic Parameters of UGT88D4 and UGT88D7. The kinetic parameters of the

383

enzymes in the catalytic glucuronidations were determined under the optimum

384

temperature and pH conditions. The Km values of UGT88D4 and UGT88D7 towards

385

hesperetin (7) were found to be 297.4 M and 253.2 M, respectively. The Vmax

386

values of UGT88D4 and UGT88D7 toward hesperetin (7) were 246.2 nmol min-1 mg-1

387

and 165.8 nmol min-1 mg-1, respectively (Figure 4).

388

Exploring the Glucuronidating Activities of UGT88D4 and UGT88D7. UGT88D4

389

(from Antirrhinum majus) and UGT88D7 (from Perilla frutescens) are flavonoid

390

7-O-glucuronosyltransferases (F7GATs), which are responsible for producing

391

specialized metabolites in Lamiales plants 2. To further utilize UGT88D4 and

392

UGT88D7 as tools for synthesizing structurally diverse and pharmacologically active

393

glucuronides, the catalytic activities of these GATs were explored. Structurally

394

diverse aglycons, including flavone (1‒2), flavonol (3‒5), flavanonol (6), flavonone

395

(7), flavan-3-ol (8), isoflavone (9), dihydrochalcone (10), coumarin (11),

396

anthraquinone (12), lignan (13), curcumin (14), naphthoquinone (15), and pentacyclic

397

triterpenoid (16‒17) derivatives, were selected as the candidate glucuronidation

398

acceptors, as shown in Figure 5C. The screening reactions under the optimal catalytic

399

conditions were analyzed by HPLC-UV/MS. The highest conversion rate of each

400

compound under the catalysis of these two GATs is shown in Figure 5A. Both

401

UGT88D4 and UGT88D7 could catalyze the reactions of compounds 1–13, which

402

have flavonoid, coumarin, anthraquinone, and lignan structural fragments. UGT88D7 17



403

showed the highest conversion rates with compounds 1, 4, 6, 8–10 and 13, while for

404

compounds 2, 3, 5, 7, 11 and 12, UGT88D4 exhibited conversion rates higher than

405

those of UGT88D7 (Figures 5A and S2‒S14, SI). UGT88D4 and UGT88D7 could not

406

only accept flavonoids (1‒10) as acceptors but also recognize substrates (11‒13) with

407

different types of skeletons. Furthermore, their catalytic promiscuity allowed these

408

two GATs to recognize different glucuronidation sites on a single acceptor. When

409

flavonoids (3–6 and 10) were used as substrates, both mono- and diglucuronides were

410

obtained as products based on HPLC-MS analysis. For example, when morin (5) was

411

used as the acceptor in UGT88D4-catalyzed glucuronidation, a total of six

412

glucuronidated products (5a, 5b, 5c, 5d, 5e and 5f) were generated (Figure 5A).

413

According to results of the HPLC-UV/ESI-MS analysis, 5a, 5e and 5f are

414

monoglucuronides while 5b, 5c and 5d are diglucuronides (Figure S6, SI). For

415

compounds 14–17, neither UGT88D4 nor UGT88D7 showed catalytic activity, which

416

was likely caused by the great differences between these structures and those of the

417

native substrate. Above all, UGT88D4 and UGT88D7, as potential enzymatic tools,

418

exhibited unprecedented catalytic promiscuity and high efficiency in the

419

glucuronidation of diverse natural products.

420

Establishing a Whole-Cell System for Production of Glucuronides. Although

421

UGT88D4 and UGT88D7 were able to produce various glucuronides, the use of

422

purified enzymes as biocatalysts in practical applications is associated with various

423

challenges. First, purified enzymes are usually unstable and easily denatured; second,

424

the sugar donor (UDPGA) is costly (USD 2000/g); finally, for the polyhydroxylated

425

acceptors, enzymatic glucuronidation lacks catalytic specificity, and multiple

426

glucuronides are produced, resulting in a low yield of the target product. Thus, to

427

overcome these challenges, a whole-cell in vivo glucuronidation catalytic system was 18


Page 18 of 35

Page 19 of 35


428

established. To supply the sugar donor UDPGA in a green and economically efficient

429

manner, an endogenous biosynthetic method for accessing UDPGA using E. coli

430

needed to be constructed 17–18. So, the UDP-glucose dehydrogenase gene (ugd), a key

431

gene in the UDPGA biosynthetic pathway of E. coli, was inserted into an expression

432

vector (pCDFDuet) carrying UGT88D4 or UGT88D7. The recombinant plasmids

433

were then individually transferred into E. coli BL21 (DE3), and the transformants

434

were designated as strain B-ugd-UGT88D4 and B-ugd-UGT88D7, respectively. E.

