Expanded investigations of the aglycon promiscuity and catalysis characteristic of flavonol 3-: O -rhamnosyltransferase AtUGT78D1 from Arabidopsis thaliana

Article abstract of DOI:10.1039/c6ra16251g

Rhamnosides usually possess better bioavailabilities and improved solubilities compared with their aglycons and are a major source of bioactive natural products. However, biosynthesis of rhamnosides is hindered by the commercially expensive UDP-rhamnose (UDP-Rha) donor and a lack of universal rhamnosyltransferases. In the present study, an efficient UDP-Rha production system via a two-step enzymatic reactions using UDP-glucose (UDP-Glc) as a substrate was constructed. Extensive in vitro enzymatic assays and preparative reactions using the obtained UDP-Rha/UDP-Glc highlighted the robust glycosylation promiscuity of the reported rhamnosyltransferase AtUGT78D1. Based on HPLC-UV and HR-MS analyses, 30 of the tested aromatic compounds belonging to 7 structural types, including flavonoids, flavonoid glycosides, phenylethyl chromones, benzophenones, coumarins, lignanoids, and anthraquinones, were accepted by AtUGT78D1 to conduct the corresponding rhamnosylation and/or glucosylation with one or more glycosyl substitutions at different positions. Further preparative reactions expanded the catalytic characteristic of AtUGT78D1 since it can catalyse the rhamnosylation at the 3-OH position of the flavonols, glucosylation at the 7-OH position of the flavone baicalein, and multiple hydroxyl substitutions for diverse types of aromatics. Interestingly, a unique reversible catalysis activity of AtUGT78D1 was observed, and it has been effectively used in one-pot rhamnosylation of the desired rhamnoside. The enzymatic rhamnosylations of diverse "drug-like" scaffolds as well as bidirectional catalysis for one-pot rhamnosylations by plant rhamnosyltransferase were rarely reported before, which indicated that AtUGT78D1 was expected to be a universal and effective tool for chemo-enzymatic synthesis of diverse bioactive rhamnosylated derivatives for drug discovery.

Full text of DOI:10.1039/c6ra16251g

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ARTICLE
Expanded investigations of the aglycon promiscuity and catalysis
characteristic of flavonol 3-O-rhamnosyltransferase AtUGT78D1
from Arabidopsis thaliana
†
ab
a
ab
a
ab
ab
ab
Ting Mo,‡ Xiao Liu,‡* Yuyu Liu, Xiaohui Wang, Le Zhang, Juan Wang, Zhongxiu Zhang,
a
a
Shepo Shi,* Pengfei Tu*
Rhamnosides, usually possess better bioavailabilities and improved solubilities compared with their aglycons, were the
major source of bioactive natural products. However, biosynthesis of these rhamnosides was hindered due to the
commercially expensive UDP-Rhamnose (UDP-Rha) donor and lack of universal rhamnosyltransferases. In the present
study, an efficient UDP-Rha production system by two-step enzymatic reactions using UDP-Glucose (UDP-Glc) as substrate
was constructed. Extensive in vitro enzymatic assays and preparative reactions using obtained UDP-Rha/UDP-Glc
highlighted the robust glycosylation promiscuity of a reported rhamnosyltransferase AtUGT78D1. Based on HPLC-UV and
HR-MS analyses, 30 of tested aromatic compounds belonging to 7 structural types including flavonoids, flavonoid
glycosides, phenylethyl chromones, benzophenones, coumarins, lignanoids, anthraquinones could be accepted by
AtUGT78D1 to conduct the corresponding rhamnosylation and/or glucosylation with one or more glycosyl substitutions at
different positions. Further preparative reactions expanded the catalytic characteristic of AtUGT78D1 since it can catalyse
not only the rhamnosylation at 3-OH position of flavonols, but also glucosylation at 7-OH position of flavone baicalein, as
well as multiple hydroxyl substitutions of diverse kinds of aromatics. Interestingly, unique reversible catalysis activity of
AtUGT78D1 was observed and it has been effectively used in one-pot rhamnosylation of desired rhamnoside. The
enzymatic rhamnosylations of diverse “drug-like” scaffolds as well as bidirectional catalysis for one-pot rhamnosylations by
plant rhamnosyltransferase were rarely reported before, which indicated that AtUGT78D1 was expected to be a universal
and effective tool for chemo-enzymatic synthesis of diverse desired bioactive rhamnosylated derivatives for drug
discovery.
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
5
,6
compared with their non-glycosylated analogs.
Thus,
1
. Introduction
exploring the rich pool of bioactive glycosylated aromatics for
the design of new candidate drugs is a compelling target for
both basic research and pharmacological applications.
However, many potentially useful glycosylated compounds
occur only in trace amounts in natural sources and are not
easily amenable to chemical synthesis. In the biosynthesis of
bioactive natural glycosides, glycosyltransferases (GTs) which
transfer one or more sugar moieties onto aromatic acceptor
,4 molecules are often involved in the last step modification of an
aglycon, leading to the corresponding ultimate bioactive
molecules. Thus, the manipulation of glycosylation reactions
catalysed by GTs might represent an advantageous shortcut
for the synthesis of glycosylated aromatic compounds.
Glycosylated aromatics which comprise multiple types of
secondary metabolites are widely distributed in natural, and
often possess various biological properties, such as anti-
cancer, anti-psychotic, anti-inflammatory, and anti-diabetic
1
,2
activeties. The remarkable properties of these compounds
are thought to reside in their increased aqueous solubilities,
stabilities, and enhanced bioavailabilities which were likely a
3
result of the presence of the bulky hydrophilic sugar moiety.
The glycosylation of aromatics not only contributes
significantly to the structural diversity of these valuable
natural products, but also be a key mechanism in determining
the structural complexity and remarkable biological activities
In plant, glycosylation is catalysed by family
І
glycosyltransferase which commonly utilize small molecular
7
compounds as acceptor substrates and UDP-sugars as donors.
This kind of enzymes are characterized by the presence of a C-
terminal consensus sequence comprising 44 amino acid
residues
termed
the
plant
8
secondary
product
glycosyltransferase (PSPG) box. In recent years, a large
number of genes encoding uridine diphosphate (UDP)-
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J. Name., 2013, 00, 1-3 | 1
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Journal Name
OH
O
3
H C
NADP+
NADPH
3
H C
O
NAD+
NADH
O
O
HO
HO
O UDP
HO
HO
HO
OH O UDP UDP-glucose-4,6-dehydratase
UDP-Glucose
OH
O
UDP UDP-4-keto-6-deoxyglucose-
OH
3
,5-epimerasel-4- reductase
UDP-4-keto-6-deoxyglucose
UDP-Rhamnose
RHM2-N
RHM2-C
Scheme 1. UDP-Rhamnose biosynthetic pathways from UDP-Glucose in A. thaliana. RHM2-N has the first activity as an UDP-D-
glucose 4,6-dehydratase, while RHM2-C has the two following UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, and UDP-4-keto-L-
2
rhamnose 4-keto-reductase activities.
