Synthesis of flavonol 3-O-glycoside by UGT78D1

Article abstract of DOI:10.1007/s10719-012-9410-5

Glycosylation is an important method for the structural modification of various flavonols, resulting in the glycosides with increased solubility, stability and bioavailability compared with the corresponding aglycone. From the physiological point of view, glycosylation of plant flavonoids is of importance and interest. However, it is notoriously complicated that flavonols such as quercetin, kaempferol and myricetin, are glucosylated regioselectively at the specific position by chemical method. Compared to the chemical method, enzymatic synthesis present several advantages, such as mild reaction condition, high stereo or region selectivity, no protection/deprotection and high yield. UGT78D1 is a flavonol-specific glycosyltransferase, responsible for transferring rhamnose or glucose to the 3-OH position in vitro. In this study, the activity of UGT78D1 was tested against 28 flavonoids acceptors using UDP-glucose as donor nucleoside in vitro, and 5 acceptors, quercetin, myricetin, kaempferol, fisetin and isorhamnetin, were discovered to be glucosylated at 3-OH position. Herein, the small-scale 3-O-glucosylated quercetin, kaempferol and myricetin were synthesized by UGT78D1 and their chemical structures were confirmed by 1H and 13C nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS). Springer Science+Business Media, LLC 2012.

Full text of DOI:10.1007/s10719-012-9410-5

Glycoconj J (2012) 29:425–432
DOI 10.1007/s10719-012-9410-5
Synthesis of flavonol 3-O-glycoside by UGT78D1
Guangxiang Ren & Jingli Hou & Qinghong Fang &
Hong Sun & Xiaoyan Liu & Lianwen Zhang &
Peng George Wang
Received: 15 April 2012 /Revised: 2 June 2012 /Accepted: 4 June 2012 /Published online: 6 July 2012
#
Springer Science+Business Media, LLC 2012
Abstract Glycosylation is an important method for the
structural modification of various flavonols, resulting in
the glycosides with increased solubility, stability and bio-
availability compared with the corresponding aglycone.
From the physiological point of view, glycosylation of plant
flavonoids is of importance and interest. However, it is
notoriously complicated that flavonols such as quercetin,
kaempferol and myricetin, are glucosylated regioselectively
at the specific position by chemical method. Compared to
the chemical method, enzymatic synthesis present several
advantages, such as mild reaction condition, high stereo or
region selectivity, no protection/deprotection and high yield.
UGT78D1 is a flavonol-specific glycosyltransferase, re-
sponsible for transferring rhamnose or glucose to the 3-OH
position in vitro. In this study, the activity of UGT78D1 was
tested against 28 flavonoids acceptors using UDP-glucose
as donor nucleoside in vitro, and 5 acceptors, quercetin,
myricetin, kaempferol, fisetin and isorhamnetin, were dis-
covered to be glucosylated at 3-OH position. Herein, the
small-scale 3-O-glucosylated quercetin, kaempferol and
myricetin were synthesized by UGT78D1 and their chemi-
Introduction
Flavonoids are a large group of plant natural products,
exhibiting bioactivities with important implications for
plant, animal and human health [1–5]. They often exist in
glycosylated forms, comprising sugar units and an aglycone
core. A large portion of this structural variability is due to
the attachment of one or more sugar moieties at the different
positions of the aglycone core as illustrated in Fig. 1 [6].
Flavonols such as quercetin, myricetin and kaempferol,
attract considerable interest due to their diverse biological
activities [7–10]. Attachment of one or more sugars units at
different positions of the core aglycone structure, can in-
crease their solubility and help to exhibit their inherent
functions such as antioxidant capability and potential me-
dicinal properties.
Glycosylated flavonols can be obtained by chemical or
enzymatic syntheses. However, chemical synthesis of
glycosylated flavonols at the specific position is notori-
ously complicated, due to several potential glycosylation
sites in the flavonol aglycone (Fig. 2). Moreover, the
chemical synthesis involves several protection and depro-
tection steps. Regioselectivity of glycosyltransferases
offers an important strategy to overcome the limitation
of chemical synthesis, and attracts a lot of attention. The
enzymes involved in transferring sugars from a range of
nucleoside donors onto acceptor molecules, glycosyltrans-
ferases (GTs), have been intensively studied recently
[11–16]. Among these enzymes, the family 1 glycosyl-
transferases has been found to predominantly recognize
low-molecular-weight compounds, such as phenolics, and
transfer sugar to the 3-OH and 7-OH of the flavonol
with high regioselectivity [6, 12, 17–22].
1
cal structures were confirmed by H and 13C nuclear mag-
netic resonance (NMR) and high resolution mass
spectrometry (HRMS).
.
.
Keywords UGT78D1 Substrate specificity UDP-
.
glucose Flavonol
Guangxiang Ren and Jingli Hou have equal contributions.
:
:
:
:
:
:
G. Ren J. Hou Q. Fang H. Sun X. Liu L. Zhang (*)
P. G. Wang (*)
College of Pharmacy and State Key Laboratory of Medicinal
Chemical Biology, Nankai University,
Tianjin 300071, People’s Republic of China
e-mail: lianwen@nankai.edu.cn
Uridine diphosphate glycosyltransferases (UGTs) catalyze
the transfer of a sugar moiety from the uridine diphosphate-
e-mail: penggwang@gmail.com