435

coli BL21 with pCDFDuet was used as the blank control. The gene ugd was

436

overexpressed to increase the level of endogenous UDPGA, which can be used as the

437

sugar donor for exogenous GATs in vivo. Therefore, by adding diverse aglycons into

438

the cell suspension culture systems, the endogenous UDPGA is directly used as the

439

sugar donor in coupling reactions, making additional UDPGA unnecessary.

440

As shown in Figure 5, comparing with the UGT88D4 and UGT88D7 catalyzing

441

reactions in vitro, the corresponding whole-cell biocatalysts also exhibited broad

442

catalytic promiscuity and high catalytic efficiency in recognizing substrates. To our

443

surprise, the whole-cell system showed high catalytic specificity in generating

444

products and only one monoglucuronidated product was generated for substrates (3, 4,

445

5, 6 and 10) with multiple potential glucuronidation sites. This catalytic specificity

446

makes whole-cell biocatalysts more efficient and applicable in synthesis of target

447

products. For flavonoids 1–7, the conversion rates in the whole-cell catalysis were

448

higher than those of in vitro enzyme catalysis. In contrast, the conversion rates of

449

compounds 8–11 in the whole-cell catalysis were significantly lower than those of

450

enzyme catalysis. What’ more, for compounds 12 and 13, no glucuronidated products

451

were detected with whole-cell in vivo catalysis. The low catalytic activities with

452

acceptors 8–13 may be caused by the permeability barrier of the cell envelope, which 19



Page 20 of 35

19.

453

leads to limited passage of the substrates through the cell envelope

454

transfer resistance of the cell envelope further makes the biocatalytic reaction difficult

455

to be accomplished

456

rates when catalyzed by the strain B-ugd-UGT88D4, and the other compounds (1, 4, 6,

457

8–10 and 13) showed higher conversion rates when catalyzed by the strain

458

B-ugd-UGT88D7. For compounds 14–17, neither enzyme catalysis nor whole-cell

459

catalysis showed catalytic activity. This means the trends in the reactivities of the

460

substrates in whole-cell catalysis were consistent with those of enzyme catalysis in

461

vitro.

462

Preparation of Novel and Bioactive Glucuronides by Utilizing a Biosynthetic

463

Method. Flavonoids are widely distributed in commonly consumed foods and in

464

traditional Chinese medicines. Their polyhydroxyl structures can promote

465

glucuronidation metabolism reactions in the organism, and further affect their

466

pharmacological activities, such as reducing toxicity, improving bioavailability and

467

changing pharmacokinetic characteristics

468

corresponding glucuronides also possess various biological activities. For instance,

469

puerarin-7-O-glucuronide, a water-soluble phase II metabolite of puerarin, prevents

470

angiotensin II-induced cardiomyocyte hypertrophy by reducing oxidative stress

471

Luteolin-7-O-glucuronide, a glucuronide isolated from plants such as Achillea

472

millefolium and Cannabis sativa, exhibited antimutagenic properties and cholagogic

473

effects

474

with sufficient regio- and stereoselectivity is necessary to the production of bioactive

475

glucuronides and drug discovery.

23–25.

20.

The mass

Compounds 2, 3, 5, 7, 11 and 12 had the highest conversion

21, 22.

What’s more, flavonoids and their

21.

