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Bioactive Constituents, Metabolites, and Functions
One-pot Multi-enzyme Cofactors Recycling (OPME-CR) System for Lactose and Non-natural Saccharide Conjugated Polyphenol Production Sumangala Darsandhari, Ramesh Prasad Pandey, Biplav Shrestha, Prakash Parajuli, Kwangkyoung Liou, and Jae Kyung Sohng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02421 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 4, 2018
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Journal of Agricultural and Food Chemistry
Journal of Agricultural and Food Chemistry One-pot Multi-enzyme Cofactors Recycling (OPME-CR) System for Lactose and Non-natural Saccharide Conjugated Polyphenol Production Sumangala Darsandhari1,Ϯ, Ramesh Prasad Pandey1,2,Ϯ, Biplav Shrestha1, Prakash Parajuli1, Kwangkyoung Liou1,2, Jae Kyung Sohng1,2,* 1
Department of Life Science and Biochemical Engineering, SunMoon University, 70 Sunmoon-ro 221, Tangjeong-
myeon, Asan-si, Chungnam 31460, Republic of Korea 2
Department of BT-Convergent Pharmaceutical Engineering, SunMoon University, 70 Sunmoon-ro 221,
Tangjeong-myeon, Asan-si, Chungnam 31460, Republic of Korea.
Ϯ
These authors contributed equally.
*Corresponding author: Prof. Jae Kyung Sohng Tel: +82(41)530-2246 Fax: +82(41)530-8229 Email:
[email protected] 1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1
ABSTRACT
2
A one-pot multi-enzyme cofactors recycling (OPME-CR) system was designed for the synthesis of UDP-α-D-
3
galactose which was combined with LgtB, a β-(1,4) galactosyltransferase from Neisseria meningitidis to modify
4
various polyphenol glycosides. This system recycles one mole of ADP and one mole of UDP to regenerate one mole
5
of UDP-α-D-galactose by consuming two moles of acetylphosphate and one mole of D-galactose in each cycle. The
6
ATP additionally used to generate UDP from UMP was also recycled at the beginning of the reaction. The
7
engineered cofactors recycling system with LgtB efficiently added a D-galactose unit to a variety of sugar units such
8
as D-glucose, rutinose, and 2-deoxy-D-glucose. The temperature, pH, incubation time, and divalent metal ions for
9
the OPME-CR system were optimized. The maximum number of UDP-α-D-galactose regeneration cycles (RCmax)
10
was 18.24 by fed batch reaction. The engineered system generated natural and non-natural polyphenol saccharides
11
efficiently and cost-effectively.
12 13
Keywords. Lactoside derivatives, Glycosylation, One pot system, Cofactors recycling
14
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INTRODUCTION
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Sugars are structural components frequently found in a large number of natural products. Important drugs such as
17
amphotericin B, avermectins, vancomycin, erythromycin, and doxorubicin, contain sugars appended to their
18
aglycone structure.1 These sugars participate in the biomolecular recognition of the cellular target by a bioactive
19
compound; their existence is therefore essential for the biological activity of many natural products.2 The vast
20
majority of the natural products (NPs) produced by microbes are naturally glycosylated and many of them are
21
utilized as drugs or drug leads owing to their biological properties.1,3 Plant secondary metabolites are also decorated
22
with different sugar units, which play distinct physicochemical and biological roles in plant physiology, interspecies
23
interactions,4 and human and animal health.5 A number of plant and animal metabolites are coated with different
24
sugars and they have their own functions. One of the metabolites of Gaultheria yunnanensis (FRANCH) REHDER
25
(G yunnanensis), methylsalicyclate 2-O-β-D-lactoside, demonstrates a potential therapeutic role, for example, in the
26
treatment of rheumatoid arthritis, swelling, and pain.6 Similarly, multifunctional human milk oligosaccharides such
27
