pH-promoted O-α-glucosylation of flavonoids using an engineered α-glucosidase mutant

Article abstract of DOI:10.1016/j.bioorg.2020.104581

Retaining glycosidase mutants lacking its general acid/base catalytic residue are originally termed thioglycoligases which synthesize thio-linked disaccharides using sugar acceptor bearing a nucleophilic thiol group. A few thioglycoligases derived from retaining α-glycosidases have been classified into a new class of catalysts, O-glycoligases which transfer sugar moiety to a hydroxy group of sugar acceptors, resulting in the formation of O-linked glycosides or oligosaccharides. In this study, an efficient O-α-glucosylation of flavonoids was developed using an O-α-glycoligase derived from a thermostable α-glucosidase from Sulfolobus solfataricus (MalA-D416A). The O-glycoligase exhibited efficient transglycosylation activity with a broad substrate spectrum for all kinds of tested flavonoids including flavone, flavonol, flavanone, flavanonol, flavanol and isoflavone classes in yields of higher than 90%. The glucosylation by MalA-D416A preferred alkaline conditions, suggesting that pH-promoted deprotonation of hydroxyl groups of the flavonoids would accelerate turnover of covalent enzyme intermediate via transglucosylation. More importantly, the glucosylation of flavonoids by MalA-D416A was exclusively regioselective, resulting in the synthesis of flavonoid 7-O-α-glucosides as the sole product. Kinetic analysis and molecular dynamics simulations provided insights into the acceptor specificity and the regiospecificity of O-α-glucosylation by MalA-D416A. This pH promoted transglycosylation using O-α-glycoligases may prove to be a general synthesis route to flavonoid O-α-glycosides.

Full text of DOI:10.1016/j.bioorg.2020.104581

Bioorganic Chemistry 107 (2021) 104581
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
pH-promoted O-
α
-glucosylation of flavonoids using an engineered
α
-glucosidase mutant
,
1
,
,
Chao Lia 2, Jetendra Kumar Roya 2, Ki-Cheul Parkb, Art E. Chob, Jaeick Leea,
,
*
Young-Wan Kima
a Department of Food and Biotechnology, Korea University, 2511 Sejong-Ro, Sejong 30019, Republic of Korea
b Department of Biotechnology and Bioinformatics, Korea University, 2511 Sejong-Ro, Sejong 30019, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Retaining glycosidase mutants lacking its general acid/base catalytic residue are originally termed thio-
glycoligases which synthesize thio-linked disaccharides using sugar acceptor bearing a nucleophilic thiol group.
Retaining glycosidase
O-glycoligase
Flavonoids
A few thioglycoligases derived from retaining α-glycosidases have been classified into a new class of catalysts, O-
glycoligases which transfer sugar moiety to a hydroxy group of sugar acceptors, resulting in the formation of O-
linked glycosides or oligosaccharides. In this study, an efficient O- -glucosylation of flavonoids was developed
using an O- -glycoligase derived from a thermostable -glucosidase from Sulfolobus solfataricus (MalA-D416A).
pH promoted O-α-glucosylation
α
α
α
The O-glycoligase exhibited efficient transglycosylation activity with a broad substrate spectrum for all kinds of
tested flavonoids including flavone, flavonol, flavanone, flavanonol, flavanol and isoflavone classes in yields of
higher than 90%. The glucosylation by MalA-D416A preferred alkaline conditions, suggesting that pH-promoted
deprotonation of hydroxyl groups of the flavonoids would accelerate turnover of covalent enzyme intermediate
via transglucosylation. More importantly, the glucosylation of flavonoids by MalA-D416A was exclusively
regioselective, resulting in the synthesis of flavonoid 7-O-
molecular dynamics simulations provided insights into the acceptor specificity and the regiospecificity of O-
-glucosylation by MalA-D416A. This pH promoted transglycosylation using O- -glycoligases may prove to be a
general synthesis route to flavonoid O- -glycosides.
α-glucosides as the sole product. Kinetic analysis and
α
α
α
1. Introduction
galactosides, rhamnosides, arabinosides, and rutinosides [10]. There-
fore, if we can simplify the sugar moiety to just glucose and invert the
stereochemistry of glycosidic bonds in flavonoid glycosides from β to
Flavonoids are widely distributed polyphenols in plants and valuable
secondary metabolites with significant roles for human health [1–3].
Importantly, flavonoids are naturally conjugated with carbohydrate
moieties, leading to significantly enhanced stability and solubility [4].
In addition, the sugar structures predominantly affect the bio-
availabilities and the adsorption in the human body [5,6]. For the
adsorption in the small intestine, it is essentially requested the degly-
cosylation of the dietary flavonoid glycosides [6–9]. However, naturally
occuring flavonoid glycosides are mostly β-glucosides which are resis-
tant to hydrolysis of human pancreatic digestive enzymes. Although the
dietary flavonoid glycosides are hydrolyzed by lactase phlorizin hy-
drolase in the small intestine [7,8], dietary flavonoids are not only
glucosides but also in the form of various glycosides, such as
α
-glucosidic linkage, the flavonoid O-α-glucosides may have improved
bioavailablity due to facile deglucosylation by intestinal digestive
hydrolases.
Several successful attempts for
α-glucosylation of flavonoids and
their glycosides with sucrose using wild type transglucosidases and
phosphorylases have been reported [11–15]. Recently, engineered en-
zymes which use sucrose as a donor sugar have been developed to
improve the yields or to expand the sugar acceptor repertories [16],
however, it is still challenging due to low yields of the desired product by
rehydrolysis of the products, uncontrollable sugar oligomerization and
poor regioselective glycosylation. Alternatively, it would be useful to use
engineered glycosidases, termed glycosynthases (Fig. 1A), which are
* Corresponding author.