Therefore, establishing a green and economical biocatalytic approach

476

To prepare novel and bioactive glucuronides, the scale-up whole-cell

477

biosynthetic reactions were performed with flavonoids 1–7 and 9 as acceptors. Based 20


Page 21 of 35


478

on the conversion rates, the strain B-ugd-UGT88D4 was used for the glucuronidation

479

of flavonoids 2, 3, 5, and 7, and the others (1, 4, 6 and 9) were glucuronidated with

480

B-ugd-UGT88D7. The reaction products were separated and characterized on the

481

basis of extensive spectroscopic analyses of MS and NMR.

482

New glucuronidated product 3a was selected as an example for structural

483

identification. MS analysis revealed that the major signal of product 3a was 176

484

atomic mass units greater than that of aglycon 3, which suggested the introduction of

485

one glucuronyl moiety. This was further confirmed by NMR spectra, which showed

486

signals characteristic of a sugar moiety (the anomeric proton (H-1'') signal at δH 5.23;

487

H-2''‒5'' signals at δH 3.16–3.96; the anomeric carbon (C-1'') signal at δC 98.7; one

488

C=O (C-6'') signal at δC 170.5 and four CH (C-2''–5'') signals at δC 71.4–75.8). The

489

location of the glucuronyl fragment was deduced from the HMBC correlation between

490

H-1′' and C-7 (δC 162.3). The large coupling constant (J = 7.3 Hz) of H-1′' indicated

491

the

492

kaempferol-7-O-β-D-glucuronide, which is a novel compound (Figures S19–S25, SI).

493

Similarly, the structures of products (4a–6a, 9a) were also confirmed to be new

494

compounds (Figures S26‒S46 and S51‒S57, SI). Products 1a, 2a and 7a were

495

identified as known compounds based on their MS, 1H NMR and

496

which were consistent with the literatures (Figures S15‒S18, S47 and S48, SI) 26–28.

presence

of

a

β-glycosidic

bond.

Thus,

3a

was

identified

13C

as

NMR data,

497

Finally, the products (1a–7a) were all identified as flavonoid-7-O-β-glucuronides,

498

which was in accordance with the fact that UGT88D4 and UGT88D7 were flavonoid

499

7-O-glucuronosyltransferases

500

stereoselectively generate β-type glycosidic bonds. It is also notable that the products

501

(3a–6a and 9a) are novel compounds (Figure 5C). The acceptors were all

502

glucuronidated at 7-OH, highlighting the high catalytic specificity of UGT88D4 and

(Figure

5).

What’s

21


more,

these

two

GATs


503

Page 22 of 35

UGT88D7 in the in vivo catalytic system.

504

UGT88D4 and UGT88D7 can also recognize epicatechin (8) and phloretin (10),

505

which possess basic structures similar to those of flavonoids (Figure 5). The external

506

enzyme UGT88D7 catalysis exhibited relatively high conversion rates and generated

507

one product (8a) from 8 and two products (10a and 10b) from 10. Finally, 8a was

508

isolated and characterized as epicatechin-7-O-β-D-glucuronide (Figures S49 and S50,

509

SI)

510

phloretin-4'-O-β-D-glucuronide and phloretin-2'-O-β-D-glucuronide, respectively

511

(Figures S58–S68, SI) 30.

29.

10a and 10b were also purified and further elucidated as new compounds,

512

To expand the applications of this biosynthetic method, different types of

513

bioactive substrates (11‒13) were also tested. 7-Hydroxy-4-methylcoumarin (11), a

514

coumarin, which is the general name for ortho-hydroxycinnamic acid lactones,

515

possesses

516

anti-hypertension activities

517

traditional Chinese herb Rhubarb (Da Huang) and has various efficacies, including

518

causing bacteriostasis, anti-inflammatory effects and protective effects on the liver

519

and kidney

520

officinalis Rehd. et Wils with special, long-lasting muscle relaxant effects 33. However,

521

the water solubilities of these natural products are low, and their oral bioavailabilities

522

are poor. To our delight, these substrates (11‒13) can be efficiently recognized by

523

UGT88D4 and UGT88D7, and their glucuronidated products were successfully

524

isolated

525

emodin-3-O-β-D-glucuronide

526

respectively (Figures S69–S79, SI)

527

concentration of emodin (12) is very low after oral administration. However, the oral

various

32.

and

biological 31.

activities

such

as

anti-HIV,

anti-tumor

and

Emodin (12) is an important component of the

Magnolol (13) is an active antibacterial component in Magnolia

characterized

as

4-methylcoumarin-7-O-β-D-glucuronide

(12a)

and 34, 35.

magnolol-O-β-D-glucuronide

(11a), (13a),

It has been reported that the plasma

22


Page 23 of 35


528

bioavailability of its glucuronidated product, emodin-3-O-glucuronide (12a), is

529

substantially

530

emodin-3-O-glucuronide (12a) is nearly 30 times larger than that of emodin (12) after

531

oral administration 36.