as 3′-sialyllactose, 6′-sialyllactose, and brain gangliosides (GD1, GT1, GQ1) also carry lactose as part of their
28
structures.7,8 The sugar unit plays a vital and wide-ranging role such as in immune system, bacterial and viral
29
infections.9,10
30
Chemo-enzymatic approaches are often employed for the synthesis of diverse types of glycoconjugates.11,12 Such
31
methods are more efficient and less expensive than the chemical methods entailing harsh chemical treatment and
32
several protection/deprotection steps before any product can be obtained.13 Glycosyltransferases (GT) catalyze the
33
formation of regio- and stereo-specific glycosidic linkages between specific sugar donors and aglycones.14-16 LgtB is
34
a β-1,4-galactosyltransferase gene from Neisseria meningitidis,
35
galactose to the terminal lacto-N-neotetraose via a β-1,4 linkage.17 LgtB also has been reported to transfer D-
36
galactose
37
galactosyltransferase/-UDP-4'-gal-epimerase fusion protein was prepared to carry out two sequential steps of an
38
important biosynthetic galactosylation pathway for the synthesis of galactosylated oligosaccharides.19 β-1,4-
39
galactosyltransferase has been used to incorporate a D-galactose unit at the terminal N-acetylglucosamine or D-
40
glucose in glycoproteins, glycolipids or other compounds (eg. carbohydrates, epothiolone, and vancomycin) with β-
41
linkage for the biosynthesis of lipo-oligolysaccharides (or their lactosylated derivatives).20-22 Recently our group
42
synthesized quercetin 3-O-β-D-lactoside and it is found to have a better anticancer activity than quercetin 3-O-β-D-
to
the
4′-OH
position
of
D-glucose
and
which transfers D-galactose from UDP-α-D-
N-acetylglucosamine.18
3 ACS Paragon Plus Environment
Previously,
the
β-1,4-
Journal of Agricultural and Food Chemistry
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glucoside.23 As far as to our knowledge, previous work about the production of other polyphenol saccharides
44
carrying lactose moiety has not been reported yet.
45
In this study, we designed a one-pot multi-enzyme cofactor recycling (OPME-CR) system for the continuous
46
regeneration of UDP-α-D-galactose re-using D-glucose-1-phosphate, UDP, and ADP in the reaction system
47
(Scheme 1), resulting in cost-effective synthesis of the derivatives of various polyphenol saccharides carrying a D-
48
galactose moiety. Thus, using the OPME-CR system, we conjugated the D-galactose moiety into various
49
polyphenolic glycosides that harbor sugars such as D-glucose, rutinose, and 2-deoxy-D-glucose in their structure.
50
One of the substrates, quercetin 3-O-β-D-glucoside, was used as a model compound and the reaction conditions
51
were optimized. Newly synthesized quercetin 3-O-β-D-lactoside was characterized by various nuclear magnetic
52
resonance (NMR) studies. The products of other substrates were characterized by ultrahigh performance liquid
53
chromatography-photo diode array (UHPLC-PDA) and high-resolution quadruple time-of-flight electrospray
54
ionization mass spectrometry (HR-QTOF-ESI/MS) analyses.
55
MATERIALS AND METHODS
56
Culture media and chemicals
57
All the compounds used in this study were either purchased from Sigma–Aldrich (St. Louis, MO, USA) or were
58
available in the laboratory library. Standard quercetin 3-O-β-D-glucoside, and DMSO-d6 were purchased from
59
Sigma-Aldrich Inc. (USA). α-D-glucose-1-phosphate, UDP-α-D-galactose, isopropyl-β-D-thiogalactopyranoside
60
(IPTG), adenine triphosphate (ATP), uridine monophosphate (UMP), and acetylphosphate were purchased from
61
GeneChem (Daejeon, South Korea). All the other chemicals and reagents used were purchased from high grade
62
commercial sources. Other substrates like α-mangostein 3-O-β-D-glucoside,24 resveratrol 3-O-β-D-glucoside,25
63
curcumin 4′-O-β-D-glucoside,26 biochanin A 7-O-β-D- glucoside, formononetin 7-O-β-D- glucoside,27 emodin 3-O-
64
β-D-glucoside, and aloe emodin 3-O-β-D-glucoside,28 used in this study were available in the laboratory.