3-25
glycosyltransferases (UGTs) have been identified in various aromatic natural products, an efficient UDP-Rha production
plant species to glycosylate multiple plant secondary system by two-step enzymatic reaction catalysed by A.
metabolites like flavonoids, terpenoids, steroids, xanthonoids, thaliana rhamnose synthase 2 (AtRHM2) using UDP-Glc as
9
et al. The sugar moieties recognized by these UGTs were substrate was constructed (scheme 1). The obtained UDP-Rha
diverse including UDP-Glucose (UDP-Glc, the most common product was subsequently used in the in vitro enzymatic assay
sugar donor), UDP-Glucuronic acid (UDP-GlcA), UDP-Rhamnose performed by AtUGT78D1 with a wide range of aromatic
(UDP-Rha), UDP-Xylose (UDP-Xyl), UDP-Galactose (UDP-Gal) substrates as glycosyl acceptor. The expanded assays revealed
and so on. the significant substrate promiscuities of AtUGT78D1 that 30
UDP-Rha is a ubiquitous sugar present in plants and many of tested aromatic compounds belonging to 7 structural types
bioactive leading compounds contain a rhamnose moiety in including flavonoids (flavones, flavonones, flavonols,
their structures, for example the antimicrobial agent quercetin isoflavones, chalcones, methylflavone), flavonoid glycosides
1
0
11
12
3
-O-rhamnoside, kaempferol 3-O-rhamnoside and icariin.
(both mono- and di-glycosides), phenylethyl chromones,
Rhamnosylation substitution played an important role in the benzophenones, coumarins, lignanoids, anthraquinones could
structural stability, solubility improvement, intracellular be accepted by AtUGT78D1 as glycosyl acceptor to conduct the
transport, and bioavailability regulation of these natural corresponding rhamnosylation using UDP-Rha as glycosyl
products. However, compared with the mostly reported donor. Among them, 10 flavonoids could also be accepted by
glucosyltransferase, rhamnosyltransferase involved in the AtUGT78D1 using UDP-Glc as glycosyl donor too, while the
enzymatic synthesis of rhamnoside products have not been rhamnosylation efficiency was much higher than the
explored extensively due to the fact that UDP-Rha is glucosylation efficiency. Besides, it was also worth to mention
commercially expensive and its chemical synthesis is multistep, that, the enzymatic rhamnosylation by recombinant proteins
1
3,14
time-consuming
and
low-yielding.
Combinatorial of methylflavone; phenylethyl chromones; coumarins;
biosynthesis of UDP-Rha synthase and rhamnosyltransferase lignanoids and anthraquinones were rarely reported before.
would offer an alternative source for bioactive rhamnosides. Considering that AtUGT78D1 has been described as a flavonol
As one of the model plants, about 120 glycosyltransferase specific glycosyltransferase at 3-OH position, two flavonol
genes belonging to the GTI family were annotated in compounds quercetin (7) and kaempferol (10) along with a
Arabidopsis thaliana. Among them, two rhamnosyltransferases flavone baicalein with relatively high conversion yield were
AtUGT78D1 and AtUGT89C1 were functionally characterized to picked out to be under the preparative scale enzymatic
catalyse the O-rhamnosylation of flavonol at 3-OH or 7-OH synthesis. The corresponding glycosylated products were
1
5
position, respectively. Further investigation of AtUGT89C1 separated and identified. Structure elucidation revealed that
revealed its donor (TDP-rhamnose and UDP-rhamnose) and AtUGT78D1 could transfer not only the rhamnose moiety onto
acceptor (various classes of flavonoids including flavonols, the 3-OH position of flavonols, but also the glucose moiety
flavones and flavanones) substrate flexibilities via microbial onto the 7-OH position of flavone baicalein (1). Besides, the
biotransformation to generate a library of flavonoid 7-O- reaction reversibility of AtUGT78D1 were observed to catalyse
rhamnosides, which offered another proof that plant UGTs the bidirectional rhamnosylation to be applied in the one-pot
may have potential catalysis promiscuities towards sugar reaction to produce diverse desired bioactive rhamnosides
donors and/or acceptors to be used in universal glycosylation without adding the expensive UDP-Rha. The remarkable
of natural products based on many identified promiscuous promiscuity of AtUGT78D1 towards diverse aromatic acceptors
1
6-18
UGTs.
endow it not only a versatile tool for chemo-enzymatic
As far as AtUGT78D1, it was identified as a flavonol-specific synthesis of diverse rhamnosylated aromatic derivatives, but
glycosyltransferase which responsible for transferring also a potentially general functional part for secondary
rhamnose or glucose to the 3-OH position of flavonol in vitro. metabolism pathway design and construction in synthetic
And in recently reports, it have been successfully used in the biology of bioactive natural and unnatural products for lead
production of flavonol 3-O-glucosides or 3-O-rhamnosides by compounds discovery in drug R&D.
1
9-22
engineered bio-catalysis system.
In this article, aiming to thoroughly investigate the substrate
flexibilities of AtUGT78D1 in enzymatic rhamnosylation of
2
| J. Name., 2012, 00, 1-3
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PleaseRd So Cn oA t da vd aj un s ct ems argins
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DOI: 10.1039/C6RA16251G
ARTICLE
The supernatants of proteins were purified by Histrap
column (GE Healthcare, Uppsala, Sweden) previously
equilibrated in binding buffer (20 mM phosphate buffer, 20
2
. Materials and methods
2.1. Chemicals
+
UDP-Glucose and NAD were purchased from Sigma-Aldrich mM imidazole, 500 mM NaCl, 3% (v/v) glycerol, pH 7.4). After
(St. Louis, USA), NADPH was purchased from Biodee loading the protein solution, the column was rinsed with
Biotechnology Co., Ltd. (Beijing, China), and various kinds of binding buffer to remove unbound contaminant proteins. The
tested substrates for enzymatic reaction were purchased from bound His-tagged protein was then eluted with elution buffer
Chengdu Push Biotechnology Co., Ltd. (Chengdu, China) and (20 mM phosphate buffer, linear gradient of 20-500 mM
Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China).
imidazole, 500 mM NaCl, 3% (v/v) glycerol, pH 7.4). Then
fractions contained targeted proteins were concentrated in
Centrifugal Filter Units (Merck KGaA, Darmstadt, Germany) at
2.2. Gene cloning and heterogenous expression of AtRHM2-N,
AtRHM2-C and AtUGT78D1
4
500 ×g, 4 ᵒC. the obtained supernatant was subsequently
Total RNA of A. thaliana were isolated by Plant RNA Kit
diluted with desalting buffer (50 mM Tris-HCl, 100 mM NaCl, 1
mM DTT, 3% (v/v) glycerol, pH 7.4) using a PD-10 column (GE
Healthcare, Uppsala, Sweden), and the obtained enzymes
were stored at -80 ᵒC. The purity of targeted protein was
detected by SDS-PAGE to be >90% (Figure S2) and protein
concentration for all studies was determined as described
before.