532

Pharmacological Activities of Biosynthesized Glucuronides. Compared with the

533

aglycons, the water solubilities of the glucuronidated products are certainly improved.

534

However, the pharmacological activities of the glucuronidated products remain

535

unknown. Hence, we evaluated the pharmacological activities of these glucuronides.

536

higher.

The

Cmax

(maximum

blood

concentration)

of

Protein tyrosine phosphatase 1B (PTP1B), a negative regulator of insulin signal 37.

537

transduction, is a target for type 2 diabetes and obesity drug discovery

PTP1B

538

inhibitors can effectively treat type 2 diabetes and obesity. The experimental results

539

indicated that kaempferol-7-O-β-D-glucuronide (3a) was a potent inhibitor of PTP1B

540

with an inhibition rate of 99.2%, while the inhibition rate of its aglycon, kaempferol,

541

was only 10.9% at a concentration of 10 M (Table S1, SM). The IC50 value of

542

O-glucuronide 3a against PTP1B was 8.02  10-6 M.

543

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was used to determine the

544

ability of the compounds to eliminate free radicals. The experimental results showed

545

that the free radical scavenging capacities of these compounds (2a, 3a, 3, 7, 8 and 12a)

546

were greater than that of vitamin C, which means that these glucuronides possess

547

significant antioxidant activities. Interestingly, glucuronides 2a and 12a had stronger

548

antioxidant activities than their corresponding aglycons 2 and 12 (Table S2, SM).

549

DISCUSSION

550

Expanding the Applications of GATs in the Biosynthesis of Glucuronides

551

UGT88D4 and UGT88D7 are flavonoid 7-O-glucuronosyltransferases (F7GAT), 23



552

which transfer glucuronic acid from UDPGA (sugar donor) to the 7-OH group of

553

flavonoids (sugar acceptor) 2. Generally, UGT88D4 and UGT88D7 are considered to

554

be highly regioselective. However, in vitro, UGT88D4 and UGT88D7 exhibit

555

catalytic promiscuity and can functionalize various hydroxyl groups with high

556

efficiency, not only the 7-OH group. In addition to the reported flavonoid acceptors,

557

these two enzymes also recognize other structurally diverse acceptors, including

558

coumarins, anthraquinones and lignans. Interestingly, UGT88D4 and UGT88D7 were

559

able to not only monoglucuronidate diverse acceptors but also diglucuronidate

560

acceptors with multiple hydroxyl groups, generating diglucuronides. Therefore,

561

according to our results, UGT88D4 and UGT88D7 exhibited catalytic promiscuity in

562

both recognizing acceptors and generating products. The applications of these two

563

GATs were expanded, and they can be utilized in the enzymatic synthesis of diverse

564

new bioactive glucuronides.

565

Construction of an Engineered Strain Supplying UDPGA for the Biosynthesis of

566

Glucuronides

567

By coexpression of ugd and GATs in the strain E. coli BL21, the level of

568

endogenous UDPGA was increased, and the exogenous GATs could then mediate

569

glucuronidation in the cells. Notably, for most flavonoid acceptors, the conversion

570

rates obtained with the whole-cell system in vivo were higher than that achieved with

571

pure enzyme in vitro. Moreover, compared with enzymatic reactions in vitro,

572

UGT88D4 and UGT88D7 exhibited higher regioselectivity in vivo, and only one

573

glucuronidated product was generated from each acceptor, and all the products were

574

flavonoid-7-O-glucuronides. The different catalytic activities of UGT88D4 and

575

UGT88D7 in vivo and in vitro might be because the level of intracellular UDPGA is