65
Recombinant enzymes expression and purification
66
The expression of proteins, acetate kinase (ACK-pET24ma, GenBank accession: WP_000095711.1), UMP kinase
67
(UMK-pET15b, GenBank accession: WP_001483307.1), α-D-glucose-1-phosphate uridylyltransferase (GalU-
68
pET24ma, GenBank accession: AIF36551.1), galactokinase (GalK, GenBank accession: YP_489030.1) and
69
galactose-1-phosphate uridylyltransferase (GalT, GenBank accession: BAA35420.1) were performed as described
70
previously.29,30 Further, the gene β-1,4-galactosyltransferase (LgtB, GenBank: AAM33872.1) was synthesized from
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GenScript (USA) with restriction sites NdeI and HindIII and cloned into pET24ma. The expression of all the genes
72
was performed in the host Escherichia coli BL21 (DE3) (Stratagene, USA). All the recombinant strains were grown
73
using respective seed cultures supplemented with the antibiotic (50 µg/mL kanamycin or 100 µg/mL ampicillin) in
74
Luria–Bertani (LB) media and incubated at 37°C until the optical density at 600 nm (OD600nm) reached ~0.8.
75
Subsequently, the cells were induced by adding IPTG to a final concentration of 0.5 mM via incubation at 20°C for
76
approximately 20 h. The cells were harvested by centrifugation at 842 x g for 15 min at 4°C and washed twice with
77
a buffer (50 mM Tris-HCl of pH 7.5, 100 mM NaCl and 10% glycerol). The cells were sonicated, and the clear
78
lysate was collected by high-speed centrifugation at 13,475 x g for 30 min at 4°C. The proteins were purified using
79
TALON metal nickel affinity resin (Takara Bio, Shiga, Japan). The resin was equilibrated by washing with a buffer
80
containing 100 mM Tris-HCl (pH 7.5) and 300 mM NaCl before adding a crude lysate of soluble proteins. The
81
mixture of protein and resin was gently agitated in ice for 30 min and eluted with various concentrations (10, 100,
82
200, and 500 mM) of imidazole. The fractions were examined using 12% sodium dodecyl sulfate-polyacrylamide
83
gel electrophoresis (SDS-PAGE). The concentrations of the enzymes were determined using Bradford’s method.
84
Regular galactosylation using LgtB enzyme
85
The regular galactosylation reaction was conducted in a 500 µL volume containing a 50 mM Tris-HCl buffer (pH
86
7.5), 10 mM MgCl2, 2 mM quercetin 3-O-β-D-glucoside, 50 µg/mL purified LgtB enzyme, and 10 mM UDP-α-D-
87
galactose. The mixture was incubated at 37°C for 3 h, followed by quenching the reaction with 400 µL chilled
88
methanol. It was vortexed, mixed well, filtered through a 0.2 µm filter and subjected to UHPLC-PDA and HR-
89
QTOF-ESI/MS analyses.
90
Galactosylation reaction using various concentration of UDP-α-D-galactose and quercetin 3-O-β-D-glucoside
91
The various concentrations of quercetin-3-O-β-D-glucoside (3 mM, 5 mM, and 10 mM) and UDP-α-D-galactose (3
92
mM, 10 mM, and 20 mM) were used for the reaction at 37°C with 50 mM Tris-HCl buffer (pH 7.5) using reaction
93
ingredients similar to the regular galactosylation reaction mixture. The 2 mM quercetin-3-O-β-D-glucoside was
94
maintained constant at different concentrations of the donor, while 10 mM UDP-α-D-galactose was held constant at
95
the different substrate concentrations.
96
Optimization of one-pot multi-enzyme cofactors recycling (OPME-CR) galactosylation reaction
97
To continuously regenerate the UDP-α-D-galactose, a donor substrate for the LgtB galactosylation reaction, an
98
OPME system was engineered using a combination of six recombinant enzymes of different origins. The UDP-α-D-
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galactose regeneration system required continuous use of two molecules of ATP cofactors. Thus, the separate
100
system of ATP regeneration was coupled with the UDP-α-D-galactose regeneration system. The OPME
101
galactosylation reaction mixture included 50 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl2, 70 mM acetylphosphate,
102
20 mM D-galactose, 1 mM α-D-glucose-1-phosphate, 0.25 mM ATP, 0.25 mM UMP, and 2 mM quercetin 3-O-β-D-
103
glucoside along with an additional six purified enzymes: UMK (50 µg/mL), ACK (50 µg/mL), GalU (50 µg/mL),
104
GalK (50 µg/mL), GalT (50 µg/mL) and LgtB (50 µg/mL). The final reaction volume was 500 µL.
105
Identical OPME reactions were carried out to optimize the temperature of the galactosylation reaction in different
106
vials and incubated at different temperatures (20°C, 30°C, 37°C, and 45°C) for 3 h. A 10 µL of the reaction sample
107
was drawn at different time intervals and quenched with 490 µL chilled methanol. The samples were analyzed using
108
UHPLC-PDA.