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Glycoconj J (2012) 29:425–432
essential to the potential acceptors of UGT78D1: 1) the 3-
hydroxyl group, 2) the double bond in the ring C, 3) the 4′-
hydroxyl group (Fig. 1). Considering the ambiguity of
UGT78D1 in recognizing sugar donors, the donor specific-
ity of UGT78D1 was also examined with UDP-galactose,
UDP-glucuronic acid and UDP-N-acetylglucosamine. No
UGT activity was observed with the three sugar donors.
To obtain a higher yield of the glycosylated products,
experiments designed to find the optimal reaction time were
conducted using myricein as the substrate acceptor. The
reaction was carried out as indicated in methods in a total
volume of 500 μl and for a time range of 20 h. The reaction
mixtures (40 μl) were sampled at some specific time points
(0 h, 0.33 h, 1 h, 2 h, 4 h, 9 h and 20 h). The same volume of
methanol was added to each sample and protein was re-
moved from the reaction mixture by filtration. To measure
the amount of glycosylated product and the remaining sub-
strate, the filtrate was monitored on HPLC. The result indi-
cates that Myricetin is quantitatively converted into the
product within 20 h, the longer the time, the higher the yield
of the product (Fig. 3).
Fig. 1 Flavonol-glycosides that accumulate in wild type arabidopsis.
R1, H (Kaempferol), OH (quercetin). R2, glucose (flavonol-3O-gluco-
side-7-O-rhamnoside), rhamnose (flavonol-3-O-rhamnoside-7-O-
rhamnoside)
sugar to low molecular weight acceptors. in vitro study
indicated that UGT78D1 is able to use UDP-glucose as sub-
strates in vitro to transfer D-glucose to 3-OH position of
quercetin [18]. Moreover, it can utilize UDP-rhamnose other
than UDP-glucose as the donor [6]. While in vitro study
indicated that quercetin and kaempferol were the substrates
for UGT78D1 with UDP-rhamnose as donor nucleoside by
screening 9 flavonoids, substrate specificity has not yet been
reported for the recombinant protein with UDP-glucose as
donor nucleoside. In this study, we tested the activity of
UGT78D1 against 28 flavonoid acceptors using UDP-
glucose as donor nucleoside in vitro, and 5 acceptors, includ-
ing quercetin, myricetin, kaempferol, fisetin and isorhamne-
tin, were discovered as the potential substrates. In addition, we
also synthesized and characterized the 3-O-glycosylated quer-
cetin, myricetin and kaempferol.
To confirm the LC-MS results, which have deduced the
generation of specifically linked glycosylated products, 3-
O-glycosylated quercetin, myricetin and kaempferol were
characterized by NMR and HRMS (Figs. 4, 5 and 6). Firstly,
these products were synthesized by scaling up the reaction
volume to 100 mL, following the indicated reaction condi-
tion for 20 h. The protein was removed by filtration and the
filtrate was freeze dried. The resulting residue was purified
to give the corresponding glucoside. Their structures were
Result and discussion
1
confirmed by H NMR, 13C NMR and HRMS.
By SDS-PAGE anaylasis, it is found that UGT78D1 was
expressed mainly in the supernatant and could be easilly
purified via the one-step nickel affinity chromatography. A
pre-experiment using the purified UGT78D1 to catalyze the
reaction between UDP-Glucose and quercetin indicated the
enzyme worked well (data not shown). To explore the
substrate specificity of UGT78D1 using UDP-glucose as
donor nucleotide, 28 flavonoids were tested in vitro and
the glycosylated product was monitored by LC-MS. Novel
compounds were formed for the substrates: quercetin, myr-
icetin, kaemferol, fisetin and isorhamnetin, according to
new LC peaks confirmed by MS data (data not shown).
But no novel compound was detected for the other sub-
strates (Table 1). A comparison of the structures among
these compounds indicates the following elements are
The HiResMALDI-FTMS analysis of the purified gluco-
sides confirmed only one glucose was transferred to the
substrates (monoglucoside of quercetin: calcd. for
C21H20NaO12 [M+Na]+ 487.0852 found 487.0842; mono-
glucoside of kaempferol: calcd. for C21H20NaO11 [M+Na]+
471.0903 found 471.0896; monoglucoside of myricetin:
calcd. for C21H20NaO13 [M+Na]+ 503.0802 found
503.0797.) The NMR data for quercetin 3-O-β-glucoside:
1H NMR (CD3OD, 400 MHz): δ 7.71 (1H, d, J02.0 Hz, H-
2′); 7.59 (1H, dd, J102.0 Hz, J208.5 Hz, H-6′); 6.87 (1H, d,
J08.5 Hz, H-5′); 6.40 (1H, d, J01.9 Hz, H-8), 6.21 (1H, d,
J01.9 Hz, H-6); 5.25 (1H, d, J07.5 Hz, H-1″); 3.19–3.73
(6H, m, H-2″, H-3″, H-4″, H-5″, H-6″); 13C NMR (CD3OD,
100 MHz): δ 179.6, 166.1, 163.1, 159.1, 158.5, 149.9,
146.0, 135.7, 123.2, 123.1, 117.6, 116.1, 105.8, 104.3,
99.9, 94.7, 78.5, 78.2, 75.8, 71.3, 62.6; the NMR data for
kaempferol 3-O-β-glucoside: 1H NMR (CD3OD,
400 MHz): δ 8.06 (2H, d, J08.6 Hz, H-2′ and H-6′), 6.89
(2H, d, J08.6 Hz, H-3′ and H-5′); 6.41 (1H, s, H-8), 6.21
(1H, s, H-6), 5.25 (1H, d, J07.0 Hz, H-1″), 3.19–3.71 (6H,
m, H-2″, H-3″, H-4″, H-5″, H-6″); 13C NMR (CD3OD,
100 MHz): δ 179.6, 166.0, 163.1, 161.6, 159.2, 158.6,
Fig. 2 The chemical structures of quercetin, mycertin and kaemferol