576

stable and relatively low in vivo, and it is not sufficient to support the glucuronidation 24


Page 24 of 35

Page 25 of 35


577

of multiple hydroxyl groups on the substrates. Therefore, the hydroxyl group at C7,

578

which is the native glucuronidation site for UGT88D4 and UGT88D7, was

579

preferentially derivatized in vivo. Finally, a total of eight flavonoid-7-O-glucuronides

580

were efficiently prepared by whole-cell catalysis without adding expensive sugar

581

donors. Unlike the enzymatic reactions in vitro, the whole-cell catalysis system was

582

more economical, selective and stable, and it provides a potential method for the

583

efficient and economical synthesis of bioactive glucuronides for drug discovery. To

584

our knowledge, this is the first reported whole-cell catalysis method of

585

glucuronidation with such broad substrate scope.

586

In this work, two biocatalytic systems, pure enzyme catalysis in vitro and

587

whole-cell catalysis in vivo, were successfully constructed. With these two

588

biocatalytic systems, a total of fourteen O-glucuronides were efficiently synthesized,

589

and seven of them were new compounds. Furthermore, one of the new compounds,

590

kaempferol-7-O--D-glucuronide (3a), potently inhibited PTP1B making it a potential

591

drug lead for the treatment of type 2 diabetes. Bio-glucuronidation mediated by GATs

592

has shown great potential in synthesizing diverse bioactive glucuronides and it will be

593

also helpful for the simulation of human drug metabolism in drug research. What’s

594

more, novel GATs with broader substrate scope and higher catalytic efficiency need

595

to be found in the future for glucuronidation of drugs and food ingredients with more

596

complicated structures.

597

Supporting Information

598

HPLC-DAD/ESI-MS spectra of the enzymatic reactions. UV, IR, 1H and 13C NMR,

599

HSQC, HMBC, and HRESIMS spectra of the glucuronides. The results of evaluating

600

the pharmacological activities of the aglycons and corresponding glucuronides.

601

Funding 25



Page 26 of 35

602

This work was financially supported by National Natural Science Foundation of

603

China (No. 21572277), CAMS Innovation Fund for Medical Sciences (No.

604

CIFMS-2016-I2M-3-012),

605

(2018ZX09711001-006), and Beijing Key Laboratory of Non-Clinical Drug

606

Metabolism and PK/PD Study (No. Z141102004414062).

607

Notes

608

The authors declare no conflicts of interest.

609

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31



Figure Legends Figure 1. Effects of temperature on the enzyme activities of UGT88D4 (A) and UGT88D7 (B). Hesperidin (7) was used as the acceptor, and UDPGA was used as the sugar donor. The error bars show the SD (n = 3). Figure 2. Effects of the pH of the buffers on the enzyme activities of UGT88D4 (A) and UGT88D7 (B). Hesperidin (7) was used as the acceptor, and UDPGA was used as the sugar donor. The error bars show the SD (n = 3). Figure 3. Effects of various divalent metal ions on the enzyme activities of UGT88D4 (A) and UGT88D7 (B). Hesperidin (7) was used as the acceptor, and UDPGA was used as the sugar donor. The error bars show the SD (n = 3). ND means not detected. Figure 4. The nonlinear regressions of the Michaelis–Menten equation for UGT88D4 (A) and UGT88D7 (B). Hesperidin (7) was used as the acceptor, and UDPGA was used as the sugar donor. Figure 5. Exploring the applications of UGT88D4 and UGT88D7 for the glucuronidation of structurally diverse substrates. (A) The conversion rates and corresponding products of substrates 1‒13 when catalyzed by the pure enzymes UGT88D4 and UGT88D7 in vitro. (B) The conversion rates and corresponding products of substrates 1‒13 when catalyzed by whole cells in vivo. (C) Structures of substrates 1‒17 and the corresponding products of the enzymatic reactions.

32


Page 32 of 35

Page 33 of 35


Figure 1

Figure 2

Figure 3

33



Figure 4

Figure 5

34


Page 34 of 35

Page 35 of 35


Graphical Abstract

35


Enzymatic synthesis of bioactive O-glucuronides using plant

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