E-mail address: ywankim@korea.ac.kr (Y.-W. Kim).
1
Current address: Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, United States.
C. L. and J. K. R. contributed equally to this work.
2
https://doi.org/10.1016/j.bioorg.2020.104581
Received 19 September 2020; Received in revised form 17 December 2020; Accepted 19 December 2020
Available online 28 December 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.

C. Li et al.
Bioorganic Chemistry 107 (2021) 104581
catalytic nucleophile-deficient retaining glycosidase mutants, catalysing
transglycosylation using glycosyl fluorides or azides with the opposite
stereochemistry to the original substrate as sugar donors [17–19]. Un-
fortunately, a few successful cases for the glycosynthase-based synthesis
of β-glycosyl flavonoids have been reported [20,21], but no report for
catalytic residue [22–24]. Interestingly, there are a few reports
regarding transglycosylation activity of the acid/base mutants toward
OH groups of normal sugars, which have been termed O-glycoligases
(Fig. 1C) [22,25,26]. Given the yields of the O-linked transfer products,
the O-glycoligase activities of
β-thioglycoligases derived from retaining
dases, respectively. The reason for the potent O-glycoligase activity of
the -thioglycoligases is not entirely clear, but the reactive skew boat
conformation of the accumulated β-glycosyl enzyme intermediate of the
acid/base -glycosidase mutants would be helpful for the coupling with
OH groups to occur [25]. Recently, the applications of O-glycoligases
α
-thioglycoligases are much higher than
α
-glycosyl flavonoid synthesis. By contrast, thioglycoligases (Fig. 1B)
α-glycosidases and β-glycosi-
that are another class of engineered retaining glycosidase mutants
generated by the mutation at its general acid/base catalytic residue are
promising. Originally, thioglycoligases catalyse the synthesis of S-linked
glycosides using thio-sugar acceptors bearing a more nucleophilic thiol
group than a hydroxyl group without the assistance of the general base
α
α
Fig. 1. Mechanism of (A) transglycosylation by α-glycosynthases toward a hydroxyl group of normal sugars; (B) transglycosylation by α-thioglycoligases toward a
thiol group of synthetic thio-sugars; (C) transglycosylation by O-glycoligases toward a hydroxyl group of normal sugars; (D) transglycosylation by O-glycoligases
toward a hydroxyl group of phenolic compounds with a pKa lower than that of normal sugars.
2

C. Li et al.
Bioorganic Chemistry 107 (2021) 104581
have been expanded to synthesis of O-
α
-glycosides through trans-
(isoflavone) and genistein 10a (isoflavone) were used as the sugar ac-
ceptors. To our surprise, all flavonoids employed in the trans-
glycosylation reacted very well, as only single transfer product from
each reaction was detected on TLC plate under UV light (Figure S1).
When the reaction mixtures were analyzed by LC-MS, only the peaks
corresponding to the glucosides with single glucosyl moiety were
detected with no peak relevant to glucosides with more than one glu-
cosyl moiety (data not shown).
glycosylation toward the hydroxyl group of simple nitrophenols and
ascorbic acid containing OH groups with a pKa value less than those of
OH groups of normal sugars (Fig. 1D) [27,28].
These results raise a question as to whether O-
α-glycoligases have
potential catalytic power for direct O-α-glycosylation of natural flavo-
noid aglycones. As flavonoids are weak acids due to presence of phenolic
groups, their water solubility is enhanced in alkaline pH, and naturally
at high temperature [29]. In addition, reaction at an alkaline pH would
promote the deprotonation of one of hydroxyl group where the glucose
moiety will be transferred (Fig. 2). Here, we investigated trans-
2.2. Time course analysis
glucosylation by an O-α-glycoligase derived from a thermostable
To determine the optimum reaction time to get maximum yield,
transglucosylation reactions using 25 mM donor (1) and 20 mM quer-
cetin (2a) as the substrates were carried out at different pHs. As expected
from the pH profile for transglucosylation activity (Fig. 2), the product
formation at pH 9 was the fastest compared to those at other pH, and the
maximum yield was 99.7% by HPLC analysis after 2 h reaction (Fig. 5).
At pH 8 and 8.5 longer reaction time than that at pH 9 was requested to
get the maximum yields (98 and 99% by HPLC analysis, respectively)
due to the slow reaction rates. After the maximum yield points, the
hydrolysis of the transfer product was observed, leading to slightly
decrease in the yield.
α
-glucosidase from Sulfolobus solfataricus (MalA-D416A) [24] using
various flavonoids as the substrate for
synthesis.
α-glucosylated flavonoid
2. Results and discussion
2.1. MalA-D416A-catalyzed transglucosylation for flavonoids
First, glucosylation of quercetin 2a (belonging to the flavonol class)
was carried out using 1 as the sugar donor. DMSO, which is commonly
used as co-solvent in enzymatic reaction, was added (30% [v/v]) to
increase the solubility of flavonoids in an aqueous buffer. The reaction
was carried out at pH 5 which was previously reported to be the opti-
mum pH for the hydrolysis activity of wide-type MalA [30]. However,
no obvious transfer product was detected by TLC. By contrast, a clear
UV-active spot corresponding to a glycosylated product (2b) appeared at
pH 7–9, and the intensity of the spot was enhanced at pH 8 and 9 (Fig. 3),
implying that the deprotonation of a OH group of 2a at the more basic
pH than pH 7 plays a significant role in the O-glucosylation by MalA-
D416A.
