Enzymatic Synthesis of Bioactive O-Glucuronides Using Plant Glucuronosyltransferases

Article abstract of DOI:10.1021/acs.jafc.9b01769

Many O-glucuronides exhibiting various pharmacological activities have been found in nature and in drug metabolism. The glucuronidation of bioactive natural products or drugs to generate glucuronides with better activity and druggability is important in drug discovery and research. In this study, by using two uridine diphosphate (UDP)-dependent glucuronosyltransferases (GATs, UGT88D4 and UGT88D7) from plants, we developed two glucuronidation approaches, pure enzyme catalysis in vitro and recombinant whole-cell catalysis in vivo, to efficiently synthesize bioactive O-glucuronides by the glucuronidation of natural products. In total, 14 O-glucuronides with different structures, including flavonoids, anthraquinones, coumarins, and lignans, were obtained, 7 of which were new compounds. Furthermore, one of the biosynthesized O-glucuronides, kaempferol-7-O-β-d-glucuronide (3a), potently inhibited protein tyrosine phosphatase (PTP) 1B with an IC₅₀ value of 8.02 × 10^-6 M. Some of the biosynthesized O-glucuronides also exhibited significant antioxidant activities.

Full text of DOI:10.1021/acs.jafc.9b01769

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Biotechnology and Biological Transformations
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
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Enzymatic synthesis of bioactive O-glucuronides using plant
glucuronosyltransferases
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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
Jungui Daia, b, c *
b
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a State Key Laboratory of Bioactive Substance and Function of Natural Medicines;
c
CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs; NHC Key
Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese
Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan
Street, Beijing, 100050, P.R. China
*
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Corresponding author. Tel: +86 10 63165762; fax: +86 10 63017757. E-mail:
keboxie@imm.ac.cn (K. X.), Tel: +86 10 63165195; fax: +86 10 63017757.
E-mail: jgdai@imm.ac.cn (J. D.).
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Abstract: Many O-glucuronides exhibiting various pharmacological activities have
been found in nature and in drug metabolism. The glucuronidation of bioactive natural
products or drugs to generate glucuronides with better activity and druggability is
important in drug discovery and research. In this study, by using two uridine
diphosphate (UDP)-dependent glucuronosyltransferases (GATs, UGT88D4 and
UGT88D7) from plants, we developed two glucuronidation approaches, pure enzyme
catalysis in vitro and recombinant whole-cell catalysis in vivo, to efficiently
synthesize bioactive O-glucuronides by the glucuronidation of natural products. In
total, fourteen O-glucuronides with different structures, including flavonoids,
anthraquinones, coumarins and lignans, were obtained, 7 of which were new
compounds. Furthermore, one of the biosynthesized O-glucuronides,
kaempferol-7-O-β-D-glucuronide (3a), potently inhibited protein tyrosine
phosphatase (PTP) 1B with an IC50 value of 8.02  106 M. Some of the
biosynthesized O-glucuronides also exhibited significant antioxidant activities.
Key words: O-glucuronides; glucuronidation; glucuronosyltransferases; enzyme
catalysis; whole-cell catalysis
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INTRODUCTION
Glucuronidation occurs during phase II conjugative drug metabolism and the
metabolism of the chemical components of foods, and these processes are catalyzed
by glucuronic acid transferases/glucuronosyltransferases (GATs) 1‒3. Usually, the
glucuronide metabolites of drugs have higher water solubility and polarity than the
parent drug compounds. Thus, glucuronidation serves as an integral step in
transforming lipophilic substrates into hydrophilic glucuronides and facilitates the
drug transport and elimination, which is generally regarded as a detoxification
reaction 4. However, some glucuronide metabolites have more potent pharmacological
activities than the corresponding aglycons, and they are the real active drug molecule
exerting the pharmacological effect. For example, morphine-3-O-glucuronide (M3G)
and morphine-6-O-glucuronide (M6G) are the two in vivo glucuronide metabolites of
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morphine . M3G is the main metabolite but without analgesic activity. In contrast,
M6G, the minor metabolite, is the potent opioid receptor agonist involved in the
analgesic effect of morphine, and the anesthetic effect of M6G is 100 times more
potent than that of morphine 6.
Natural glucuronides are widely distributed in plants, and many natural
glucuronides with various pharmacological activities have been isolated from plants.
For instance, the main component of the clinical injectable drug breviscapine is
scutellarin-7-O-glucuronide, which has been used in the clinic for more than ten years
to treat cardio-cerebral vascular diseases 7, 8. Apigenin-7-O-glucuronide is distributed
in plants such as Scutellaria baicalensis and Chrysanthemum morifolium and has a
variety of pharmacological activities, including anti-atherosclerosis, antioxidant,
9‒12
anti-inflammatory, anti-complement, and aldose reductase inhibitory activities
.
Unlike mammalian GATs, which are generally considered physiological
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membrane-bound enzymes, plant GATs are soluble and able to recognize extremely
diverse sugar acceptors. Chemical routes to the synthesis of glucuronides face
challenges associated with regioselectivity, stereoselectivity, protection and
deprotection of functional groups. Therefore, enzymatic glucuronidations catalyzed
by soluble plant GATs are a potential approach for generating target bioactive
glucuronides in drug discovery.
Hence, to construct efficient methods for the synthesis of bioactive glucuronides,
two plant GATs (UGT88D4 and UGT88D7) were employed and developed as
enzyme tools in this study. Two kinds of catalytic systems, pure enzyme catalysis in
vitro with high efficiency and whole-cell catalysis in vivo without adding expensive
sugar donors, were both successfully established and utilized to synthesize novel and
structurally diverse bioactive glucuronides.