(
OMEQA Bio-Tek, Inc. GA, USA). All PCR were done using high
fidelity KOD-Plus-Neo DNA Polymerase (TOYOBO, Osaka,
Japan). The AtRHM2-N, AtRHM2-C and AtUGT78D1 genes were
amplified by RT-PCR based on reported mRNA sequences as
described before and using cDNA of A. thaliana as template
with primer pairs as follows: AtRHM2-N-BamHI-F (5′-GGA TCC
ATG GAT GAT ACT ACG TAT AAG C-3′), AtRHM2-N-XhoI-R (5′-
CTC GAG TCA TAC AAC CGT AAA TGT CTG G-3′); AtRHM2-C-
BamHI-F(5′-GGA TCC ATG ACA CCT AAG AAT GGT G-3′),
2
.4. Chemo-enzymatic synthesis, HPLC-UV and HR-MS analysis of
UDP–Rha
AtRHM2-C-XhoI-R (5′-CTC GAG GGT TCT CTT GTT TGG TTC-3′); UDP-Rha was enzymatically synthesized by a two-step reaction
AtUGT78D1-EcoRI-F (5′-GAA TTC ATG ACC AAA TTC TCC GAG using UDP-Glc as substrate. For this purpose, the UDP-glucose-
CC-3′), AtUGT78D1-XhoI-R (5′-CTC GAG AAC TTT CAC AAT TTC 4,6-dehydratase (AtRHM2-N) and the UDP-4-keto-6-
GTC C-3′). Underlining indicates restriction sites (BamHI, EcoRI deoxyglucose-3,5-epimerase and UDP-4-keto-rhamnose-4-
and XhoI, respectively). The amplified DNA fragments were keto-reductase domains (AtRHM2-C) of A. thaliana UDP-Rha
digested with corresponding restriction enzymes (Takara, synthase were heterogenous expressed. For the first step of
Dalian, China) and inserted into the counterpart sites in pET- the reaction, UDP-4-keto-6-deoxy-glucose was synthesized
+
2
8a vector (Novagen). The fragments of vector and genes were using 1 mM UDP-Glc, 30 μg of AtRHM2-N, 0.5 mM NAD , and
recovered with Nucleic Acid Purification Kit (Axygen, Union 10 mM Tris-HCl (pH 8.5) in a total volume of 2.0 mL. After
City, USA) and then ligated with T DNA ligase (BioLabs, Beijing, overnight incubation at 25 ᵒC, the reaction was stopped by
4
China) at 16 ᵒC for 6 hours. The recombinant plasmid was heat inactivation for 10 min at 95 ᵒC. The volume of the
transformed into Escherichia coli BL21 (DE3) (TransGen, reaction mixture was then adjusted to 10.0 mL, containing 1
Beijing, China) before being confirmed.
mM NADPH, 60 μg of AtRHM2-C, and 10 mM Tris-HCl (pH 8.5).
After overnight incubation at 25 ᵒC, the reaction was stopped
by heat inactivation for 10 min at 95 ᵒC.
2.3. Expression and purification of recombinant proteins of
AtRHM2-N, AtRHM2-C and AtUGT78D1
The obtained UDP-Rha product was subsequently analysed
using a CAPCELL PAK ADME S5 column (4.6 mm I.D.×250 mm,
SHISEIDO Co., Ltd, Tokyo, Japan) eluted with ammonium
A single isolated bacterial colony was inoculated into liquid LB
-1
medium supplemented with kanamycin (50 mg·L ), and grown
for 5 h at 37 ᵒC, 200 rpm. A portion of the cultured cells was
transferred into fresh LB liquid medium supplemented with
the same antibiotics at a ratio of 1:100 and then grown at 37
ᵒC, 200 rpm until the optical density at 600 nm reached 0.4–
-1
formate (5 mM) over 10 min at a flow rate of 0.3 mL·min ,
using an Agilent 1260 Series HPLC system equipped with an
autosampler, diode array detector and ChemStation software.
During chromatographic runs, the injection volume was 5.0 µL,
plate cooler temperature was set to 4 ᵒC, and column
compartment was 25 ᵒC. Nucleotides sugars were detected at
0.6. Recombinant protein expression was induced by addition
of 1.0 mM isopropyl βꢀD-thiogalactopyranoside (IPTG), and
grown for 16 h at 25 ᵒC, 200 rpm. Cells were harvested by
centrifugation at 7000 ×g for 20 min before being suspended
in lysis buffer (20 mM phosphate buffer, 20 mM imidazole, 500
mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol,
pH 7.4). Cells were lysed in ice bath with Ultrasonic Processor
2
60 nm and coupled directly to the mass spectrometer for
analysis. Ultra-high purity helium (He) was used as the collision
gas, and high purity nitrogen (N ) was used as the nebulizing
gas. The optimized ESI source parameters were as follows:
2
-1
sheath gas flow rate, 1.5 mL·min ; auxiliary gas flow rate, 1.5
(VERNON HILLS, Illinois, USA) equipped with microtip probe
-1
mL·min ; spray voltage, 4.5 kV; capillary temperature, 200 ᵒC.
The spectra were recorded in the 100-1000 m/z range for a full
scan MS analysis.
using 200 sonication. Supernatants containing soluble RNM2-
N, RNM2-C and AtUGT78D1 proteins were filtered through
0.45 µm Millex Syringe-driven Filter Units (Merck KGaA,
Darmstadt, Germany) after centrifuging at 10000 ×g for 40 min
at 4 ᵒC.
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.5. Expanded enzymatic assays for substrate flexibility of
Journal Name
2
(1H, d, J=5.0 Hz, 3″-OH), 5.08 (1H, d, J=5.5 Hz, 4″-OH), 5.02 (1H,
d, J=7.5 Hz, H-1″), 4.64 (1H, d, J=6.0 Hz, 6″-OH), 3.76 (1H, m, H-
AtUGT78D1
6
″a), 3.50 (2H, m, H-6″b, H-3″), 3.37-3.19 (3H, m, H-2″, 4″, 5″).
The reaction mixture routinely contained 0.4 mM aromatic
substrate, 0.8 mM UDP-Glc or UDP-Rha and 60 µg AtUGT78D1
proteins in 200 μL reaction buffer (50 mM Tris-HCl, 100 mM
NaCl, 1 mM DTT, 1.5% (v/v) glycerol, pH 7.4). Assays with heat-
inactivated enzyme, were used as negative controls. After
incubation for 16 h at 30 °C, the reaction was terminated by
1
3
C-NMR (DMSO-d
6
, 125 MHz) δ 182.52 (C-4), 163.44 (C-2),
1
51.58 (C-7), 149.17 (C-9), 146.47 (C-5), 132.01 (C-4'), 130.81
(
C-6), 130.58 (C-1'), 129.12 (C-3', 5'), 126.34 (C-2', 6'), 106.07
C-10), 104.70 (C-3), 100.95 (C-1″), 94.24 (C-8), 77.32 (C-5″),
(
7
5.86 (C-3″), 73.16 (C-2″), 69.67 (C-4″), 60.63 (C-6″).