2.3. Determination of structures of flavonoid O-α-glucosides
Next, glucosylation reactions of flavonoids by MalA-D416A (5 mg)
were carried out at pH 9 on a preparative scale using 1 (0.125 mmol) and
one of flavonoids 2a-10a (0.1 mmol in 5 mL). The corresponding
transfer products (2b-10b) were then purified by flash silica gel chro-
matography with the isolation yields over 90% (Fig. 6).
Then, 2D (COSY, NOESY, HSQC and HMBC) NMR analyses were
carried out to identify the structure of these transfer products
(Figure S3, S4). Given the NMR data, all signals for anomeric protons
(H1′′) appeared around 5.3–5.6 ppm as typical doublet peaks, indicating
the MalA-D416A synthesized a glycosidic linkage between flavonoid
and sugar moieties. Because of the low values of coupling constants J1,2
(3.2–4.0 Hz) of H1′′, the anomeric configurations of all the synthetic
The initial rate of the reactions determined by HPLC using the iso-
lated transfer product as the standard was the highest at pH 9 (Fig. 4)
and 45 ◦C. The reduction of the rates at pH 10 could be explained by the
instability of sugar donor 1, due to the enhancement of its spontaneous
hydrolysis at basic conditions [31]. In order to investigate the possibility
of the action of another amino acid residue in the active site as the
general base catalyst to activate 7-OH group of the flavonoids, the
linkages were α-anomers. Next, the regioselectivity of MalA-D416A was
determined by heteronuclear interactions HMBC. The C7 and H8 of
flavonoids coupled with the anomeric proton (H1′′) and carbon (C1′′) of
glucose moiety, respectively, revealing that MalA-D416A catalyzes
highly regioselective glucosylation towards hydroxyl group of flavo-
noids only at O7. Another evidence of the specific regioselectivity of
MalA-D416A comes from the NOESY spectra, as H1′′ of glucose moiety
showed interaction only with H8 of the flavonoids, instead of other
proton on the phenolic rings.
control reactions using para-nitrophenyl α- and β-D-glucophyranosides
as the sugar acceptors were conducted, resulting in no transfer product.
Therefore, there results revealed that MalA-D416A is able to transfer
glucosyl moiety of the glucosyl intermediate to deprotonated hydroxy
groups of aryl compounds [27] and flavonoids at pH 9 without the
assistance of general base catalytic residue.
With the aim of investigating the substrate spectrum of MalA-D416A
in the synthesis of flavonoid O-α-glucosides, another diverse 8 congeners
including kaempferol 3a (belonging to flavonol), hesperetin 4a (flava-
none), naringenin 5a (flavanone), taxifolin 6a (flavanonol), (+)-cate-
chin 7a (flavanol), (-)-epicatechin 8a (flavanol), daidzein 9a
2.4. Kinetics of transglycosylation reaction catalyzed by MalA-D416A
To investigate the substrate specificity of MalA-D416A, apparent
Fig. 2. Scheme of flavonoid O-
α
-glucosides synthesis catalyzed by MalA-D416A using α-D-glucopyranosyl fluoride (1) and flavonoids as the sugar donor and ac-
ceptors, respectively.
3

C. Li et al.
Bioorganic Chemistry 107 (2021) 104581
Fig. 3. TLC analysis of transglycosylation reaction using
α
-glucosyl fluoride (1) and quercetin (2a) as the substrate at different pH. (A) under UV254nm; (B) visualized
by H2SO4 and charring. Lane 1, 1 and 2a (control); lane 2, 1 and 2a (blank reaction); lane 3, reaction mix at pH 5.0; lane 4, at pH 6.0; lane 5, at pH 7.0; lane 6, at pH
8.0; lane 7, at pH 9.0.
were classified into the good acceptors. The relatively high catalytic
efficiency for these acceptors were mainly caused from the higher kcat
values than those for 2a and 3a. Of the acceptors, catechins (7a and 8a)
8
lacking the oxo group at C4 and isoflavones (9a and 10a) bearing the B
ring at C3 exhibited lower kcat/KM than the others due to the relatively
6
high KM values ranging from 0.5 to 1.1 mM. The kinetics analyses
revealed that the hydroxyl group at 5-OH and the non-aromatic C ring
with the oxo group at C4 give positive effects on the transglucosylation.
4
Upon comparing 6a and 7a, indeed, the only difference is whether the
oxo group at C4. In addition, an isoflavone 10a without the OH group at
2
0
C5 exhibited 2-fold higher KM value than the other isoflavone 9a. The
inappropriate orientation of the ring B in isoflavones would also result in
weak affinity toward MalA-D416A. The KM values for the donor (1)
during transglucosylation at a fixed acceptor concentration were lower
than that for 4-nitrophenyl
α-D-glucopyranoside in hydrolysis by the
5
6
7
8
9
10
11
wild-type MalA enzyme (KM = 2.0 mM) [32], which is consistent with
the kinetic reports for acid/base mutants derived from retaining glyco-
sidases [33,34]. Such low KM of MalA-D416A for the sugar donor 1
pH
implies that flavonoid O-α-glycoside synthesis using O-α-glycoligases
Fig. 4. Effect of pH on transglucosylation activities using
(1) and quercetin (2a) as the substrates.
α-glucosyl fluoride
would be more potent than that using glycosynthases with much higher
KM values for fluoride sugars as the donors in the transglycosylation
reactions [35].