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MATERIALS AND METHODS
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General. Uridine diphosphate glucuronic acid (UDPGA) was purchased from
Sigma-Aldrich (St Louis, USA). Compounds 1‒17 (Figure 5C) were purchased from
Nanjing Zelang Biological Technology Co., Ltd. (Nanjing, China). All restriction
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Optical rotations were acquired with a Perkin-Elmer Model-343 digital polarimeter
(PerkinElmer Inc., USA). UV spectra were measured with a Jasco J-815 circular
dichroism (CD) spectrometer (Jasco Corp., Japan). IR spectra were obtained on a
Nicolet 5700 FT-IR spectrometer (Thermo Electron Scientific Instrument Corp.,
USA). Compounds were characterized by 1D and 2D NMR on Bruker 400/500/600
spectrometers (Bruker BioSpin AG, Switzerland). High-resolution electrospray
ionization mass spectrometry (HRESIMS) data were measured with an
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ESI-FTICR-MS (LTQ-FT Ultra, ThermoFisher Scientific, USA). The products of
enzymatic reactions were analyzed on an Agilent 1260 series HPLC system (Agilent
Technologies, Germany) coupled with an LCQ Fleet ion trap mass spectrometer
(Thermo Electron Corp., USA) equipped with an ESI source. Samples were analyzed
on a Shiseido Capcell pak C18 MG III column (4.6 mm × 250 mm; particle size, 5 μm,
Shiseido Co., Ltd., Japan). Purified products were obtained on a Thermo SRD-3200
series HPLC with a Shiseido Capcell pak C18 MG II column (10 mm × 250 mm;
particle size, 5 μm, Shiseido Co., Ltd., Tokyo, Japan).
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Gene Cloning and Plasmid Construction. The genes of UGT88D4 (AmUGT10;
Gene ID AB362988), UGT88D7 (PfUGT50; Gene ID AB362991), and UDP-glucose
dehydrogenase (ugd; Gene ID 946571) were chemically synthesized (Sangon Biotech,
Shanghai, China) with optimized codon usage for expression in Escherichia coli.
AmUGT10 and PfUGT50 were subcloned into the EcoRI/NotI site of the pCDFDuet-1
(Novagen, Germany) expression vector, resulting in plasmids named
pCDF-UGT88D4 and pCDF-UGT88D7, respectively. Subsequently, ugd was
subcloned into the NdeI/XhoI site of pCDF-UGT88D4 and pCDF-UGT88D7,
resulting in plasmids named pCDF-UGT88D4-ugd and pCDF-UGT88D7-ugd,
respectively. Then, the recombinant plasmids were introduced into E. coli BL21 (DE3)
(TransGen Biotech, China) for heterologous expression after verification of the
sequences.
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Expression and Purification of UGT88D4 and UGT88D7. The confirmed E. coli
BL21 harboring pCDF-UGT88D4/pCDF-UGT88D7 was cultured in 400 mL of
Luria-Bertani (LB) medium containing 40 μg/mL streptomycin sulfate. Cells were
grown at 37 °C with shaking at 200 rpm until the OD600 reached 0.6. Then, the
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expression of UGT88D4 and UGT88D7 was performed by induction with 0.2 mM
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isopropyl β-D-thiogalactoside (IPTG) for 20 h at 18 °C. The cells were collected by
centrifugation at 8,000 g for 10 min at 4 °C and resuspended in 20 mL of binding
buffer (20 mM imidazole, 0.5 M NaCl, 20 mM phosphate buffer, pH 7.4) containing 1
mM phenylmethylsulfonyl fluoride (PMSF) and 1 mg/mL lysozyme and were
disrupted by sonication. The supernatants containing soluble proteins were filtered
0.45 μm syringe filter units after centrifuging at 10,000 g for 40 min at 4 °C. The
obtained supernatants were immediately loaded onto a column of Ni-NTA resin (GE,
USA) which was pre-equilibrated with binding buffer. Subsequently, the resin was
washed with binding buffer to remove unbound proteins. The elution was performed
with elution buffers containing 50–500 mM imidazole, and the purified protein was
desalted with desalting buffer (50 mM NaCl, 1 mM dithiothreitol, 4% glycerol, 50
mM Tris-HCl buffer, pH 7.4) by concentration and dilution using an Amicon-Ultra 30
centrifugal filter unit (Millipore, USA). The protein purity was verified by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the
concentration of pure protein was determined by a Protein Quantitative Kit (TransGen
Biotech, China) 13.
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Effects of Temperature, pH and Divalent Metal Ions on the Catalytic Activities of
UGT88D4 and UGT88D7. To optimize the reaction temperature, the reactions were
performed at different temperatures ranging from 15–65 °C. To evaluate the effects of
pH, reactions were carried out in reaction buffers with pH values of 4.0–6.0 (50 mM
citric acid-sodium citrate buffer), 6.0–8.0 (50 mM Na2HPO4-NaH2PO4 buffer),
8.0–9.0 (50 mM Tris-HCl buffer) and 9.0–11.0 (50 mM Na2CO3-NaHCO3 buffer). To
investigate the dependence of divalent metal ions for UGT88D4 and UGT88D7, 5
mM solutions of BaCl2, CaCl2, CoCl2, CuCl2, FeCl2, MgCl2, MnCl2, NiCl2, ZnCl2 and
EDTA were added to reactions with the final concentration of 5 mM, respectively.
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Reactions without adding metal ions were used as a control. All assays were carried
out with UDPGA as the sugar donor and hesperetin (7) as the acceptor.
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Kinetic Parameters of UGT88D4 and UGT88D7. Kinetic analysis of recombinant
UGT88D4 and UGT88D7 towards hesperetin (7) was performed in 100 l aliquots
with 0.05 mg/mL purified enzyme, 50 mM Na2HPO4-NaH2PO4 buffer (pH 7.0), 5
mM UDPGA and varying concentrations of 7 (100–800 M). For UGT88D4, the
reactions were incubated at 45 °C for 30 min; for UGT88D7, the reactions were
incubated at 40 °C for 30 min and then stopped by adding 200 l of methanol.
Subsequently, the reaction mixtures were centrifuged to collect the supernatant, and
aliquots were analyzed by HPLC-UV. The kinetic parameters were calculated by
nonlinear regression of the Michaelis–Menten equation using Origin 8.0 14.
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Glucuronidation Reactions Catalyzed by UGT88D4 and UGT88D7 in vitro. The
reaction mixtures contained 0.5 mM aglycons (1‒17), 1 mM UDPGA and 1 mg/mL
purified enzyme in a final volume of 100 μL. Activity assays, initiated by the addition
of enzymes, were performed at 30 °C for 12 h and terminated by addition of 200 μL
ice cold methanol. Subsequently, the mixtures were centrifuged at 15,000 g for 30
min to collect the supernatant, and aliquots were analyzed by HPLC-UV/ESIMS.