1
the addition of 400 μL of ethyl acetate and vortexed. The Quercetin-3-O-rhamnoside (7a). Yellow powder, H-NMR
supernatant were separated and evaporated to dryness under (Methanol-d , 500 MHz) δ 7.34 (1H, d, J=2.0 Hz, H-2'), 7.31 (1H,
4
reduced pressure. The resulted residues were re-dissolved in dd, J=8.5, 2.0 Hz, H-6'), 6.91 (1H, d, J=8.5 Hz, H-5'), 6.37 (1H, d,
methanol and centrifuged at 12,000 ×g for 30 min. The J=2.0 Hz, H-8), 6.20 (1H, d, J=2.0 Hz, H-6), 5.35 (1H, brs, H-1''),
supernatant was analysed by HPLC-UV and HR-ESI-MS. HPLC 4.21 (1H, brd, J=1.5 Hz, H-3''), 3.74 (1H, dd, J=9.5, 3.0 Hz, H-2''),
analyses were performed on a CAPCELL PAK C18 column (4.6 3.42 (1H, m, H-5''), 3.34 (1H, m, H-4''), 0.94 (3H, d, J=6.0 Hz, H-
1
3
mm I.D.×250 mm, SHISEIDO Co., Ltd, Tokyo, Japan) at a flow 6''). C-NMR (Methanol-d
4
, 125 MHz) δ 179.80 (C-4), 166.04
rate of 1 mL·min , and column temperature was maintained at (C-7), 163.45 (C-5), 159.46 (C-9), 158.68 (C-2), 149.96 (C-4'),
0 °C. The mobile phase was methanol (A) and 0.1 % formic 146.58 (C-3'), 136.38 (C-3), 123.10 (C-1'), 122.99 (C-6'), 117.05
acid in H O (B). The gradient elution A:B (v/v) was as follows: 0 (C-5'), 116.50 (C-2'), 106.03 (C-10), 103.69 (C-1''), 99.9 (C-6),
min (30:70)~25 min (90:10)~27 min (100:0). 94.84 (C-8), 73.39 (C-4''), 72.25 (C-3''), 72.19 (C-2''), 72.05 (C-
''), 17.81 (C-6'').
-1
3
2
5
2.6. In vitro preparation of enzymatic products by AtUGT78D1
1
Kaempferol-3-O-rhamnoside (10a). Yellow powder, H-NMR
The preparative scale reactions contained 0.03 mmol aromatic
substrate (baicalein , quercetin and kaempferol 10), 0.06
mmol UDP-Glc or UDP-Rha and 35 mg AtUGT78D1 proteins in
0 mL reaction buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM
DTT, 1.5% (v/v) glycerol, pH 7.4). After incubation for 16 h at
0 °C, the reaction supernatant were separated by column
(
Methanol-d
2H, d, J=8.5 Hz, H-3', 5'), 6.38 (1H, s, H-8), 6.20 (1H, s, H-6),
.38 (1H, brs, H-1''), 4.22 (1H, brs, H-2''), 3.71 (1H, d, J=5.5 Hz,
4
, 500 MHz) δ 7.76 (2H, d, J=8.5 Hz, H-2', 6'), 6.93
1
7
(
5
5
1
3
H-3''), 3.33 (2H, m, H-4'', 5''), 0.92 (3H, d, J=4.5 Hz, H-6''). C-
NMR (Methanol-d , 125 MHz) δ 179.61 (C-4), 165.86 (C-7),
63.22 (C-5), 161.58 (C-4'), 159.27 (C-9), 158.54 (C-2), 136.20
4
3
1
chromatography with macroporous resin (MCI GEL CHP) after
centrifuged at 12,000 ×g for 30 min. The mobile phase was a
gradient elution of 100 % water to 100% methanol, then each
aliquot were analysed by HPLC-UV. Aliquots including targeted
(
C-3), 131.89 (C-2', 6'), 122.61 (C-1'), 116.51 (C-3', 5'), 105.92
C-10), 103.49 (C-1''), 99.81 (C-6), 94.73 (C-8), 73.17 (C-4''),
(
7
2.10 (C-2''), 72.03 (C-3''), 71.92 (C-5''), 17.65 (C-6'').
1
product were evaporated to dryness under reduced pressure. Quercetin-3-O-glucopyranoside (18a). Yellow powder, H-
The resulted residues were re-dissolved in methanol and NMR (Methanol-d , 500 MHz) δ 7.72 (1H, d, J=2.0 Hz, H-2'),
4
purified by reverse phase semi-preparative HPLC, which was 7.59 (1H, dd, J=8.5, 2.0 Hz, H-6'), 6.87 (1H, d, J=8.5 Hz, H-5'),
performed on a YMC-Pack ODS-A HPLC column (10 mm 6.39 (1H, d, J=1.5 Hz, H-8), 6.20 (1H, d, J=1.5 Hz, H-6), 5.26 (1H,
I.D.×250 mm
，S-5 µm, 12 nm, Co., Ltd, Tokyo, Japan) at a flow d, J=7.5 Hz, H-1''), 3.72 (1H, brd, J=12.0 Hz, H-6''a), 3.58 (1H,
-1
rate of 3 mL·min . Column temperature was maintained at 25 dd, J=12.0, 5.0 Hz, H-6''b), 3.52-3.42 (2H, m, H-2″, 3″), 3.36
1
3
°
C. The mobile phase was methanol (A) and water (B). The (2H, m, H-4″, 5″). C-NMR (Methanol-d
gradient elution A:B (v/v) was as follows: 0 min (30:70)~10 min (C-4), 166.11 (C-7), 163.17 (C-5), 159.15 (C-9), 158.58 (C-2),
90:10)~20 min (100:0). Then, the obtained products were 149.97 (C-4'), 146.03 (C-3'), 135.76 (C-3), 123.33 (C-1'), 123.21
4
, 125 MHz) δ 179.62
(
1
13
confirmed by H and C nuclear magnetic resonance or (C-6'), 117.70 (C-5'), 116.13 (C-2'), 105.83 (C-10), 104.46 (C-1''),
followed by HMBC (heteronuclear multiple bond correlation). 100.01 (C-6), 94.84 (C-8), 78.52 (C-5''), 78.26 (C-3''), 75.87 (C-
''), 71.36 (C-4''), 62.70 (C-6'').
2
2
.7. One-pot reactions catalysed by AtUGT78D1
The one-pot reaction was composed of 0.4 mM quercitrin (18),
.4 mM kaempferol (10), 0.4 mM UDP and 60 μg purified
AtUGT78D1 in a total volume of 200 μL. After incubated at 30
C for 12 h, the reaction was terminated with 400 μL ice-cold
0
3
. Result and discussion
3
.1. Gene cloning and heterogenous expression of AtRHM2
°
methanol and centrifuged at 15,000 ×g for 1 h. The obtained A. thaliana contains three bi-functional enzymes that catalyse
supernatant was analysed by HPLC-UV and HR-MS as the synthesis of UDP-L-rhamnose from UDP-D-glucose:
described above.
AtRHM1 (At1g78570; GeneBank accession AY042833),
AtRHM2 (At1g53500; GeneBank accession AY328518) and
1
13
2
.8. H and C NMR data of prepared glycosylated products
2
AtRHM3 (At3g14790; GeneBank accession AY078958). As
3
1
-glucopyranoside (1a). Yellow powder, H-NMR
2
3-25
Baicalein-7-O-
β
illustrated in scheme 1,
functional domain analysis
(
DMSO-d
6
, 500 MHz) δ 12.56 (1H, s, OH-5), 8.56 (1H, s, OH-6),
.07 (2H, dd, J =7.0, 1.0 Hz, H-2', 6'), 7.61 (3H, m, H-3', 4', 5'),
revealed that the N-terminal region of RHM2 (RHM2-N; amino
8
7
acids 1-370) has the activity as an UDP-D-glucose-4,6-
.05 (1H, s, H-8), 7.00 (1H, s, H-3), 5.40 (1H, brs, 2″-OH), 5.11
4
| J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6RA16251G
ARTICLE
Inten. (x100,000)
D
549.0515
4
.0
3
.0
.0
.0
208.7663
2
311.0396
1
343.9665 426.0290
100
200
300
400
500
600
700
800
900
m/z
Inten. (x100,000)
322.9814
4.0
3
.0
.0
2
1.0
100
200
300
400
500
600
700
800
900
m/z
Inten. (x1,000,000)
E
565.0455
5.0
2.5
100
200
300
400
500
600
700
800
900
m/z
Inten. (x1,000,000)
322.9817
5
.0
.5
565.0456
2
100
200
300
400
500
600
700
800
900
m/z
Figure 1. HPLC-UV and HR-ESI-MS analysis of chemo-enzymatic synthesis reaction of UDP-Rha by UDP-Glc.
chromatogram and UV spectra of authentic UDP-Glc; (
reaction of UDP-Rha by UDP-Glc; ( ), HPLC chromatogram of negative control; (
(
A
), HPLC
B
), HPLC chromatogram and UV spectra of chemo-enzymatic synthesis
C
D
), HR-ESI-MS (negative) spectrum and typical
2
2
negative ion MS spectrum of UDP-Rha product; (
authentic UDP-Glc.