100
80
60
40
20
0
2.5. in-silico modelling of complexes of MalA-D416A and flavonoids
It is worth noting that the transglucosylated products of catechin and
epicatechin whose 3′–OH groups on B ring have been proposed as the
most acidic OH groups (pKa1 = 8.7 and 8.9, respectively) [36], also
possessed the glucosyl moiety at their 7-OH groups. In addition, the
second pKa values of other flavonoids are around pH 8–9 that would be
able to ionize under the basic condition (pH 9), so it could be very likely
to generate a mixture of regioisomers. However, MalA-D416A produced
only one transfer product, suggesting that the O-α-glucosylation by
MalA-D416A would be controlled by not only the pH-promoted nucle-
ophilicity of the 7-OH groups of the flavonoids, but also the steric
tolerance for the orientation of the 7-OH groups to direct the glucosyl
moiety of the glucosyl-enzyme intermediate.
0
60
120
180
240
300
Reaction mine (min)
The docking simulations for the model structure of the covalent
glucosyl enzyme intermediate with the flavonoids were conducted. The
Glide standard precision docking scores of the flavonoids agree with the
experimental catalytic efficiencies yielding R2 = 0.872 (Fig. 7A), sug-
gesting that the computational modelling is mostly likely reliable. The
complex models exhibited a consistent binding mode for the flavonoids.
Given the complex models, it was predicted that the A and C rings of the
flavonoids would bind in the subsite + 1 which has been proposed by the
crystal structure analysis of the wild type MalA [37]. In addition, the in-
Fig. 5. Time course analysis of the transglucosylation of quercetin (2a) cata-
○
lyzed by MalA-D416A. Each reaction was conducted at pH 7(●), pH 7.5 ( ), pH
8 (▾), pH 8.5 (△), pH 9 (■), and pH 10 (□).
kinetic parameters were determined at a fixed concentration of either
the donor (1) or an acceptor (Table 1 and Figure S2). Given kcat/KM
values for the flavonoids, the flavonoids with the non-aromatic C ring
with both oxo group at C4 and hydroxyl group at C5 (4a, 5a, and 6a)
4

C. Li et al.
Bioorganic Chemistry 107 (2021) 104581
Fig. 6. Structures and isolation yields of flavonoid 7-O- α-glucosides.
acceptors, and the absence of 5-OH in daidzein (10a) made a synergistic
negative effect on the catalytic efficiency (Table 1). Unfortunately, these
docking complex models did not give clear insight for the role of the oxo
group at C4 and non-aromatic C ring for the transglucosylation, which
were discussed in section 2.4.
Table 1
Kinetic parameters of MalA-D416A.
Substrates
varieda)
kcat (minꢀ 1
)
KM (mM)
kcat/KM (minꢀ 1 mMꢀ 1
)
fixedb)
2a
3a
4a
5a
6a
7a
8a
9a
10a
1
1
1.3 ± 0.06
1.5 ± 0.1
2.3 ± 0.1
2.5 ± 0.1
3.1 ± 0.9
1.8 ± 0.1
1.6 ± 0.07
1.5 ± 0.09
1.8 ± 0.1
1.4 ± 0.08
1.7 ± 0.05
2.4 ± 0.1
2.6 ± 0.1
2.5 ± 0.1
1.7 ± 0.08
1.5 ± 0.06
1.5 ± 0.1
1.4 ± 0.1
0.37 ± 0.04
0.34 ± 0.04
0.39 ± 0.03
0.39 ± 0.04
0.25 ± 0.02
0.77 ± 0.09
0.65 ± 0.08
0.52 ± 0.05
1.10 ± 0.10
0.52 ± 0.05
0.49 ± 0.04
0.53 ± 0.05
0.50 ± 0.04
0.53 ± 0.03
0.47 ± 0.04
0.47 ± 0.03
0.53 ± 0.04
0.46 ± 0.02
3.6
4.5
5.8
6.4
12.4
2.4
2.5
2.9
1.6
2.8
3.4
4.6
5.2
6.6
3.5
3.2
2.7
3.1
1
3. Conclusion
1
1
The regioselective 7-O-
α
-glucosylation for flavonoids using an O-
1
α
-glycoligase has been successfully accomplished for the first time.
1
1
Although the detail reaction mechanism of O-
α-glucosylation in this
1
study is not fully clear, our findings in this work suggest that the pH-
promoted nucleophilicity enhancement of the sugar acceptor at alka-
line pH and the steric tolerance of the binding site for flavonoids in the
active site of MalA-D416A make it possible to progress the efficient and
1
2a
3a
4a
5a
6a
7a
8a
9a
10a
1
1
1
regioselective O-
α
-glucosylation for flavonoids. This strategy could be
1
applied to other
α
-glycosidases to synthesis of various O- -glycosides
α
1
derived from phenols, flavonoids, and their analogues. Recently, the
1
synthesis of 1-O-acyl
α-L-arabinofuranoses using a thioglycoligase
1
1
derived from an arabinofuranosidase has been reported [38]. It would
therefore be interesting to carry out synthesis of glycosyl esters using O-
glycoligases using phenolic acids as acceptors.
Reaction condition was 0.2 M Tris-HCl buffer (pH 9.0) at 45 ◦C.
a)
The concentrations were varied up to 2 mM.
b)
The concentration was fixed at 2 mM.