Glycoconj J (2012) 29:425–432
427
Table 1 Quantitative substrate specificity of recombinant UGT78D1 using UDP-glucose as sugar nucleotide as analyzed by LC-MS (CR:
Conversion rate)

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Fig. 3 Changes in the level of
Myricetin-3-O-glucoside and
aglycone in the reaction mixture
over a time-course of 0–20 h
135.5, 132.3, 122.8, 116.1, 105.8, 104.1, 99.9, 94.8, 78.5,
78.1, 75.8, 71.4, 62.7; Myricetin-3-O-β-glucoside: 1H NMR
(CD3OD, 400 MHz): δ 7.30 (s, 2H, H-2′ and H-6′), 6.39
(1H, d, J02.0 Hz, H-8), 6.21 (1H, d, J02.0 Hz, H-6), 5.26
(1H, d, J07.7 Hz, H-1″), 3.21–3.78 (6H, m, H-2″, H-3″, H-
4″, H-5″, H-6″); 13C NMR (CD3OD, 100 MHz): δ 179.5,
166.1, 163.1, 1589.1, 158.5, 146.5, 138.1, 135.9, 121.9,
110.0, 105.7, 104.5, 99.9, 94.7, 78.4, 78.2, 75.7, 71.1, 62.5.
The NMR data were in agreement with those available in
the literature [18, 23, 24], and some of them have been
reported for the first time in this work (1H and 13C NMR
data for kaempferol 3-O-glucoside and myricetin 3-O-
Fig. 4 Characterization of quercetin-3-O-glucoside with HRMS and NMR(1H-NMR, 13C-NMR)