E), HR-ESI-MS (negative) spectrum and typical negative ion MS spectrum of
2 4
dehydratase, while the C-terminal region of RHM2 (RHM2-C; have been used. KH PO or ion-pairing reagents such as tetra-,
amino acids 371–667) has the two following UDP-4-keto-6- octa- and hexadecyltrimethyl-ammonium bromide in the
deoxy-D-glucose 3,5-epimerase, and UDP-4-keto-L-rhamnose presence of borate and tetrabutyl-ammonium salts have been
2
-keto-reductase activities. Here, we amplified the coding used in mobile phases.
3
24, 26-29
4
Thus the post-treatment of the
region of RHM2-N and RHM2-C separately based on the known enzymatic products such as desalting or ion-exchange et al
sequence to obtain the 1113 bp and 897 bp open reading were needed since the existed irons were not suitable for
frame by using PCR from the cDNA of A. thaliana before being direct mass spectrometry analysis. Here, the obtained UDP-
cloned into the expression vector pET-28a, respectively (Fig. Rha product was directly subjected to the HPLC-UV and HR-
S1). Soluble proteins were obtained from transformants of E. ESI-MS analysis using ammonium formate (5 mM) as mobile
-1
coli after overnight induction with 1.0 mM IPTG at 25 °C. phase at a flow rate of 0.3 mL·min . Nucleotide sugars were
Purified polypeptide was obtained by Ni-NTA-agarose to detected at 260 nm. The retention time for standard UDP-Glc
generate a product with apparent homogeneity by SDS-PAGE. was 9.774 min (Fig. 1A), and compared with blank control (Fig.
The observed molecular mass of AtRHM2-N and AtRHM2-C 1C), the reaction sample had a new peak at 10.570 min which
were 42.52 kDa (Fig. S2A) and 35.32 kDa (Fig. S2B) with the was suggested as an enzymatic reaction product (Fig. 1B). By
-1
-1
yield of 571 mg·L culture and 908 mg·L culture, respectively. HR-ESI-MS analyses in the negative ion mode (de-protonated
-
M-H] ), total MS ions of UDP-Glc standards gave [M-H] ion at
-
[
3
.2. Chemo-enzymatic synthesis of UDP-Rha by AtRHM2 protein
24 2 17 2
m/z 565.0455 (calcd 565.0477 for C15H N O P ), and the CID-
The UDP-Rha synthetic activity of AtRHM2 was assayed by a
two-step reaction using the homogeneous protein of AtRHM2-
+
N and AtRHM2-C as catalyst, UDP-Glc as substrate, NAD and
fragments formed from the parent ion gave a product ion at
-
m/z at 322.9817, corresponding to UMP [M-H] (Fig. 1E).
-
Similarly, Total MS ions of enzymatic product gave [M-H] ion
NADPH as co-factors. Control reaction was performed under
the same condition using the high temperature deactivated
protein of AtRHM2.
at m/z 549.0515 (the theoretical molecular weight of UDP-Rha
was 549.0528 for C15
24 2 16 2
H N O P ), and the CID-fragments
formed from the parent ion gave a product ion at m/z at
-
In the previously reported HPLC analysis of nucleotide 322.9814, corresponding to UMP [M-H] (Fig. 1D). The mass
sugars, anion exchange column and reversed phase columns spectrometry behaviour of the new peak at 10.570 min was
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Journal Name
consistent with UDP-Rha, confirmed that the enzymatic the reported optimum substrate flavonols (7–10), as well as
product was UDP-Rha with the yield of 39.08%. flavones , isoflavones 11, and flavonoid glycosides 14
1
.
3
.3. Gene cloning and heterogenous expression of AtUGT78D1
Considering the flexibility of AtUGT78D1 in recognizing
sugar acceptors, the donor specificity of AtUGT78D1 was also
examined with UDP-Glc, UDP-Gal, UDP-GlcA and UDP-N-
acetylglu-cosamine (UDP-GlcNAc). When using UDP-Glc as
glycosyl donor, the HPLC-UV chromatogram generated a single
product peak from all the flavonols (7–10) reaction mixtures,
The coding region of AtUGT78D1 (At1g30530; GeneBank
accession AY056312) was amplified using PCR from cDNA of
A.thaliana. For gene expression, the 1362 bp coding sequence
of AtUGT78D1 was cloned into the expression vector pET-28a.
E. coli cells harboring the expression plasmid were induced by
as well as flavones
1
, flavonones
4, isoflavones 12, and
1
.0 mM IPTG at 25 ᵒC. Protein induction was clearly observed
flavonoid glycosides 14
,
18, 19. These reaction mixtures were
and subsequently purified on nickel-NTA agarose to apparent
homogeneity as judged by SDS-PAGE (Fig. S2C). A yield of 668
then subjected to high-resolution mass analyses using HR-
QTOF-ESI-MS. We found the exact mass spectrum matching
with the calculated mass of each flavonoid glucoside (Table 2).
Among the ten positive substrates, the glucosylated product
mg purified His
6
-tagged AtUGT78D1 per litre of culture was
obtained. The observed molecular mass was corresponded
reasonably to the calculated mass of 51.74 kDa for His
AtUGT78D1.
6
-
yield of compound
1, 8, 9, 14 were high to 64.96%, 100%,
8
7.34%, and 96.84% respectively. Meanwhile, no obvious
3.4. Expanded enzymatic assays for substrate flexibility of
catalysis activity was observed with the three sugar donors of
UDP-Gal, UDP-GlcA and UDP-GlcNAc.