4. Materials and methods
silico modelling showed another subsite (subsite + 2) for the binding of
ring B, consisting of Y184, A482, T483, D484, and the peptide backbone
from F449 to N453 (Fig. 7B). The D87 and D484 of the enzyme would
interact with 5-OH and 4′–OH of most of ligands, respectively, through
H-bonds (Fig. 7B and 7C). The H-bonding of D87 and D484 with fla-
vonoids and the hydrophobic platform for the binding of the B ring
would allow to locate the 7-OH of flavonoids toward glucose moiety of
the intermediate, leading to the highly regioselective transglucosylation
at 7-OH position. In the case of the isoflavones (9a and 10a), binding of
the B-ring in the subsite + 2 would engage in the misorientation of the A
and C rings, leading to moving of 7-OH further away from the glucosyl
moiety in the intermediate; the distance from the anomeric centre of the
glucosyl moiety to 7-OH of 6a and 10a would be 3.3 and 4.6 Å,
respectively (Fig. 7C). Indeed, 9a and 10a were one of the poor
4.1. General experiments
MalA-D416A was produced and purified as described previously
[24]. Flavonoids and silica gel (200–425 mesh) for flash chromatog-
raphy were purchased from Sigma-Aldrich. HPLC was carried out with
YL9100 HPLC system (Younglin, Anyang, Korea) equipped with a Sun-
Fire™ C18 5 μL column (4.6 × 150 mm; Waters, Milford, MA). The
products were eluted at flow rate 0.8 mL minꢀ 1 with acetonitrile/water
(30:70, v/v) containing formic acid (0.1%) and analyzed with an ultra-
violet (UV) detector under the optimized wavelengths of respective
flavonoid product (varied from 220 to 380 nm). 1H and 13C NMR spectra
were recorded on a 400 MHz spectrometer (JEOL, Tokyo, Japan) with
methanol‑d4 as the solvent. Mass data for transfer products were
5

C. Li et al.
Bioorganic Chemistry 107 (2021) 104581
Fig. 7. In-silico modelling for complex structures of the glucosyl enzyme intermediate of MalA-D416A with flavonoids. (A) Plots of Glide SP scores against
experimental catalytic efficiency (kcat/KM) for nine flavonoids, (B) Interaction between amino acid residues of the glucosyl enzyme intermediate of MalA-D416A with
taxifolin (6a). Green arrows and numbers represent H-bonds and their lengths. The residues around 6a within 4 Å and glucosyl moiety in the intermediate are
represented. (C) Superposition of acceptors, 6a, genistein (9a), or daidzein (10a) in the complex models of the glucosyl enzyme intermediate. Side chains for the
residues are depicted in bond style coloring with carbon atoms in grey. Flavonoids are represented in bond style coloring with carbon atoms in purple (6a), cyan (9a),
or green (10a). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
recorded with an LTQ XL linear ion trap mass spectrometer (Thermo
Scientific). TLC was performed on aluminum-backed 0.2 mm silica gel
60F254 sheets (Whatman/GE Healthcare) with ethyl acetate/methanol/
water (17:2:1, v/v/v) containing formic acid (0.1%). The plates were
visualized under UV (254 nm) and/or exposure to sulfuric acid (10%) in
methanol followed by charring.
(C-3′), 147.84 (C2), 137.03 (C3), 122.41 (C-1′), 121.03 (C-6′), 116.96
(C-5′), 116.50 (C-2′), 104.66 (C-10), 97.76 (C-6), 94.40 (C-8) (Ph-C);
101.39 (C-1′’), 76.85 (C-3′’), 74.42 (C-4′’), 73.83 (C-5′’), 73.17 (C-2′’),
61.87 (C-6′’). HMBC shows H1′’ coupled with C7 and H8 coupled with
C1′’. NOESY shows H8 coupled with H1′’. ESI-MS: calc. for [C21H20O12
H+]– = 463.4; found 463.4.
–
4.3.2. Kaempferol 7-α-O-glucoside (3b)
4.2. Catalytic properties of MalA-D416A
Yellow powder (40.4 mg, isolation yield 90%). 1H NMR (meth-
anol‑d4, 400 MHz): 8.05 (m, 2H, Ph-H2′ and 6′), 6.83 (m, 2H, Ph-H3′ and
5′), 6.68 (m, 1H, Ph-H6), 6.39 (m, 1H, Ph-H8), 5.49 (d, 1H, J = 3.2 Hz,
The reactions were carried out using 25 mM donor sugar (1) and 20
mM quercetin (2a) in a buffer (200 mM) containing 30% DMSO and
MalA-D416A (1 mg/mL). To determine optimum pH, the reactions were
conducted at a pH ranging from pH 6 to pH 10 and 45 ◦C. Upon varying
temperature, the pH was fixed at pH 9 using 0.2 M Tris-HCl buffer. At
each time point the samples were aliquoted and boiled to terminate the
reaction. The amount of the glucosyl product (2b) was determined by
HPLC using a calibration curve which was generated using the purified
compound from the preparative scale reaction as the standard. The
initial rates of the reactions were determined by linear regression with
the data within the 10% consumption of the initial flavonoid. In the case
of time course analysis, the reaction was conducted in 200 mM Tris/HCl
(pH 9) at 45 ◦C, and the reaction yield was calculated based on the
subjected 2a as the substrate.