Glycoconj J (2012) 29:425–432
429
Fig. 5 Characterization of kaemferol-3-O-glucoside with HRMS and NMR(1H-NMR, 13C-NMR)
glucoside). The shielding parameters for hydrogen atoms in
the substrate flavonol, when compared to the corresponding
signals for the aglycon moiety of the product, supported the
fact that the glycosidic linkage is by the C-3 position (Table 2).
The measured value for the coupling constant (J HH) between
the sugar protons H-1″ and H-2″ ranging within 7–8 Hz for
the three glucosides, indicated β-configuration for the 3-O-
glycosidic linkage. In addition, the UV–vis spectra are also
consistent with the literature [23] (Table 3).
keampferol 3-O-glucoside and myricetin 3-O-glucoside
were synthesized using UGT78D1, and their structures were
confirmed by HRMS and NMR.
Material and methods
General information
Flavonol chemicals were purchased from Chengdu PuRuiFa
Technology & Develop. Co. Ltd., China. Other materials
were purchased from Beijing Dingguo Changsheng Bio-
technology Co. Ltd., China.
Conclusion
In summary, the substrate specificity for UGT78D1 using
UDP-glucose as sugar donor was studied extensively by
screening 28 flavonoids. Five flavonols, quercetin, myrice-
tin, kaempferol, fisetin and isorhamnetin, were identified as
the preferred substrates. In addition, quercetin 3-O-glucose,
Expression and purification of UGT78D1
UGT78D1 (GI: 839933) was codon optimized for E. coli
heterologous expression and synthesized in Beijing Augct

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Fig. 6 Characterization of myricetin-3-O-glucoside with HRMS and NMR(1H-NMR, 13C-NMR)
1
Table 2 The H NMR spectral data of Quercetin, Kaempferol, Myricetin and their monoglucosides
Position Quercetin
Quercetin 3-O-glucoside Myricetin
Myricetin 3-O-
Kaempferol
Kaempferol 3-O-
glucoside
glucoside
C2′
C6′
7.73(1H, d, J02.1 Hz 7.71(1H, d, J02.0 Hz)
7.63(1H, d, J102.1 Hz, 7.59(1H, dd, J102.0 Hz,
J208.5 Hz J208.5 Hz)
7.34 (2H,s)
7.30(2H, s)
8.05(2H, d, J08.5 Hz) 8.06(2H, d, J08.6 Hz)
6.88 (2H, d, J08.5 Hz) 6.89 (2H, d, J08.6 Hz)
C3′
—
—
—
—
—
—
C5′
6.88 (1H, d, J08.5 Hz) 6.87 (1H, d, J08.5 Hz)
C8
6.38 (1H, d, J02.0 Hz) 6.40 (1H, d, J01.9 Hz) 6.36 (1H, d, J02.0 Hz) 6.39 (1H, d, J02.0 Hz) 6.35 (1H,s)
6.17(1H, d, J02.0 Hz) 6.21(1H, d, J01.9 Hz) 6.16 (1H, d, J02.0 Hz) 6.21(1H, d, J02.0 Hz) 6.15(1H,s)
6.41 (1H,s)
C6
6.21(1H, s)
Glc-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6
—
—
—
—
—
—
5.25 (1H, d, J07.5 Hz)
3.19–3.73 (6H, m)
—
—
—
—
—
—
5.26(1H,d, J07.7 Hz)
3.21–3.78 (6H, m)
—
—
—
—
—
—
5.25 (1H, d, J07.0 Hz)
3.19–3.71 (6H, m)

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Table 3 UV–VIS of flavonol 3-O-glycosides
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