AtUGT78D1
Acceptor library with structurally diverse “drug-like” aromatics
scaffolds was established to investigate the substrate flexibility
and synthetic utility of AtUGT78D1. Totally, 30 different
members (their structures were shown in Fig. 2B), including
Our extensive enzymatic reactions expanded the substrate
spectrum of AtUGT78D1 effectively, and make
a
comprehensive clarification of its catalysis behavior towards
both sugar donor and acceptor. In addition to flavonols,
diverse flavones, flavonones, isoflavones, chalcones, flavonoid
flavones (baicalein
1
, diosmetin
hesperetin
, morin hydrate
2
, chrysin
naringenin
, fisetin , kaempferol 10);
genistein 12); chalcones
isoliquiritigenin 13); flavonoid glycosides (vincetoxicoside B
3
); flavonones
(
pinocembrin
quercetin
4,
5
,
6); flavonols
glycosides,
di-glycosides,
methylflavone,
phenylethyl
(
7
8
9
chromones, benzophenones, coumarins, lignanoids and
anthraquinones with active hydroxyl groups in their structures
could also be accepted by AtUGT78D1 to yield the
corresponding rhamnosylated products. According to HPLC-UV
isoflavones (formononetin 11
,
(
14, isovitexin 15, hyperin 16, swertisin 17, quercitrin 18,
scutellarin 19, sopho-ricoside 20); di-glycosides (rhoifolin 21);
methylflavone (methylophiopogonone A 22); phenylethyl
chromones (7-hydroxy-2-(2-phenylethyl) chromen-4-one 23, 8-
chloranyl-6-hydroxy-2-(2-phenylethyl) chromen-4-one 24);
benzophenones (4,4′-di-hydroxybenzophenone 25, 2,4-
dihydroxybenzophenone 26); coumarins (7-hydroxycoumarin
n
and HR-QTOF-ESI-MS analyses, all of the generated aromatic
rhamnosides were presumed to be O-rhamnosides. For
instance, as can be seen in Fig. S9, the parent-molecule ion
+
M+H] of the rhamnosylated product of 7-hydroxycoumarin
2
27) at m/z 309.0966 yielded MS fragment ions at m/z
[
(
1
63.0401, corresponding to the loss of a rhamnosyl moiety
2
7
, 7-hydroxy-4-methylcoumarin 28); lignanoids (magnolol
); anthraquinones (emodin 30) were used for expanded
(
146 amu). Since only one active hydroxyl group were existed
29
in 27, the rhamnosylation substitution were inferred to be
occurred at 7-OH group in product 27a. Similar situations were
also observed towards substrate 23 (Fig. S7), 24 (Fig. S8) and
enzymatic assays while both UDP-Rha and UDP-Glc were
tested as sugar donors, respectively.
From HPLC-UV analyses, it was revealed that when using the 28 (Fig. S10), and their rhamnosylated products were
optimum UDP-Rha as sugar donor, AtUGT78D1 could catalyse presumed to be 7-O-rhamnosyloxy-2-(2-phenylethyl) chromen-
the rhamnosylation of all 30 tested aromatic compounds 4-one (23a), 8-chloranyl-6-O-rhamnosyloxy-2-(2-phenylethyl)
belonging to 7 structural types except for 19. All the predicted chromen-4-one
(
24a
)
and
7-O-rhamnosyloxy-4-
rhamnosylated products were further identified by high methylcoumarin (28a) respectively.
resolution quadruple time-of-flight electrospray ionization
Besides, our extensive enzymatic reactions also revealed
that not only UDP-Rha but also UDP-Glc could also be accepted
by AtUGT78D1 to catalyse the glycosylation of 10 flavonoids
while the rhamnosylation efficiency was much higher than the
glucosylation efficiency, indicated that UDP-Rha was the
optimum sugar donor. Similarly, the rhamnosylation and/or
glucosylation efficiency of flavonols were much higher than
other substrates structure types, which indicated that
flavonols were the optimum sugar acceptor of AtUGT78D1.
mass spectrometry (HR-QTOF-ESI-MS). The found molecular
mass of the novel peaks matched with their calculated mass
exactly (Table 1). Besides, HR-MS analysis confirmed that both
mono-rhamnoside and di-rhamnoside were produced when
using baicalein (
1
) as sugar acceptor with UDP-Rha as sugar
[M-
H] 415.1035, found 415.1027, with the yield of 50.74%; di-
donor (mono-rhamnoside of baicalein: calcd. for C21
-
20 9
H O
+
rhamnoside of baicalein: calcd. for C27
found 563.1754, with the yield of 37.01%, Table 1.).
Moreover, AtUGT78D1 exhibited high substrate
conversion rate (>40%) with 7 out of 30 substrates, including
30
H O13 [M+H] 563.1759,
Of particular note is that the enzyme exhibited the
capability to glycosylate natural “drug-like” scaffolds including
a
methylflavone
(
22); phenylethyl chromones
(23-24);
6
| J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6RA16251G
ARTICLE
coumarins (27-28); magnolol (29) and emodin (30). Besides, best of our knowledge, the rhamnosylations of these “drug-
the rhamnosylation of diverse flavonoid glycosides including like” scaffolds by plant GTs were rarely reported before, and
both mono- and di-glycosides resulted in the formation of the unusual substrate promiscuity rendered AtUGT78D1 a
polysaccharides with one or more glycosyl substitutions at promising enzyme for the creation of structurally diverse
different positions which usually lead to the improved aqueous bioactive glycosides especially for various rhamnosides.
solubilities, stabilities, and enhanced bioavailabilities. To the
A
R
R
6
5
R
6
R
R
5
4
B
R
R
2
1
R
R
4
3
O
O
R
R
5
4
R
3
O
O
HO
O
O
R
R
3
2
O
R
1
R
1
R
2
R
2
OH
1 2 5 6 3 4
14 R =R =R =R =OH; R =H; R =ORha
15 R =R =H; R =R =R =OH; R =Glc
R
1
O
7
7
8
9
R =R =R =R =R =OH; R =H
1
1
1
2
2
3
4
5
=R
6
1
1
1
1
5
2
4
6
3
a
R
R
R
=ORha; R
2
=R
=R
=R =R
3
4
=R
5
6
=OH; R =H
1
1
2
3
R
a R
R
1
1
1
1
=R
=R
=R
=R
2
2
3
3
=R
3
=OH; R
=OH; R
=R =OH; R
=OH; R
O
4
=R
=OGlc; R
=H; R
=R
5
=H
=R
=OCH
=H
4 R =R =H
16
17
18
R
R
R
=OGal; R
=R
=ORha; R
2
=R
=R
=R
4
=R
=OH; R
=R =R
5
=R
6
=OH; R
=Glc; R
=OH; R
3
=H
=OCH
=H
1
2
=R
=R
2
=R
3
=R
5
6
=OH; R
=R =OH
5
4
=H
=H
5 R =OH; R =OCH
3
6 R =H; R =OH
5
=H; R
2
6
3
4
3
3
4
5
1
2
6
=H; R
1
3
4
4
2
4
5
3
1
2
2
4
5
6
3
10 R =R =R =R =OH; R =R =H
1
1
2
3
5
=R
4
6
R
2
=R
5
18a R =OGlc; R =R =R =R =OH; R =H
1
1
2
4
5
6
3
1
0a R
=ORha; R
2
3
=R
5
=OH; R
4
=R
6
=H
1
9
R
=H; R
2
=R
3
=R
6
=OH; R
4
5
=OGlcA; R =H
OH
OH
HO
OH
HO
O
O
O
O
HO
HO
HO
O
O
R
1
O
R
2
OH
O
OH
13
O
R
ΗΟ
OH
O
HO
1
1
1 R
2 R
1
1
=H,R
=R
2
=OCH
3
OH
2
0 R=OGlc
21
2
=OH
R
3
R2
O
OH
R
2
O
R
1
R
2
R
3
HO
O
O
O
O
R
1
R1
O
O
O
OH
HO
29
2
7
R
1
=OH; R
=OGlc; R
=OH; R =CH
=OGlc; R =CH
2
=H
2
2
2
2
3
3a R
4
4a R
R
1
1
1
1
=R
=R
3
=H; R
=H; R
2
=OH
=OGlc
=Cl
=H; R =Cl
1 3 2
25 R =R =OH; R =H
26 R =H; R =R =OH
1 2 3
2
2
27a R
2
2
1
2
=H
3
2
8
8a R
R
1
2
3
R
=OH; R
=OGlc; R
2
=H; R
3
1
2
3
2
3
O
O
H
3
C
OH
OH
OH
O
OH
HOOC
HO
HO
O
O
ΗΟ
O
HO
HO
HO
HO
OH
OH
OH
OH
OH
OH
3
0
Glc=β-D-Glucosyl
GlcA=β-D-Glucuronosyl
Gal=β-D-Galactosyl
Rha=α-L-Rhamnosyl
Figure 2. Exploring the aglycon promiscuity of AtUGT78D1 using UDP-Glc and UDP-Rha as sugar donor respectively. (A),
Conversions of glycosylated products catalysed by AtUGT78D1. Members are listed with the number which corresponding to the
structures listed in part B. The columns in dark color represent the percent conversion using UDP-Rha as sugar donor, while the
light color represents the percent conversion using UDP-Glc as sugar donor. “*” represents the glycosylated products that were
prepared and confirmed by HPLC-UV, HR-MS, and NMR spectroscopic data analyses (1a, 7a and 10a). ‘‘#” represents the
2
glycosylated products that were structurally identified by HPLC-UV, HR-MS, and typical ion MS data analyses (23a
,
24a
,
27a and
were
), The structures of tested aglycons and corresponding
28a), or by comparison with authentic sample (18a). “§” represents that a mono-rhamnoside and di-rhamnoside of
1
produced with the yield of 50.74% and 37.01% respectively. (
B
glycosylated products.