Control reactions without adding the enzyme or UDPGA were performed. The elution
conditions of HPLC analysis were as follows: solvent A (0.1% formic acid aqueous
solution), solvent B (methanol); flow rate (1 mL/min); gradient (15−100% B in 30
min followed by 100% B for 5 min for 1−11 and 13−17; 50−100% B in 20 min
followed by 100% B for 5 min for 12).
Glucuronidation Reactions Catalyzed by Whole-Cell Biocatalyst in vivo. An
overnight culture of E. coli BL21 (DE3) harboring pCDF-ugd-UGT88D4 or
pCDF-ugd-UGT88D7 was inoculated into 50 mL of fresh LB medium containing an
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appropriate antibiotic (40 μg/mL streptomycin sulfate). Cells were grown at 37 °C
with shaking (200 rpm). When the OD600 reached 0.6, genes ugd and UGT in E. coli
were induced by adding IPTG to a final concentration of 0.2 mM. Subsequently, the
cultures were shaken at 18 °C for 20 h. Cells were harvested by centrifugation and
resuspended in fresh M9 medium (Na2HPO4 6.8 g/L, KH2PO4 3.0 g/L, NaCl 0.5 g/L,
NH4Cl 1.0 g/L, MgSO4 1.2 g/L, and CaCl2 1.1 g/L) containing 2% glucose and
antibiotics. Cell density was maintained at OD600 = 3.0, and the substrates (1–17) for
the biotransformation were added into the culture at a final concentration of 0.7 mM,
respectively and the total volume of each reaction was 1 mL. Then, the cultures were
incubated at 30 °C for 24 h, and the supernatants were analyzed by HPLC-UV/MS. E.
coli BL21 (DE3) with pCDFDuet served as the blank control.
Preparing the Representative Glucuronides by Biocatalysis. For products prepared
by whole cells in vivo, the synthesis reactions were performed as follows. Chrysin (1,
10.7 mg) was dissolved in 50 l of dimethyl sulfoxide (DMSO), and this solution was
combined with 60 mL of a M9 cell suspension culture of strain B-ugd-UGT88D7 at a
final concentration of 0.7 mM with a cell density of OD600 = 3.0. The reaction was
performed at 30 °C with shaking at 200 rpm for 24 h, and the reaction mixture was
centrifuged at 10,000 g for 30 min to remove precipitated cells. The supernatant
containing product 1a was passed through an Amberlite XAD-16 macroporous resin
column (50 mL, Rohm & Haas Corp., USA). Aliquots (250 mL) of ultrapure water,
50% (v/v) ethanol and 100% (v/v) ethanol were sequentially loaded into the column
and then eluted at a flow rate of 1 mL/min. The product 1a was eluted with 50% (v/v)
ethanol, and the fraction was concentrated under reduced pressure. The residue
containing 1a was further purified by reversed-phase semipreparative HPLC (solvent
A: 0.1% formic acid aqueous solution; solvent B: methanol; flow rate: 3 mL/min;
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gradient: 30−90% B in 20 min followed by 100% B for 5 min) to afford 1a.
Following the above procedure, the other substrates, including diosmetin (2),
kaempferol (3), fisetin (4), morin (5), (2R, 3R)-dihydroquercetin (6), hesperetin (7)
and biochanin A (9), were also glucuronidated by whole cells in vivo, and
corresponding glucuronides 2a, 3a, 4a, 5a, 6a, 7a and 9a were successfully obtained.
For products prepared by purified enzymes in vitro, the reactions were performed
as follows. Epicatechin (8, 10.4 mg) was dissolved in 50 l of DMSO, and this
solution along with UDPGA (35 mg) were added to 10 mL of desalting buffer
containing 1 mg/mL purified UGT88D7 extracted from 4 g (wet weight) of induced E.
coli cells containing pCDF-UGT88D7. The reactions were performed at 30 °C with
shaking at 90 rpm for 48 h. After centrifugation, the supernatant containing product
8a was passed through an Amberlite XAD-16 macroporous resin column. Target
product 8a was eluted with 50% (v/v) ethanol, and this fraction was concentrated. The
residue containing 8a was further purified by reversed-phase semipreparative HPLC
(solvent A: 0.1% formic acid aqueous solution; solvent B: methanol; flow rate: 3
mL/min; gradient: 15−58% B in 15 min followed by 100% B for 5 min) to give 8a.
Following the above procedure, the other substrates, including phloretin (10),
7-hydroxy-4-methylcoumarin (11), emodin (12) and magnolol (13), were also
glucuronidated by purified UGT88D4 and UGT88D7 in vitro, and corresponding
glucuronides 10a, 10b, 11a, 12a and 13a were obtained.
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The Spectroscopic Data of Prepared Glucuronides. Chrysin-7-O-β-D-glucuronide
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(1a, isolated yield 36%). ESI-MS m/z 429.30 [M  H], 859.11 [2M−H]; H NMR
(500 MHz, DMSO-d6): δH 12.82 (1H, s, 5-OH), 8.19 (2H, d, J = 7.6 Hz, H-2', 6'),
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
(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'',
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4'', 5''); C NMR (125 MHz, DMSO-d6): δC 182.2 (C-4), 171.0 (C-6''), 163.7 (C-2),
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'),
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
(C-5''), 74.5 (C-3''), 72.9 (C-2''), 71.7 (C-4'').
Diosmetin-7-O-β-D-glucuronide (2a, isolated yield 67%). ESI-MS m/z 475.48
[MH], 951.21 [2MH]; H NMR (500 MHz, DMSO-d6): δH 12.95 (1H, s, OH-5),
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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 =
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 =
13
7.2 Hz, H-1''), 3.87 (3H, s, -OCH3), 3.53-3.11 (4H, m, H-2'', 3'', 4'', 5''); C NMR
(100 MHz, DMSO-d6): δC 182.0 (C-4), 171.0 (C-6''), 164.1 (C-2), 163.0 (C-7), 161.5
(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'),
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
(C-5''), 72.9 (C-2''), 71.7 (C-4'').