H1′’), 3.62 (m, 1H, H4′’), 3.42 (m, 1H, H3′’), 3.36 (m, 3H, H6′’ and 2′’),
13
3.18 (m, 1H, H5′’). C NMR (methanol‑d , 100 MHz): 177.70 (C O);
–
–
4
167.31 (C-7), 159.72 (C-5), 158.29 (C-9), 155.74 (C-4′), 147.37 (C2),
138.31 (C3), 131.98 (2C), 124.04 (C-1′), 117.33 (2C), 105.17 (C-10),
99.92 (C-6), 95.29 (C-8) (Ph-C); 100.03 (C-1′’), 76.21 (C-3′’), 74.86 (C-
4′’), 74.16 (C-2′’), 73.69 (C-5′’), 62.81 (C-6′’). HMBC shows H1′’
coupled with C7 and H8 coupled with C1′’. NOESY shows H8 coupled
with H1′’. ESI-MS: calc. for [C21H20O11 – H+]– = 447.4; found 447.4.
4.3.3. Hesperetin 7-α-O-glucoside (4b)
White powder (46.0 mg, isolation yield 99%). 1H NMR (methanol‑d4,
400 MHz): 6.97 (m, 1H, Ph-H2′), 6.82 (m, 2H, Ph-H5′ and 6′), 6.36 (m,
1H, Ph-H6), 5.72 (m, 1H, Ph-H8), 5.37 (d, 1H, J = 3.6 Hz, H1′’), 5.08 (t,
1H, J = 4.7 Hz, H2), 3.92 (s, 3H, OCH3), 3.86 (m, 1H, H4′’), 3.67 (m, 1H,
4.3. Preparative scale reaction and structure analysis of transfer products
H3′’), 3.59 (m, 2H, H6′’), 3.44 (m, 1H, H2′’), 3.31 (m, 2H, H3), 3.19 (m,
13
1H, H5′’). C NMR (methanol‑d , 100 MHz): 198.50 (C O); 165.19 (C-
–
–
4
A mixture of donor sugar (1, 0.12 mmol) and acceptor (0.1 mmol) in
Tris-HCl buffer (5 mL; 0.2 M; pH 9.0) containing 30% DMSO was treated
with MalA-D416A (1 mg), and the mixture was incubated for 2 h at
7), 162.45 (C-5), 161.99 (C-9), 151.24 (C-4′), 148.17 (C-3′), 132.64 (C-
1′), 121.53 (C-6′), 118.95 (C-2′), 116.28 (C-5′), 105.66 (C-10), 98.51 (C-
6), 96.46 (C-8) (Ph-C); 97.29 (C-1′’), 81.48 (C-2), 76.92 (C-3′’), 75.86
(C-4′’), 74.68 (C-2′’), 73.22 (C-5′’), 62.27 (C-6′’), 57.24 (OCH3), 43.52
(C-3). HMBC shows H1′’ coupled with C7 and H8 coupled with C1′’.
◦
45 C. The flavonoid glucosides were purified on a C18 SEP PAK car-
tridge (Waters) to remove unreacted sugar, DMSO, proteins and salts.
After the solvent was evaporated under reduced pressure, transfer
products were isolated by flash silica gel chromatography by solvent
gradient elution (ethyl acetate/methanol/water, 17:2:1 to 7:2:1). The
structures of purified product were characterized by LC-MS, 1D (1H and
13C) and 2D (COSY, HSQC, HMBC, and NOESY) NMR spectra, succes-
sively. The isolation yield of each transfer product was calculated using
the weight of the isolated product based on the subjected flavonoid as
the substrate.
NOESY shows H8 coupled with H1′’. ESI-MS: calc. for [C22H24
= 463.4; found 463.4.
O
11 – H+]–
4.3.4. Naringenin 7-α-O-glucoside (5b)
White powder (42.9 mg, isolation yield 99%). 1H NMR (methanol‑d4,
400 MHz): 7.23 (m, 2H, Ph-H2′ and 6′), 6.76 (m, 2H, Ph-H3′ and 5′),
6.18 (m, 2H, Ph-H6 and 8), 5.47 (d, 1H, J = 3.4 Hz, H1′’), 5.19 (t, 1H, J
= 5.5 Hz, H2), 3.71 (m, 1H, H4′’), 3.56 (m, 1H, H3′’), 3.43 (m, 3H, H6′’
and 2′’), 3.28 (m, 2H, H3), 3.16 (m, 1H, H5′’). 13C NMR (methanol‑d4,
4.3.1. Quercetin 7-α-O-glucoside (2b)
–
100 MHz): 197.29 (C O); 168.42 (C-7), 161.86 (C-5), 160.42 (C-9),
–
Yellow powder (42.7 mg, isolation yield 92%). 1H NMR (meth-
anol‑d4, 400 MHz): 7.16 (m, 1H, Ph-H2′), 6.92 (m, 1H, Ph-H5′), 6.87 (m,
1H, Ph-H6′), 6.23 (m, 1H, Ph-H6), 5.77 (m, 1H, Ph-H8), 5.52 (d, 1H, J =
3.6 Hz, H1′’), 3.56 (m, 1H, H4′’), 3.50 (m, 1H, H3′’), 3.41 (m, 3H, H6′’
154.21 (C-4′), 132.60 (C-1′), 130.65 (2C), 118.37 (2C), 104.28 (C-10),
97.18 (C-6), 95.83 (C-8) (Ph-C); 101.29 (C-1′’), 79.63 (C-2), 76.10 (C-
4′’), 75.89 (C-3′’), 74.52 (C-2′’), 72.77 (C-5′’), 64.13 (C-6′’), 42.96 (C-
3). HMBC shows H1′’ coupled with C7 and H8 coupled with C1′’. NOESY
and 2′’), 3.32 (m, 1H, H5′’). 13C NMR (methanol‑d4, 100 MHz): 176.47
shows H8 coupled with H1′’. ESI-MS: calc. for [C21H22O10 – H+]–
=
′
–
(C O); 166.94 (C-7), 158.32 (C-5), 157.74 (C-9), 150.83 (C-4 ), 148.25
–
6

C. Li et al.
Bioorganic Chemistry 107 (2021) 104581
433.4; found 433.4.