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n
Table 1. Characterization of enzymatic rhamnosylated products by HPLC–ESI–MS analysis.
Substrate Substrate RT Rha-product Rha-product Measured m/z
Predicted
formula
Theoretical
Error
(ppm)
-1.93
-0.89
-1.28
0.67
(
No)
(min)
RT (min)
yield %
50.74
37.01
24.28
22.02
14.38
11.94
6.17
m/z
-
1
9.5
0.6
415.1027 [M-H]
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
21
27
22
21
21
22
21
21
21
21
21
22
21
21
27
27
27
28
27
H
20
H
30
H
22
H
20
H
22
H
24
H
22
H
20
H
20
H
20
H
20
H
22
H
20
H
22
H
30
H
30
H
30
H
32
H
30
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
9
415.1035
563.1759
469.1105
455.1140
425.1207
477.1297
441.1156
447.0933
471.0898
455.0949
431.0984
437.1207
461.1089
401.1242
595.1657
577.1563
609.1461
591.1719
593.1512
1
21.1
+
2
563.1754 [M+H]
13
10
8
+
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
*
20.9
24.0
22.5
18.4
17.8
17.8
17.1
15.6
20.2
21.3
18.9
20.4
16.8
12.3
13.4
12.4
15.1
13.4
13.8
14.2
27.4
22.9
26.6
21.4
14.2
10.8
13.4
27.2
29.1
19.8
22.8
21.7
17.8
17.3
15.0
15.1
12.8
17.0
19.6
16.9
19.5
13.5
10.9
11.0
10.6
12.8
N.P.*
11.4
13.2
26.2
21.6
24.9
20.8
13.4
10.0
12.4
25.8
27.3
469.1099 [M+Na]
-
445.1143 [M+HCOO]
+
425.1205 [M+Na]
8
-0.47
-4.70
1.36
-
447.1276 [M-H]
10
9
+
441.1162 [M+Na]
-
96.38
95.63
79.25
40.01
45.32
25.65
3.99
447.0930 [M-H]
11
11
10
10
8
-0.67
4.67
+
471.0920 [M+Na]
+
455.0969 [M+Na]
4.39
-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
431.0974 [M-H]
-2.32
4.12
+
437.1225 [M+Na]
-
461.1074 [M+HCOO]
9
-3.25
-4.49
0.84
-
401.1224 [M-H]
8
+
98.86
4.45
595.1662 [M+H]
15
14
16
14
15
-
577.1532 [M-H]
-5.37
-3.78
-3.89
-0.34
-
8.00
609.1348 [M-H]
-
6.27
591.1696 [M-H]
-
5.83
593.1510 [M-H]
+
13.09
9.85
579.1689 [M+H]
C
C
C
C
C
C
C
C
C
C
C
27
33
25
23
23
19
19
15
16
24
21
H
30
H
40
H
26
H
24
H
23
H
20
H
20
H
16
H
18
H
28
H
20
O
O
O
O
O
O
O
O
O
O
O
14
18
10
7
579.1708
725.2287
509.1418
435.1414
491.1114
383.1101
361.1282
309.0969
323.1125
457.1868
439.1000
-3.28
3.59
4.12
10.34
-2.85
2.35
-1.66
-0.97
4.02
-5.03
1.37
+
725.2313 [M+H]
+
9.61
509.1439 [M+Na]
+
12.08
4.82
435.1459 [M+Na]
-
491.1100 [M+HCOO]
7
7
7
7
7
6
9
Cl
+
20.59
14.68
22.58
22.47
25.23
13.59
383.1110 [M+Na]
+
361.1276 [M+H]
+
309.0966 [M+H]
+
323.1138 [M+H]
-
457.1845 [M+HCOO]
+
439.1006 [M+Na]
N.P. represents as no product.
n
Table 2. Characterization of enzymatic glucosylated products by HPLC–ESI–MS analysis.
Substrate Substrate
No)
Glc-product
RT (min)
19.6
N.P.*
N.P.
Glc-product
yield %
Measured m/z
Predicted
formula
Theoretical
m/z
Error
(ppm)
-2.55
(
RT (min)
21.1
20.9
24.0
22.5
18.4
17.8
17.8
17.1
15.6
20.2
21.3
18.9
20.4
16.8
12.3
13.4
12.4
15.1
13.4
---
-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
*
64.96
431.0973 [M-H]
C
21 20
H O
10
431.0984
-
16.9
N.P.
N.P.
1.92
463.1233 [M+HCOO]
C
21 22
H O
9
463.1246
-2.81
+
13.5
13.4
11.7
36.28
100.00
87.34
22.24
465.1038 [M+H]
C
C
C
C
21 20
H O
21 20
H O
21 20
H O
21 20
H O
12
12
11
11
465.1028
463.0882
471.0898
447.0933
2.15
-5.40
4.03
-2.91
-
463.0857 [M-H]
+
471.0917 [M+Na]
-
0
1
2
15.2
N.P.
11.6
447.0920 [M-H]
-
5.03
477.1016 [M+HCOO]
C
C
21
H
20
O
O
10
16
477.1038
609.1461
-4.61
-4.43
3
4
5
N.P.
11.0
N.P.
-
96.84
609.1434 [M-H]
H
27 30
6
7
8
N.P.
N.P.
13.5
+
8.09
7.11
465.1043 [M+H]
C
C
21
H
H
20
O
O
12
17
465.1028
623.1254
3.23
-
9
0-30
10.8
N.P.
623.1218 [M-H]
27
28
-5.78
N.P. represents as no product.
8
| J. Name., 2012, 00, 1-3
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3
.5. In vitro preparation of enzymatic products by AtUGT78D1
It is important to note that most glycosylated products
exhibited improved biological activities or water solubilities
compared to the parent molecules. For instance, in vivo
quercetin 3-O-rhamnoside (7a) can be a more important
As mentioned above, our extensive enzymatic reactions
expanded the recognition of AtUGT78D1 as a flavonol-3-O-
rhamnosyltransferase. To further reveal the catalytic
antioxidant and neuro-protective agent than quercetin (
because its high bioavailability in the digestive tracks.