Kaempferol-7-O-β-D-glucuronide (3a, novel, isolated yield 57%). Yellow powder;
[]25 D −84 (c 0.40, DMSO); UV (MeOH) λmax (log ε) 366 (3.52), 323 (3.25), 267
(3.49), 207 (3.61) nm; IR νmax: 3345, 2924, 1601, 1010, 950, 831 cm-1; ESI-MS m/z
461.50 [MH], 923.13 [2MH]; HRESIMS m/z 461.0508 [MH] (calcd for
C21H17O12, 461.0715); 1H NMR (600 MHz, DMSO-d6): δH 12.50 (1H, s, OH-5), 10.18
(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'),
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,
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
MHz, DMSO-d6): δC 176.1 (C-4), 170.5 (C-6''), 162.3 (C-7), 160.4 (C-5), 159.4 (C-4'),
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'),
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
(C-2''), 71.4 (C-4'')
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Fisetin-7-O-β-D-glucuronide (4a, novel, isolated yield 68%). Yellow powder; []25
D −63 (c 1.46, DMSO); UV (MeOH) λmax (log ε) 363 (3.52), 316 (3.23), 251 (3.45),
207 (3.78) nm; IR νmax: 3296, 2925, 1611, 1257, 1019, 952, 770 cm-1; ESI-MS m/z
461.63 [MH], 923.27 [2MH]; HRESIMS m/z 463.0876 [M+H]+ (calcd for
C21H19O12, 463.0871); 1H NMR (400 MHz, DMSO-d6): δH 9.30 (2H, br.s, OH-3', 4'),
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,
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),
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,
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H-5''), 3.66-3.30 (3H, m, H-2'', 3'', 4''); C NMR (100 MHz, DMSO-d6): δC 171.9
(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'),
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
(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''),
72.9 (C-4'').
Morin-7-O-β-D-glucuronide (5a, novel, isolated yield 78%). Yellow powder; []25 D
−113 (c 0.48, DMSO); UV (MeOH) λmax (log ε) 371 (3.14), 293 (3.06), 256 (3.34),
210 (3.57) nm; IR νmax: 3356, 2920, 1601, 1292, 952, 710 cm-1; ESI-MS m/z 477.49
[MH], 955.29 [2MH]; HRESIMS m/z 479.0814 [M+H]+ (calcd for C21H19O13,
1
479.0820); H NMR (400 MHz, DMSO-d6): δH 7.71 (1H, d, J = 1.9 Hz, H-3'), 7.56
(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
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
= 9.5 Hz, H-5''), 3.51-3.18 (3H, m, H-2'', 3'', 4''); 13C NMR (150 MHz, DMSO-d6): δC
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
(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'),
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
(C-2''), 71.7 (C-3'').
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(2R, 3R)-Dihydroquercetin-7-O-β-D-glucuronide (6a, novel, isolated yield 50%).
Yellow powder; []25 D −88 (c 1.16, DMSO); UV (MeOH) λmax (log ε) 375 (3.25),
285 (3.76), 209 (3.95) nm; IR νmax: 3385, 3309, 2916, 1643, 1024, 827, 709 cm-1;
ESI-MS m/z 479.37 [M  H]; HRESIMS m/z 481.0977 [M + H]+ (calcd for
C21H21O13, 481.0977); 1H NMR (500 MHz, DMSO-d6): δH 11.81 (1H, s, OH-5), 9.07
(2H, s, OH-3', 4'), 6.90 (1H, s, H-2'), 6.78-6.73 (2H, overlapped, H-5', 6'), 6.17 (2H, s,
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,
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,
m, H-2'', 3'', 4''); 13C NMR (100 MHz, DMSO-d6): δC 198.7 (C-4), 170.5 (C-6''),
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
(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),
75.8 (C-5''), 74.8 (C-3''), 72.7 (C-2''), 71.7 (C-3), 71.4 (C-4'').
Hesperetin-7-O-β-D-glucuronide (7a, isolated yield 50%). ESI-MS m/z 477.34 [M 
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H]; H NMR (500 MHz, DMSO-d6): δH 12.04 (1H, s, OH-5), 9.16 (1H, s, OH-3'),
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 =
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,
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,
H-3axial, 2'',3'',4''), 2.75 (1H, dd, J =17.1 Hz, 2.0 Hz, H-3equatorial); 13C NMR (125 MHz,
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
(C-4'), 146.5 (C-3'), 130.9 (C-1'), 117.8 (C-6'), 114.4 (C-2'), 112.0 (C-5'), 103.3
(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
(C-2''), 71.5 (C-3''), 55.7 (-OCH3), 42.2 (C-3).
Epicatechin-7-O-β-D-glucuronide (8a, isolated yield 61%). ESI-MS m/z 465.46 [M 
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H]; 1H NMR (400 MHz, Acetone-d6): δH 7.07 (1H, s, H-2'), 6.86 (1H, d, J = 8.7 Hz,
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),
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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 =
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,
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
MHz, Acetone-d6): δC 170.2 (C-6''), 157.9 (C-7), 157.5 (C-5), 157.1 (C-9), 145.3
(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
(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
(C-4''), 66.7 (C-3), 29.1 (C-4).
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Biochanin A-7-O-β-D-glucuronide (9a, novel, isolated yield 35%). White powder;
[]25 D −150 (c 0.46, DMSO); UV (MeOH) λmax (log ε) 326 (3.01), 261 (3.93), 207
(3.73) nm; IR νmax: 3359, 2916, 1657, 1026, 828, 703, 566 cm-1; ESI-MS m/z 459.41
[M  H], 919.33 [2M  H]; HRESIMS m/z 461.1067 [M + H]+ (calcd for C22H21O11,
461.1078); 1H NMR (400 MHz, DMSO-d6): δH 12.91 (1H, s, OH-5), 8.47 (1H, s, H-2),
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'),
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
=9.2 Hz, H-5''), 3.79 (3H, s, -OCH3), 3.38-3.27 (3H, m, H-2'', 3'', 4''); 13C NMR (150
MHz, DMSO-d6): δC 180.4 (C-4), 170.4 (C-6''), 162.7 (C-7), 161.7 (C-5), 159.2 (C-4'),
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'),
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
(C-4''), 71.4 (C-3''), 55.2 (-OCH3).