H3′’), 3.45 (m, 3H, H6′’ and 2′’), 3.37 (m, 2H, H3), 3.24 (m, 1H, H5′’).
13
–
C NMR (methanol‑d , 100 MHz): 178.26 (C O); 166.73 (C-7), 158.34
–
4
4.3.5. Taxifolin 7-
α
-O-glucoside (6b)
(C-9), 157.59 (C-4′), 153.27 (C2), 130.69 (2C), 129.57 (C-5), 125.49 (C-
1′), 123.74 (C3), 118.69 (C-10), 117.73 (2C), 115.48 (C-6), 99.36 (C-8)
(Ph-C); 105.83 (C-1′’), 76.35 (C-3′’), 74.93 (C-4′’), 73.74 (C-2′’), 72.02
(C-5′’), 61.40 (C-6′’). HMBC shows H1′’ coupled with C7 and H8
coupled with C1′’. NOESY shows H8 coupled with H1′’. ESI-MS: calc. for
[C21H20O9 – H+]– = 415.4; found 415.4.
White powder (46.2 mg, isolation yield 99%). 1H NMR (methanol‑d4,
400 MHz): 7.01 (m, 1H, Ph-H2′), 6.86 (m, 1H, Ph-H5′), 6.80 (m, 1H, Ph-
H6′), 6.39 (m, 1H, Ph-H6), 5.84 (m, 1H, Ph-H8), 5.45 (m, 1H, H2), 5.41
(m, 1H, H3), 5.36 (d, 1H, J = 3.2 Hz, H1′’), 3.72 (m, 1H, H4′’), 3.63 (m,
1H, H3′’), 3.55 (m, 2H, H6′’), 3.42 (m, 1H, H2′’), 3.34 (m, 2H, H3), 3.26
13
(m, 1H, H5′’). C NMR (methanol‑d , 100 MHz): 197.58 (C O); 167.29
–
–
4
(C-7), 163.77 (C-5), 162.27 (C-9), 148.86 (C-4′), 147.41 (C-3′), 130.37
(C-1′), 120.65 (C-6′), 116.33 (C-2′), 115.24 (C-5′), 101.48 (C-10), 99.85
(C-6), 97.28 (C-8) (Ph-C); 98.26 (C-1′’), 86.53 (C-2), 75.73 (C-3′’), 74.51
(C-4′’), 74.10 (C-2′’), 73.74 (C-5′’), 70.81 (C-3), 63.62 (C-6′’). HMBC
shows H1′’ coupled with C7 and H8 coupled with C1′’. NOESY shows H8
coupled with H1′’. ESI-MS: calc. for [C21H22O12 – H+]– = 465.4; found
465.4.
4.4. Kinetic analysis of transglycosylation catalyzed by MalA-D416A
All kinetic studies were carried out at 45 ◦C in Tris-HCl (0.2 M, pH
9.0). The amount of released fluoride ion was detected by using a
fluoride electrode (Thermo Scientific). The concentration of the donor or
acceptor sugar was fixed (2 mM), while that of the counterpart was
varied. For fluoride electrode analysis, a standard curve was generated
by using various concentrations of fluoride ion solution containing
DMSO (30%). All enzymatic rates were corrected for the spontaneous
hydrolysis rate of 1. The apparent KM and kcat values were determined
by fitting the initial velocity curves to the Michaelis-Menten equation by
nonlinear regression in GraFit (Erithacus Software, UK).
4.3.6. (+)-Catechin 7-α-O-glucoside (7b)
White powder (40.7 mg, isolation yield 90%). 1H NMR (methanol‑d4,
400 MHz): 7.14 (m, 1H, Ph-H2′), 6.95 (m, 1H, Ph-H5′), 6.87 (m, 1H, Ph-
H6′), 5.81 (m, 1H, Ph-H6), 5.67 (m, 1H, Ph-H8), 5.48 (d, 1H, J = 4.0 Hz,
H1′’), 5.03 (m, 1H, H2), 4.52 (m, 1H, H3), 3.82 (m, 1H, H4′’), 3.64 (m,
1H, H3′’), 3.58 (m, 3H, H6′’ and 2′’), 3.48 (m, 2H, H3), 3.25 (m, 1H,
H5′’), 2.68 (m, 2H, H4). 13C NMR (methanol‑d4, 100 MHz): 159.35 (C-
7), 158.21 (C-5), 157.83 (C-9), 145.26 (C-4′), 145.87 (C-3′), 130.29 (C-
1′), 120.93 (C-6′), 116.39 (C-5′), 115.35 (C-2′), 99.52 (C-10), 94.34 (C-
6), 92.64 (C-8) (Ph-C); 99.01 (C-1′’), 85.49 (C-2), 77.53 (C-3′’), 75.28
(C-4′’), 73.96 (C-5′’), 73.43 (C-2′’), 67.36 (C-3), 62.74 (C-6′’), 29.28 (C-
4). HMBC shows H1′’ coupled with C7 and H8 coupled with C1′’. NOESY
4.5. Model structure preparation
The flavonoid structures were generated by the 2D Sketcher in
¨
¨
Maestro (Schrodinger Release 2019-3, Schrodinger, LLC), after which
¨
LigPrep (Schrodinger Release 2019-3) was used to convert them into 3D
structures. During the preparation, the force field was set to OPLS3, and
all the combinations of stereoisomers were generated. MalA cocrystal
complex was downloaded from Protein Data Bank (PDB ID: 2G3M).
shows H8 coupled with H1′’. ESI-MS: calc. for [C21H24O11 – H+]–
=
¨
451.4; found 451.4.