109
To study the effect of pH on the galactosyltransferase activity of LgtB enzyme, identical reaction mixtures with 3
110
mM quercetin 3-O-β-D-glucoside were prepared in a 50 mM of different buffers (carbonate-bicarbonate buffer,
111
citrate buffer, phosphate buffer, Tris-HCl buffer and glycine buffer) at various pH (pH 4 to pH 10.5) and incubated
112
at 37°C for 3 h. The sample preparation method was similar to the method described above.
113
For a preparative scale reaction, a 20 mL reaction volume containing quercetin 3-O-β-D-glucoside was treated under
114
the optimized conditions of temperature (37°C), pH (8), and divalent metal ions (Mg2+). The starting concentration
115
of quercetin 3-O-β-D-glucoside was 2 mM. After 0.5 h of the reaction, 1 mM quercetin 3-O-β-D-glucoside was
116
added to the reaction mixture. Similarly, additional quercetin 3-O-β-D-glucoside was added during 1.5 h and 2.5 h
117
of the reaction. The pH of the reaction mixture was maintained throughout the reaction with 0.1 M NaOH. Other
118
reaction ingredients used were 70 mM acetylphosphate, 20 mM D-galactose, 10 mM Mg2+, 1 mM α-D-glucose-1-
119
phosphate, 0.25 mM ATP, 0.25 mM UMP, and six purified enzymes as mentioned above. The progress of the
120
reaction was monitored by UHPLC-PDA for up to 12 h. The maximum number regeneration cycles (RCmax) of
121
UDP-α-D-galactose was calculated as the total amount of quercetin 3-O-β-D-lactoside (mM) produced per 0.25 mM
122
of UMP.
123
OPME-CR galactosylation reaction with other polyphenolic glycosides
124
After optimizing the conditions, the OPME reaction mixture (500 µL) for the attachment of a D-galactose unit to
125
various compounds was prepared by using 50 mM glycine buffer (pH 8), 10 mM MgCl2, 70 mM acetylphosphate,
126
20 mM D-galactose, 1 mM α-D-glucose-1-phosphate, 0.25 mM ATP, 0.25 mM UMP, and 2 mM substrate
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(kaempferol 3-O-β-D-glucoside, biochanin A 7-O-β-D-glucoside, formononetin 7-O-β-D-glucoside, resveratrol 3-O-
128
β-D-glucoside, curcumin 4′-O-β-D-glucoside, emodin 3-O-β-D-glucoside, aloe emodin 3-O-β-D-glucoside, rutin,
129
diosmin, and α-mangostein 3-O-β-2-deoxy-D-glucoside), and the purified enzymes (UMK, ACK, GalU, GalK, GalT,
130
and LgtB) at similar concentrations as described above.
131
The reactions were incubated at 37°C for 2 h, and reaction samples were extracted at different time intervals and
132
quenched by adding a double volume of chilled methanol and vortexed. After centrifugation at 13,475 x g for 5 min
133
and filtering with a 0.2 µm filter, the supernatants of the reaction mixture were analyzed by UHPLC-PDA and LC-
134
QTOF-ESI/MS in positive ion mode. Appropriate dilutions were made as needed for the UHPLC-PDA analysis.
135
Analytical methods
136
The samples were analyzed using a Thermo Scientific Dionex Ultimate 3000 UHPLC-PDA system consisting of
137
High Pressure Gradient Rapid Separation HPG-3200RS series pumps, a Thermo Scientific™ Dionex™ UltiMate™
138
ACC-3000 autosampler, and a UV-Vis absorbance diode array detector, operated using Thermo Scientific™
139
Dionex™ Chromeleon™ 7.2 Chromatography Data System (CDS) software. The reverse-phase UHPLC-PDA
140
analysis was performed using a C18 column (Mightysil RP-18 GP (4.6 mm × 250 mm, 5-µm particle size) (Kanto
141
Chemical, Japan) with HPLC-grade water containing 0.05% trifluoroacetic acid (TFA) (A) and acetonitrile (B) from
142
Mallinckrodt Baker (Phillipsburg, NJ, USA) at a flow rate of 1 mL/min for 25 min. The elution protocol was as
143
follows: starting with 10% B, a linear gradient from 10 to 30% B for 0−5 min, 30−50% B for 5−10 min, 50−90% B
144
for 10−15 min, 90−70% B for 15−18 min, 70−10% B for 18−25 min was performed, followed by washing and
145
equilibration of the column. Absorbance of the eluent was monitored from 200 nm to 600 nm. The maximum UV
146
absorbance of each compound was used to monitor the chromatograms. Calibration curves of the standard quercetin
147
3-O-β-D-glucoside and the purified quercetin 3-O-β-D-lactoside were prepared for the quantification of quercetin 3-
148
O-β-D-lactoside, whereas conversion percentage of other polyphenols was determined using integrated peak area of
149
substrate and product in HPLC chromatogram.