Phloretin-4'-O-β-D-glucuronide (10a, novel, isolated yield 58%). White powder;
[]25 D −91 (c 0.45, DMSO); UV (MeOH) λmax (log ε) 280 (3.41), 225 (3.44) nm; IR
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νmax: 3282, 2921, 1630, 1261, 1083, 946, 740, 583 cm-1; ESI-MS m/z 449.41 [M  H],
899.23 [2M  H]; HRESIMS m/z 433.1128 [M + H  H2O]+ (calcd for C21H21O10,
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433.1129); H NMR (600 MHz, DMSO-d6): δH 12.37 (2H, s, OH-2', 6'), 9.15 (1H, s,
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,
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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,
m, H-2'', 3'', 4'', α-CH2), 2.78 (2H, t, J = 7.8 Hz, β-CH2); 13C NMR (150 MHz,
DMSO-d6): δC 205.1 (C=O), 170.3(C-6''), 163.8 (C-2', 6'), 163.0 (C-4'), 155.4 (C-4),
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'),
75.7 (C-5''), 75.3 (C-3'') , 72.8 (C-2''), 71.4 (C-4''), 45.8 (C-α), 29.3 (C-β).
Phloretin-2'-O-β-D-glucuronide (10b, isolated yield 9%). ESI-MS m/z 449.42 [M 
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H]; H NMR (400 MHz, DMSO-d6): δH 13.52 (1H, s, OH-6'), 9.10 (1H, s, OH-4),
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
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
H, m, H-2'', 3'', 4'', 5'', α-CH2), 2.78 (2H, t, J = 7.3 Hz, β-CH2); 13C NMR (150 MHz,
DMSO-d6): δC 204.6 (C=O), 171.0 (C-6''), 165.4 (C-6'), 164.6 (C-4'), 160.6 (C-2'),
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''),
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),
29.0 (C-β).
4-Methylcoumarin-7-O-β-D-glucuronide (11a, isolated yield 75%). ESI-MS m/z
351.47 [M  H], 703.36 [2M  H]; 1H NMR (400 MHz, DMSO-d6): δH 7.70 (1H, d,
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,
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,
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-CH3); C NMR (100 MHz, DMSO-d6): δC 170.5 (C-6'), 160.1 (C-2), 159.7 (C-7),
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
(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).
Emodin-3-O-β-D-glucuronide (12a, novel, isolated yield 24%). Yellow powder;
[]25 D −34 (c 0.27, DMSO); UV (MeOH) λmax (log ε) 430 (3.22), 284 (3.44), 262
(3.49), 225 (3.62) nm; IR νmax: 3387, 2921, 1629, 1086, 761 cm-1; ESI-MS m/z 445.25
[M  H], 891.08 [2M  H]; HRESIMS m/z 445.0562 [M  H] (calcd for C21H17O11,
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445.0765); H NMR (400 MHz, DMSO-d6): δH 7.50 (1H, s, H-5), 7.24 (1H, s, H-4),
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 =
6.5 Hz, H-5'), 3.48-3.25 (3H, m, H-2', 3', 4'), 2.42 (3H, s, -CH3); 13C NMR (100 MHz,
DMSO-d6): δC 190.1 (C-9), 181.1 (C-10), 170.4 (C-6'), 164.0 (C-3), 163.6 (C-8),
161.5 (C-1), 148.6 (C-6), 134.9 (C-4a), 132.8 (C-10a), 124.2 (C-7), 120.6 (C-5),
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
(C-4'), 72.8 (C-2'), 71.4 (C-3'), 21.6 (-CH3).
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Magnolol-O-β-D-glucuronide (13a, isolated yield 79%). ESI-MS m/z 441.36 [M 
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H], 883.00 [2M  H]; H NMR (400 MHz, DMSO-d6): δH 7.09- 6.80 (6H, m, H-3,
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
(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''),
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3.08 (1H, m, H-2''); C NMR (100 MHz, DMSO-d6): δC 170.7 (C-6''), 152.5 (C-2'),
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
(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
(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''),
40.15 (C-7'), 38.89 (C-7).
Protein Tyrosine Phosphatase Inhibitory Activity Assay. Para-nitrophenyl
phosphate (pNPP) was used as the substrate of recombinant human protein tyrosine
phosphatase (PTP1B). The compounds were preincubated with the enzyme PTP1B at
room temperature for 5 min. The hydrolysis of pNPP catalyzed by PTP1B was
measured in a 100 l reaction system containing 50 mM HEPES, 5 mM DTT, 150
mM NaCl, 2 mM EDTA and 2 mM pNPP at pH 7.0. After incubation at 30 °C for 10
min, the PTP1B enzyme reaction was stopped by adding 50 l of NaOH (3 M). The
hydrolysate of pNPP has a strong absorption at 405 nm, and the OD values were used
to calculate the inhibition rates against PTP1B. A similar system without PTP1B
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protein was used as a blank, and compound CC06240, which is a benzofuran, was
used as a positive control 15.
Antioxidative activity assay. The antioxidative activities of these compounds were
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evaluated by means of a 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay .
Briefly, 10 µl of the sample (200 M in DMSO) was added to 96-well plates, and 190
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µl of DPPH (200 M in anhydrous ethanol) was added. The reactions were incubated
at 37 °C without light for 30 min. Then, the absorbance values (A values) were
determined at 517 nm. The scavenging capacity for the free radical was Sc (%) =
(1−Asample/Acontrol)100%. The absorbance value of the reaction without the test
compounds was used as a control.