7)
characteristic of AtUGT78D1 in vitro, a flavone baicalein (
without hydroxyl group at C-3 position as well as two flavonols
quercetin and kaempferol 10) with hydroxyl group at C-3
1)
3
0
Kaempferol 3-O-rhamnoside (10a) showed higher inhibitory
activities against aldose reductase (AR) than flavonoid
aglycone kaempferol (10) to be a useful natural source in the
development of a novel AR inhibitory agent against diabetic
(
7
position were picked out as sugar acceptors and subjected to
the preparative scale enzymatic reactions using UDP-Glc and
UDP-Rha as sugar donor, respectively. When using baicalein (1)
3
1
complications.
The solubility of baicalein 7-O-β-
glucopyranoside (1a) in water (50 mg·L ) was 10 times greater
as sugar acceptor, UDP-Glc as sugar donor, one glucosylated
1
product (1a) was obtained, and structurally analysed by H and
-1
-1
1
3
than baicalein (
1, 5.4 mg·L ), which implies that the
C-NMR followed by HMBC (heteronuclear multiple bond
1
attachment of a glucosyl residue to baicalein enhanced the
3
water solubility of the original compound effectively.
correlation). While comparing the H NMR of standard
baicalein ( ) and reaction product (1a), signals at δ 12.56 (1H,
s, 5-OH) and δ 8.56 (1H, s, 6-OH) were detected but 7-OH was
2
1
In a word, our results expanded the catalytic characteristic
missing in reaction product (Fig. S45), reminded that the sugar of AtUGT78D1 in vitro. It can catalyse not only the
moiety could have replaced proton of this hydroxyl group. The rhamnosylation at 3-OH position of flavonols, but also
anomeric proton (H-1″) was consistent with δ 5.02 (d, J=7.5 Hz, glucosylation at 7-OH position of baicalein, as well as multiple
1H), and the anomeric proton coupling constant (J=7.5 Hz) hydroxyl substitutions of diverse kinds of aromatics (discussed
confirms the conjugation of glucose moiety to be in β in section 3.4) to afford various glycosylated products which
-
configuration. To determine the position of sugar conjugation, always exhibited improved pharmacological activities
HMBC data of 1a were further analysed. Correlation of compared with their precursors. These finding provide strong
anomeric proton H-1″ (δ 5.02) with C-7 (δ 151.58) in HMBC hints that AtUGT78D1 could be used as a potential powerful
confirmed the attachment of glucose moiety at 7-OH position tool for the glycosylation of diverse aromatic for structural
of baicalein (
determined to be baicalein-7-O-
The rhamnosylation of two flavonols (quercetin
1
, Fig. 3). Therefore, the final product was modifications and drugs discovery.
β
-glucopyranoside.
3
.6. One-pot reaction catalysed by AtUGT78D1
In our aforementioned extensive enzymatic assays for
substrate flexibility of AtUGT78D1, when quercitrin (18
quercetin 3-O-rhamnoside, C H O ) was tested as sugar
7
and
kaempferol 10) resulted in two rhamnosylated products 7a
and 10a. Their structures were identified as quercetin 3-O-
rhamnoside (7a) and kaempferol 3-O-rhamnoside (10a) with
,
2
1 20 11
acceptor with UDP-Glc as sugar donor, a glycosylated product
8a was formed compared with the negative control at t 13.5
1
13
HR-MS, H and C NMR spectroscopic data analysis (Figs. S48–
S51, NMR data shown in section 2.8), which confirmed that the
rhamnosylation were specifically occurred at 3-OH position
when using flavonols as substrates.
1
R
min in the yield of 8.09%. However, HR-MS analysis revealed
that the molecular formula of 18a was predicted to be
+
C H O (calcd. for C H O [M+H] 465.1028, found
2
1
20 12
21 20 12
4
65.1043), which possess an extra oxygen atom compared
with substrate 18. Meanwhile, as can be seen in Fig. S6, the
-
parent-molecule ion [M+H] of 18a at m/z 465.1028 yielded
2
MS fragment ions at m/z 303.0458, corresponding to the loss
6 10
of the glucosyl moiety (C H O5, 162 amu). These results
indicated that the glucose moiety was transferred into
quercetin instead of quercitrin. Based on further
chromatographic and spectral analysis, as well as comparison
and co-HPLC with the authentic samples (Fig. S43), product
1
8a was identified to be quercetin 3-O-glucopyranoside, (Fig.
S52-S53, NMR data shown in section 2.8), confirmed that de-
rhamnosylation of quercitrin (18) was occurred firstly in the
reaction to give the aglycon quercetin (
glucosylation at 3-OH position.
7) before subsequent
Generally, GTs are perceived as unidirectional catalysts that
drive the formation of glycosidic bonds from NDP-sugar donors
Figure 3. HMBC of glucosylated product of baicalein (1),
baicalein-7-O-β-glucopyranoside (1a) showing correlation of
anomeric proton and C-7 of baicalein.
3
3
and aglycon acceptors. However, more and more reported
GTs were observed to catalyse reversible even bidirectional
3
4-37
reactions.
The observed de-rhamnosylation of quercitrin
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Journal Name
18 reminded us that AtUGT78D1 may be employed for de- characteristic of AtUGT78D1 respectively. Combined with the
glycosylation in certain cases to transfer sugar moiety from obvious reaction reversibility for one-pot production,
rhamnoside scaffolds to aglycons. To prove this speculation, AtUGT78D1 was expected to be a universal and effective tool
the reverse assays of AtUGT78D1 catalysed reactions were for chemo-enzymatic synthesis of diverse desired bioactive
conducted in the presence of UDP and quercitrin (18). As rhamnosylated derivatives. Our studies also provide useful
expected, the de-rhamnosylated product quercetin (
7) was hints for further enzyme engineering to develop powerful
detected by HPLC-UV analysis (Fig. S44A), confirming the biocatalysts in the combinatorial biosynthesis of leading
reaction reversibility of AtUGT78D1.
compounds for drug R&D.
Removal of rhamnose unit from 3-O position of quercitrin
(
18) affords AtUGT78D1 the availability for molecule “one-pot”
Acknowledgements
reaction. Simple aromatic substrate kaempferol (10) was
selected as an example to subjected to the coupled reactions We are thankful to the financial supports by the National
with UDP and quercitrin (18) to further exploit the application Natural Science Foundation of China (Grant Nos. 81402809)
of AtUGT78D1 in generating bioactive rhamnosides through and graduate independent project in Beijing University of
one-pot reactions. Interestingly, in the coupled reaction (Fig. Chinese Medicine (No. 2016-JYB-XS077).
4), UDP-Rha was generated by AtUGT78D1 catalysed de-
rhamnosylation of 18 with the presence of UDP, and the
rhamnose moiety was intermediately transferred to
kaempferol (10) through subsequent rhamnosylation catalysed
by AtUGT78D1 to generate the product kaempferol-3-O-
rhamnoside (10a) with the high yield of 50.17% (Fig. S44). This
one-pot reaction, providing desired products via reusing the
byproduct without adding the expensive UDP-Rha, was
economic and environmental-friendly. As far as we know, it is
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