150
The HR-QTOF ESI/MS analysis was performed using an ACQUITY (UPLC, Waters Corp., Billerica, MA, USA)
151
column coupled with a SYNAPT G2-S (Water Corp.) and the separation was performed with water (A) and
152
acetonitrile (B) as mobile phases. The column temperature was set to 35°C, the injection volume was 5 µL, and the
153
flow rate was 0.3 mL/min. The total analysis time per sample was 12 min. The gradient program was as follows: 0–
154
7 min, 0–100% B; 7–9.5 min, 100% B; 9.5–12 min, 0% B. The conditions for HR-QTOF ESI/MS measurement
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155
were
156
600 L/h; capillary voltage, 3 kV; cone voltage, 40 V; source temperature, 120°C. Collision energy of the MS was set
157
4eV and the mass spectra were recorded from m/z 50 to 1500. Data acquisition was managed by the MassLynx
158
software.
159
The purification of quercetin 3-O-β-D-lactoside was performed using preparative HPLC equipped with a C18 column
160
(YMC-Pack ODS-AQ) (250×20 mm I.D., 10 µm) connected to a UV detector (SPD-20A) with a 40 min binary
161
program. The percentage of acetonitrile used was as follows: 10−40% (0−15 min), 40−90% (15−30 min), and 90−10%
162
(30−40 min) at a flow rate of 10 mL/min. The solvent of collected sample was evaporated. The product was
163
lyophilized to remove water molecules and dissolved in DMSO-d6 for nuclear magnetic resonance (NMR) analysis
164
using a Bruker-BioSpin Avance 700 MHz NMR Spectrometer (Germany) using a Cryogenic TCi probe (5 mm) for
165
elucidation of the structure. One-dimensional NMR (1H-NMR,
166
(heteronuclear single quantum correlation (HSQC), and heteronuclear multiple-bond correlation (HMBC)) were
167
performed to elucidate the structure of the quercetin 3-O-β-D-lactoside.
168
RESULTS
169
Expression and preparation of enzymes
170
The expressions of individual recombinant proteins were examined using SDS-PAGE. The protein marker was used
171
to verify the size of the proteins by 12% SDS-PAGE analysis which was in good agreement with those of the
172
calculated values obtained for each protein such as LgtB (~32 kDa), UMK (~26 kDa), ACK (~40 kDa), GalU (~38
173
kDa), GalK (~35 kDa), and GalT (~35 kDa) (Fig. S1). The soluble fractions of the enzymes were purified and used
174
for the reactions.
175
Galactosylation of quercetin 3-O-β-D-glucoside
176
Galactosylation by LgtB enzyme was verified using the reaction mixture containing UDP-α-D-galactose as the sugar
177
donor and quercetin 3-O-β-D-glucoside as the acceptor substrate as described in the Methods. UHPLC-PDA of this
178
reaction mixture yielded a product peak at a retention time (tR) of ~ 7.8 min (Fig. 1A). The UV spectra of the
179
substrate and product were similar (Fig. 1B). The product peak was further analyzed by LC-QTOF-ESI/MS. In the
180
positive-ion mode, a mass fragment of m/z+ 627.1569 was found, which matched with [quercetin 3-O-β-D-lactoside
181
+ H]+, the exact calculated mass of which was 627.1561 Da (Fig. 1C). In addition, other product ions of the
182
synthesized compound were analyzed to confirm the presence of sugar units (Fig. 1D). A distinct fragment ion of
as
follows:
ionization
mode,
positive; desolvation temperature,
13
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300°C;
desolvation
gas
flow,
C-NMR) and two-dimensional NMR
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183
the disaccharide sugar unit was found (m/z+ 325.1132) along with other mass spectra characterizing fragments of
184
quercetin 3-O-β-D-glucoside and quercetin aglycone.