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RESULTS
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Effects of Temperature, pH and Divalent Metal Ions on the Activities of
UGT88D4 and UGT88D7. The genes for UGT88D4 and UGT88D7 were
successfully heterologously expressed in E. coli BL21 (DE3) as N-terminal
His6-tagged proteins. The two recombinant GATs were purified to homogeneity
through Ni-NTA metal affinity chromatography and used for the catalytic reactions
(Figure S1, Supporting Information, SI). To obtain the highest catalytic efficiency of
UGT88D4 and UGT88D7, the catalytic conditions, including the temperature, pH and
divalent metal ions, were optimized. As shown in Figure 1A, the catalytic activity of
UGT88D4 increased with increasing temperature in the 15‒45 °C interval but
decreased with increasing temperature in the 45‒65 °C interval, which is likely due to
denaturation of the enzyme. Thus, the optimum temperature was 45 °C. Similarly, for
UGT88D7, the optimum temperature was 40 °C (Figure 1B). The analysis of the
enzyme activity over the pH range 4.0 to 11.0 revealed that for both UGT88D4 and
UGT88D7, the maximum activity was observed at pH 7.0, while no activity was
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observed at pH <5.0 or >9.0 (Figure 2). The assay of divalent cations showed that
both UGT88D4 and UGT88D7 were independent of the presence of metal ions, while
the catalytic activities of these two GATs were enhanced by the presence of Mg2+,
Ca2+, Fe2+ and Ba2+ (Figure 3).
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Kinetic Parameters of UGT88D4 and UGT88D7. The kinetic parameters of the
enzymes in the catalytic glucuronidations were determined under the optimum
temperature and pH conditions. The Km values of UGT88D4 and UGT88D7 towards
hesperetin (7) were found to be 297.4 M and 253.2 M, respectively. The Vmax
values of UGT88D4 and UGT88D7 toward hesperetin (7) were 246.2 nmol min-1 mg-1
and 165.8 nmol min-1 mg-1, respectively (Figure 4).
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Exploring the Glucuronidating Activities of UGT88D4 and UGT88D7. UGT88D4
(from Antirrhinum majus) and UGT88D7 (from Perilla frutescens) are flavonoid
7-O-glucuronosyltransferases (F7GATs), which are responsible for producing
2
specialized metabolites in Lamiales plants . To further utilize UGT88D4 and
UGT88D7 as tools for synthesizing structurally diverse and pharmacologically active
glucuronides, the catalytic activities of these GATs were explored. Structurally
diverse aglycons, including flavone (1‒2), flavonol (3‒5), flavanonol (6), flavonone
(7), flavan-3-ol (8), isoflavone (9), dihydrochalcone (10), coumarin (11),
anthraquinone (12), lignan (13), curcumin (14), naphthoquinone (15), and pentacyclic
triterpenoid (16‒17) derivatives, were selected as the candidate glucuronidation
acceptors, as shown in Figure 5C. The screening reactions under the optimal catalytic
conditions were analyzed by HPLC-UV/MS. The highest conversion rate of each
compound under the catalysis of these two GATs is shown in Figure 5A. Both
UGT88D4 and UGT88D7 could catalyze the reactions of compounds 1–13, which
have flavonoid, coumarin, anthraquinone, and lignan structural fragments. UGT88D7
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showed the highest conversion rates with compounds 1, 4, 6, 8–10 and 13, while for
compounds 2, 3, 5, 7, 11 and 12, UGT88D4 exhibited conversion rates higher than
those of UGT88D7 (Figures 5A and S2‒S14, SI). UGT88D4 and UGT88D7 could not
only accept flavonoids (1‒10) as acceptors but also recognize substrates (11‒13) with
different types of skeletons. Furthermore, their catalytic promiscuity allowed these
two GATs to recognize different glucuronidation sites on a single acceptor. When
flavonoids (3–6 and 10) were used as substrates, both mono- and diglucuronides were
obtained as products based on HPLC-MS analysis. For example, when morin (5) was
used as the acceptor in UGT88D4-catalyzed glucuronidation, a total of six
glucuronidated products (5a, 5b, 5c, 5d, 5e and 5f) were generated (Figure 5A).
According to results of the HPLC-UV/ESI-MS analysis, 5a, 5e and 5f are
monoglucuronides while 5b, 5c and 5d are diglucuronides (Figure S6, SI). For
compounds 14–17, neither UGT88D4 nor UGT88D7 showed catalytic activity, which
was likely caused by the great differences between these structures and those of the
native substrate. Above all, UGT88D4 and UGT88D7, as potential enzymatic tools,
exhibited unprecedented catalytic promiscuity and high efficiency in the
glucuronidation of diverse natural products.
Establishing a Whole-Cell System for Production of Glucuronides. Although
UGT88D4 and UGT88D7 were able to produce various glucuronides, the use of
purified enzymes as biocatalysts in practical applications is associated with various
challenges. First, purified enzymes are usually unstable and easily denatured; second,
the sugar donor (UDPGA) is costly (USD 2000/g); finally, for the polyhydroxylated
acceptors, enzymatic glucuronidation lacks catalytic specificity, and multiple
glucuronides are produced, resulting in a low yield of the target product. Thus, to
overcome these challenges, a whole-cell in vivo glucuronidation catalytic system was
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established. To supply the sugar donor UDPGA in a green and economically efficient
manner, an endogenous biosynthetic method for accessing UDPGA using E. coli
needed to be constructed 17–18. So, the UDP-glucose dehydrogenase gene (ugd), a key
gene in the UDPGA biosynthetic pathway of E. coli, was inserted into an expression
vector (pCDFDuet) carrying UGT88D4 or UGT88D7. The recombinant plasmids
were then individually transferred into E. coli BL21 (DE3), and the transformants
were designated as strain B-ugd-UGT88D4 and B-ugd-UGT88D7, respectively. E.
coli BL21 with pCDFDuet was used as the blank control. The gene ugd was
overexpressed to increase the level of endogenous UDPGA, which can be used as the
sugar donor for exogenous GATs in vivo. Therefore, by adding diverse aglycons into
the cell suspension culture systems, the endogenous UDPGA is directly used as the
sugar donor in coupling reactions, making additional UDPGA unnecessary.