Downloaded files were prepared with Schrodinger’s Protein Preparation
Wizard. Restrained minimization was performed to relax only the added
hydrogens using Impact with OPLS3 force field. The skew boat confor-
mation of glucosyl moiety was attached to Asp320, the nucleophilic
residue of MalA. And then, mutated Asp416 to Ala and stabilized the
4.3.7. (-)-Epicatechin 7-α-O-glucoside (8b)
White powder (44.3 mg, isolation yield 98%). 1H NMR (methanol‑d4,
400 MHz): 7.06 (m, 1H, Ph-H2′), 6.82 (m, 1H, Ph-H5′), 6.73 (m, 1H, Ph-
H6′), 5.72 (m, 1H, Ph-H6), 5.60 (m, 1H, Ph-H8), 5.45 (d, 1H, J = 3.9 Hz,
H1′’), 5.01 (m, 1H, H2), 4.46 (m, 1H, H3), 3.78 (m, 1H, H4′’), 3.60 (m,
1H, H3′’), 3.53 (m, 3H, H6′’ and 2′’), 3.44 (m, 2H, H3), 3.19 (m, 1H,
H5′’), 2.62 (m, 2H, H4). 13C NMR (methanol‑d4, 100 MHz): 158.37 (C-
7), 158.02 (C-5), 157.29 (C-9), 144.83 (C-4′), 144.17 (C-3′), 129.66 (C-
1′), 119.18 (C-6′), 114.76 (C-5′), 114.23 (C-2′), 98.24 (C-10), 94.39 (C-
6), 92.20 (C-8) (Ph-C); 100.16 (C-1′’), 85.08 (C-2), 76.36 (C-3′’), 76.02
(C-4′’), 74.59 (C-5′’), 73.29 (C-2′’), 66.33 (C-3), 60.22 (C-6′’), 26.36 (C-
4). HMBC shows H1′’ coupled with C7 and H8 coupled with C1′’. NOESY
¨
structure through energy minimization by Prime (Schrodinger
Release 2019-3).
4.6. Molecular docking
¨
Schrodinger’s Glide [39] was used to predict poses for a given pro-
tein–ligand complex. Glide generated the possible binding modes of
ligand–protein complexes and scores them with GlideScore, a mixture of
interaction energy and parameter-based penalty functions that roughly
represents binding energy. The docking algorithm in Glide utilizes a
hierarchical search protocol. In the first stage, 5000 poses for each
ligand docking were kept from initial generation for refinement. After
the refinement 400 poses were kept for minimization using grids during
which a maximum of 100 steps were imposed. Finally, the one pose was
scored and ranked after minimizations. Selection of the final ligand pose
for Glide is done with GlideScore, which is an extension of an empiri-
cally based Chem-Score function of Eldridge et al.[40].
shows H8 coupled with H1′’. ESI-MS: calc. for [C21H24O11 – H+]–
=
451.4; found 451.4.
4.3.8. Genistein 7-α-O-glucoside (9b)
White powder (41.0 mg, isolation yield 95%). 1H NMR (methanol‑d4,
400 MHz): 8.12 (s, 1H, C = CH), 7.37 (m, 2H, Ph-H2′ and 6′), 6.83 (m,
2H, Ph-H3′ and 5′), 6.72 (m, 1H, Ph-H6), 6.58 (m, 1H, Ph-H8), 5.48 (d,
1H, J = 3.6 Hz, H1′’), 3.52 (m, 1H, H4′’), 3.46 (m, 1H, H3′’), 3.39 (m,
3H, H6′’ and 2′’), 3.31 (m, 2H, H3), 3.14 (m, 1H, H5′’). 13C NMR
–
–
(methanol‑d , 100 MHz): 180.74 (C O); 167.21 (C-7), 161.63 (C-5),
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
157.73 (C-94), 156.23 (C-4′), 153.28 (C2), 131.82 (2C), 125.89 (C3),
124.44 (C-1′), 117.12 (2C), 104.63 (C-10), 97.28 (C-6), 94.51 (C-8) (Ph-
C); 100.64 (C-1′’), 75.51 (C-3′’), 74.68 (C-4′’), 74.07 (C-2′’), 72.68 (C-
5′’), 61.33 (C-6′’). HMBC shows H1′’ coupled with C7 and H8 coupled
with C1′’. NOESY shows H8 coupled with H1′’. ESI-MS: calc. for
[C21H20O10 – H+]– = 431.4; found 431.4.
This work was supported by a Research Fellowship Program funded
by Korea University, Republic Korea.
4.3.9. Daidzein 7-α-O-glucoside (10b)
White powder (38.3 mg, isolation yield 92%). 1H NMR (methanol‑d4,
400 MHz): 8.28 (s, 1H, C = CH), 8.01 (m, 1H, H5), 7.32 (m, 2H, Ph-H2′
and 6′), 7.14 (m, 1H, Ph-H6), 7.03 (m, 1H, Ph-H8), 6.78 (m, 2H, Ph-H3′
and 5′), 5.56 (d, 1H, J = 3.6 Hz, H1′’), 3.63 (m, 1H, H4′’), 3.56 (m, 1H,
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104581.
7

C. Li et al.
Bioorganic Chemistry 107 (2021) 104581
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