185
Structural analysis
186
The compound dissolved in NMR grade DMSO-d6 was analyzed using 1H, 13C, HSQC, and HMBC-NMR analyses.
187
The 1H-NMR spectrum of the product showed two anomeric protons with doublets at δ 5.49 (J=7.769 Hz) and δ
188
4.21 (J=7.2 Hz), representing the beta (β) configuration of the two sugar moieties, whereas other proton signals for
189
sugar moieties (D-glucose and D-galactose) were observed in the region from δ (3.0–4.0) ppm (Table 1; Fig. S2).
190
The anomeric carbons of D-glucose and D-galactose were obtained at δ 101.22 ppm and δ 104.29 ppm, respectively
191
(Table 2; Fig. S3), and their correlations were measured in HSQC for confirmation of the position of sugar
192
conjugation (Fig. S4). The correlation between 1″-H at δ 5.49 ppm and the 3rd position carbon of quercetin (C-3) at δ
193
133.56 ppm confirmed the conjugation of D-glucose moiety at the 3-OH position of quercetin Fig. S5. The
194
correlation between 1′′′-H of δ 4.21 ppm and 4″-C at δ 80.94 (4th carbon of glucose moiety) confirmed the
195
conjugation of D-galactose via (1-4) β-linkage obtained from the HMBC (Figs. S4 and S5). The 1H and 13C-NMR
196
data of quercetin 3-O-β-D-glucoside, and quercetin 3-O-β-D-lactoside are compared in Tables 1 and 2.14,31 The
197
spectra obtained here for lactose moiety are consistent with the previously published results. 20,22
198
Reaction using various concentration of UDP-α-D-galactose and quercetin 3-O-β-D-glucoside
199
The conversion percentage of quercetin 3-O-β-D-glucoside to quercetin 3-O-β-D-lactoside was observed at varying
200
concentrations of UDP-α-D-galactose (3 mM, 10 mM, and 20 mM) while keeping the concentration of quercetin 3-
201
O-β-D-glucoside constant (Fig. 2). Similarly, quercetin 3-O-β-D-glucoside concentration was varied from 3 mM to
202
10 mM while keeping the concentration of UDP-α-D-galactose constant in an another set of reactions. When 3 mM
203
of the donor UDP-α-D-galactose was used, only 42% of the 2 mM quercetin 3-O-β-D-glucoside was converted to
204
the product (0.84 mM) in 2 h. After increasing the UDP-α-D-galactose concentration to 10 mM, almost all the
205
substrate was converted into the product (~1.99 mM). When 20 mM UDP-α-D-galactose was used, all the substrate
206
was converted to the product within an hour of the reaction. Similarly, the maximum amount of quercetin 3-O-β-D-
207
glucoside that was galactosylated by LgtB was 3 mM in the presence of 10 mM UDP-α-D-galactose as a donor. At 5
208
mM concentration of quercetin 3-O-β-D-glucoside, the conversion was 39% (quercetin 3-O-β-D-lactoside, 1.95 mM)
209
in the first hour of reaction, which increased slightly to 42% (2.1 mM) in 2 h. Similarly, the conversion was also
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limited to 30% (quercetin 3-O-β-D-lactoside, 3 mM) when10 mM quercetin 3-O-β-D-glucoside was used as initial
211
substrate concentration (Fig. 2).