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As shown in Figure 5, comparing with the UGT88D4 and UGT88D7 catalyzing
reactions in vitro, the corresponding whole-cell biocatalysts also exhibited broad
catalytic promiscuity and high catalytic efficiency in recognizing substrates. To our
surprise, the whole-cell system showed high catalytic specificity in generating
products and only one monoglucuronidated product was generated for substrates (3, 4,
5, 6 and 10) with multiple potential glucuronidation sites. This catalytic specificity
makes whole-cell biocatalysts more efficient and applicable in synthesis of target
products. For flavonoids 1–7, the conversion rates in the whole-cell catalysis were
higher than those of in vitro enzyme catalysis. In contrast, the conversion rates of
compounds 8–11 in the whole-cell catalysis were significantly lower than those of
enzyme catalysis. What’ more, for compounds 12 and 13, no glucuronidated products
were detected with whole-cell in vivo catalysis. The low catalytic activities with
acceptors 8–13 may be caused by the permeability barrier of the cell envelope, which
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leads to limited passage of the substrates through the cell envelope 19. The mass
transfer resistance of the cell envelope further makes the biocatalytic reaction difficult
20
to be accomplished . Compounds 2, 3, 5, 7, 11 and 12 had the highest conversion
rates when catalyzed by the strain B-ugd-UGT88D4, and the other compounds (1, 4, 6,
8–10 and 13) showed higher conversion rates when catalyzed by the strain
B-ugd-UGT88D7. For compounds 14–17, neither enzyme catalysis nor whole-cell
catalysis showed catalytic activity. This means the trends in the reactivities of the
substrates in whole-cell catalysis were consistent with those of enzyme catalysis in
vitro.
Preparation of Novel and Bioactive Glucuronides by Utilizing a Biosynthetic
Method. Flavonoids are widely distributed in commonly consumed foods and in
traditional Chinese medicines. Their polyhydroxyl structures can promote
glucuronidation metabolism reactions in the organism, and further affect their
pharmacological activities, such as reducing toxicity, improving bioavailability and
changing pharmacokinetic characteristics 21, 22. What’s more, flavonoids and their
corresponding glucuronides also possess various biological activities. For instance,
puerarin-7-O-glucuronide, a water-soluble phase II metabolite of puerarin, prevents
21
angiotensin II-induced cardiomyocyte hypertrophy by reducing oxidative stress
.
Luteolin-7-O-glucuronide, a glucuronide isolated from plants such as Achillea
millefolium and Cannabis sativa, exhibited antimutagenic properties and cholagogic
effects 23–25. Therefore, establishing a green and economical biocatalytic approach
with sufficient regio- and stereoselectivity is necessary to the production of bioactive
glucuronides and drug discovery.
To prepare novel and bioactive glucuronides, the scale-up whole-cell
biosynthetic reactions were performed with flavonoids 1–7 and 9 as acceptors. Based
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on the conversion rates, the strain B-ugd-UGT88D4 was used for the glucuronidation
of flavonoids 2, 3, 5, and 7, and the others (1, 4, 6 and 9) were glucuronidated with
B-ugd-UGT88D7. The reaction products were separated and characterized on the
basis of extensive spectroscopic analyses of MS and NMR.
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New glucuronidated product 3a was selected as an example for structural
identification. MS analysis revealed that the major signal of product 3a was 176
atomic mass units greater than that of aglycon 3, which suggested the introduction of
one glucuronyl moiety. This was further confirmed by NMR spectra, which showed
signals characteristic of a sugar moiety (the anomeric proton (H-1'') signal at δH 5.23;
H-2''‒5'' signals at δH 3.16–3.96; the anomeric carbon (C-1'') signal at δC 98.7; one
C=O (C-6'') signal at δC 170.5 and four CH (C-2''–5'') signals at δC 71.4–75.8). The
location of the glucuronyl fragment was deduced from the HMBC correlation between
H-1′' and C-7 (δC 162.3). The large coupling constant (J = 7.3 Hz) of H-1′' indicated
the presence of
a
β-glycosidic bond. Thus, 3a was identified as
kaempferol-7-O-β-D-glucuronide, which is a novel compound (Figures S19–S25, SI).
Similarly, the structures of products (4a–6a, 9a) were also confirmed to be new
compounds (Figures S26‒S46 and S51‒S57, SI). Products 1a, 2a and 7a were
1
identified as known compounds based on their MS, H NMR and 13C NMR data,
which were consistent with the literatures (Figures S15‒S18, S47 and S48, SI) 26–28
.
Finally, the products (1a–7a) were all identified as flavonoid-7-O-β-glucuronides,
which was in accordance with the fact that UGT88D4 and UGT88D7 were flavonoid
7-O-glucuronosyltransferases (Figure 5). What’s more, these two GATs
stereoselectively generate β-type glycosidic bonds. It is also notable that the products
(3a–6a and 9a) are novel compounds (Figure 5C). The acceptors were all
glucuronidated at 7-OH, highlighting the high catalytic specificity of UGT88D4 and
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UGT88D7 in the in vivo catalytic system.
UGT88D4 and UGT88D7 can also recognize epicatechin (8) and phloretin (10),
which possess basic structures similar to those of flavonoids (Figure 5). The external
enzyme UGT88D7 catalysis exhibited relatively high conversion rates and generated
one product (8a) from 8 and two products (10a and 10b) from 10. Finally, 8a was
isolated and characterized as epicatechin-7-O-β-D-glucuronide (Figures S49 and S50,
SI) 29. 10a and 10b were also purified and further elucidated as new compounds,
phloretin-4'-O-β-D-glucuronide and phloretin-2'-O-β-D-glucuronide, respectively
(Figures S58–S68, SI) 30.
To expand the applications of this biosynthetic method, different types of
bioactive substrates (11‒13) were also tested. 7-Hydroxy-4-methylcoumarin (11), a
coumarin, which is the general name for ortho-hydroxycinnamic acid lactones,
possesses various biological activities such as anti-HIV, anti-tumor and
anti-hypertension activities 31. Emodin (12) is an important component of the
traditional Chinese herb Rhubarb (Da Huang) and has various efficacies, including
causing bacteriostasis, anti-inflammatory effects and protective effects on the liver
and kidney 32. Magnolol (13) is an active antibacterial component in Magnolia
officinalis Rehd. et Wils with special, long-lasting muscle relaxant effects 33. However,
the water solubilities of these natural products are low, and their oral bioavailabilities
are poor. To our delight, these substrates (11‒13) can be efficiently recognized by
UGT88D4 and UGT88D7, and their glucuronidated products were successfully
isolated and characterized as 4-methylcoumarin-7-O-β-D-glucuronide (11a),
emodin-3-O-β-D-glucuronide (12a) and magnolol-O-β-D-glucuronide (13a),
34,
respectively (Figures S69–S79, SI)
35. It has been reported that the plasma
concentration of emodin (12) is very low after oral administration. However, the oral
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bioavailability of its glucuronidated product, emodin-3-O-glucuronide (12a), is
substantially higher. The Cmax (maximum blood concentration) of
emodin-3-O-glucuronide (12a) is nearly 30 times larger than that of emodin (12) after
oral administration 36.
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541
Pharmacological Activities of Biosynthesized Glucuronides. Compared with the
aglycons, the water solubilities of the glucuronidated products are certainly improved.
However, the pharmacological activities of the glucuronidated products remain
unknown. Hence, we evaluated the pharmacological activities of these glucuronides.
Protein tyrosine phosphatase 1B (PTP1B), a negative regulator of insulin signal
transduction, is a target for type 2 diabetes and obesity drug discovery 37. PTP1B
inhibitors can effectively treat type 2 diabetes and obesity. The experimental results
indicated that kaempferol-7-O-β-D-glucuronide (3a) was a potent inhibitor of PTP1B
with an inhibition rate of 99.2%, while the inhibition rate of its aglycon, kaempferol,
was only 10.9% at a concentration of 10 M (Table S1, SM). The IC50 value of
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O-glucuronide 3a against PTP1B was 8.02  10-6 M.
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was used to determine the
ability of the compounds to eliminate free radicals. The experimental results showed
that the free radical scavenging capacities of these compounds (2a, 3a, 3, 7, 8 and 12a)
were greater than that of vitamin C, which means that these glucuronides possess
significant antioxidant activities. Interestingly, glucuronides 2a and 12a had stronger
antioxidant activities than their corresponding aglycons 2 and 12 (Table S2, SM).
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DISCUSSION
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Expanding the Applications of GATs in the Biosynthesis of Glucuronides
UGT88D4 and UGT88D7 are flavonoid 7-O-glucuronosyltransferases (F7GAT),
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which transfer glucuronic acid from UDPGA (sugar donor) to the 7-OH group of
2
flavonoids (sugar acceptor) . Generally, UGT88D4 and UGT88D7 are considered to
be highly regioselective. However, in vitro, UGT88D4 and UGT88D7 exhibit
catalytic promiscuity and can functionalize various hydroxyl groups with high
efficiency, not only the 7-OH group. In addition to the reported flavonoid acceptors,
these two enzymes also recognize other structurally diverse acceptors, including
coumarins, anthraquinones and lignans. Interestingly, UGT88D4 and UGT88D7 were
able to not only monoglucuronidate diverse acceptors but also diglucuronidate
acceptors with multiple hydroxyl groups, generating diglucuronides. Therefore,
according to our results, UGT88D4 and UGT88D7 exhibited catalytic promiscuity in
both recognizing acceptors and generating products. The applications of these two
GATs were expanded, and they can be utilized in the enzymatic synthesis of diverse
new bioactive glucuronides.
Construction of an Engineered Strain Supplying UDPGA for the Biosynthesis of
Glucuronides
By coexpression of ugd and GATs in the strain E. coli BL21, the level of
endogenous UDPGA was increased, and the exogenous GATs could then mediate
glucuronidation in the cells. Notably, for most flavonoid acceptors, the conversion
rates obtained with the whole-cell system in vivo were higher than that achieved with
pure enzyme in vitro. Moreover, compared with enzymatic reactions in vitro,
UGT88D4 and UGT88D7 exhibited higher regioselectivity in vivo, and only one
glucuronidated product was generated from each acceptor, and all the products were
flavonoid-7-O-glucuronides. The different catalytic activities of UGT88D4 and
UGT88D7 in vivo and in vitro might be because the level of intracellular UDPGA is
stable and relatively low in vivo, and it is not sufficient to support the glucuronidation
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of multiple hydroxyl groups on the substrates. Therefore, the hydroxyl group at C7,
which is the native glucuronidation site for UGT88D4 and UGT88D7, was
preferentially derivatized in vivo. Finally, a total of eight flavonoid-7-O-glucuronides
were efficiently prepared by whole-cell catalysis without adding expensive sugar
donors. Unlike the enzymatic reactions in vitro, the whole-cell catalysis system was
more economical, selective and stable, and it provides a potential method for the
efficient and economical synthesis of bioactive glucuronides for drug discovery. To
our knowledge, this is the first reported whole-cell catalysis method of
glucuronidation with such broad substrate scope.
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In this work, two biocatalytic systems, pure enzyme catalysis in vitro and
whole-cell catalysis in vivo, were successfully constructed. With these two
biocatalytic systems, a total of fourteen O-glucuronides were efficiently synthesized,
and seven of them were new compounds. Furthermore, one of the new compounds,
kaempferol-7-O--D-glucuronide (3a), potently inhibited PTP1B making it a potential
drug lead for the treatment of type 2 diabetes. Bio-glucuronidation mediated by GATs
has shown great potential in synthesizing diverse bioactive glucuronides and it will be
also helpful for the simulation of human drug metabolism in drug research. What’s
more, novel GATs with broader substrate scope and higher catalytic efficiency need
to be found in the future for glucuronidation of drugs and food ingredients with more
complicated structures.
Supporting Information
HPLC-DAD/ESI-MS spectra of the enzymatic reactions. UV, IR, 1H and 13C NMR,
HSQC, HMBC, and HRESIMS spectra of the glucuronides. The results of evaluating
the pharmacological activities of the aglycons and corresponding glucuronides.
Funding
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This work was financially supported by National Natural Science Foundation of
China (No. 21572277), CAMS Innovation Fund for Medical Sciences (No.
CIFMS-2016-I2M-3-012),
the
Drug
Innovation
Major
Project
(2018ZX09711001-006), and Beijing Key Laboratory of Non-Clinical Drug
Metabolism and PK/PD Study (No. Z141102004414062).
Notes
The authors declare no conflicts of interest.
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