212
Development of one-pot multi-enzyme cofactors recycling (OPME-CR) system
213
A one-pot reaction system was engineered for the continuous production of the activated nucleotide sugar donor,
214
UDP-α-D-galactose, in a single vessel while regenerating ATP cofactor using simple and inexpensive starting
215
materials such as UMP, acetylphosphate, and D-galactose (Scheme 1). The use of NDP-sugars for in vitro
216
production of a large quantity of NP glycosides at an industrial scale is limited because of their high cost and lack of
217
commercial availability. In the proposed system, the final ATP and α-D-glucose 1-phosphate concentrations
218
required are very low (1 mM each) as both are regenerated economically. The reaction system converts UMP to
219
UDP (Scheme 1) at the expense of a molecule of ATP catalyzed by a UMP kinase enzyme (UMK). Similarly, D-
220
galactose is converted to α-D-galactose-1-phosphate by a galactokinase enzyme (GalK). The byproduct ADP is
221
utilized by ACK and converted to ATP by consuming a molecule of acetylphosphate. Thus, the galactose 1-
222
phosphate generated along with the UDP-α-D-glucose in the reaction by GalU is utilized by GalT for the synthesis
223
of UDP-α-D-galactose. The LgtB enzyme uses UDP-α-D-galactose produced in the pathway to attach D-galactose to
224
the various substrates containing D-glucose, 2-deoxy-D-glucose, or rutinose. The byproduct UDP, which is
225
produced in the galactosylation reaction, is re-utilized to yield UTP at the expense of one molecule of
226
acetylphosphate catalyzed by ACK in the system. Overall, UDP is recycled whereas D-galactose and
227
acetylphosphate are consumed in this OPME-CR system to generate a continuous supply of UDP-α-D-galactose
228
required for LgtB activity as shown in Scheme 1.
229
Optimization of the OPME-CR system
230
The effect of pH, divalent cofactors, incubation temperature, and incubation time on the reaction process was
231
observed in the conversion of quercetin 3-O-β-D-glucoside to quercetin 3-O-β-D-lactoside. Initially, the OPME-CR
232
reactions were carried out at four different temperatures: 20°C, 30°C, 37°C, and 45°C. At both 20°C and 30°C, the
233
concentration of the product increased with the incubation time to its maximum amount in 3h. The highest
234
conversion of quercetin 3-O-β-D-glucoside to quercetin 3-O-β-D-lactoside was observed at 37°C within 2 h. Almost
235
all (99%) of the 2 mM quercetin 3-O-β-D-glucoside added to the reaction mixture was converted to a product (1.99
236
mM) at this temperature (Fig. 3A). At a higher temperature of 45°C, 81% conversion (1.62 mM) was achieved in 30
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min. The prolonged incubation of the reaction mixture at this temperature resulted in a slight reduction in the
238
amount of product to 79% (1.58 mM) within 3 h.
239
Similarly, the divalent metal ion co-factor requirement for the LgtB enzyme was determined with various divalent
240
cations. Among the nine different divalent cations tested at a 10 mM concentration, Cu2+, Fe2+, and Zn2+ rendered
241
LgtB virtually inactive. As shown in fig. 3B, the enzyme also showed limited activity with Co2+ and Ni2+. LgtB
242
showed a conversion of 48% of quercetin 3-O-β-D-glucoside with Pb2+, which was higher than the level attained
243
with Ca2+. With Ca2+, 35% of quercetin 3-O-β-D-glucoside was converted to quercetin 3-O-β-D-lactoside. The
244
enzyme activity with Pb2+ was almost half compared with that of Mg2+ or Mn2+. Mg2+ and Mn2+ played equal roles
245
in activating the conversion of nearly all of the substrate into product (Fig. 3B).
246
A pH profile of the OPME-CR galactosylation reactions of quercetin 3-O-β-D-glucoside measured in carbonate-
247
bicarbonate, citrate, phosphate, Tris-HCl and glycine buffers within their appropriate pH ranges revealed that its
248
catalytic activity was optimum at pH 7.5 (Tris-HCl buffer and phosphate buffer) to pH 8 (glycine buffer) (Fig. 3C).
249
The 15% conversion (0.45 mM) observed at the lowest pH tested (pH 4) in citrate buffer increased to 96% (2.88
250
mM) at pH 8 in glycine buffer. Similar conversion was recorded in phosphate buffer (pH 7.5). A significant decrease
251
to less than 60% molar conversion of quercetin 3-O-β-D-glucoside was observed when the pH of the reaction
252
mixture was slightly basic (pH 9.0). Further lowering the pH to 10.5 produced only 30% of product (0.9 mM).
253
Preparative scale production of quercetin 3-O-β-D-lactoside using OPME-CR system
254
To obtain a maximum yield of galactosylated product from the engineered OPME-CR system, the reaction was
255
performed on a preparative scale of 20 mL reaction volume starting with 2 mM quercetin 3-O-β-D-glucoside,
256
followed by the addition of 1 mM quercetin 3-O-β-D-glucoside three times up to 2.5 h of reaction. The progress of
257
the reaction was observed for up to 12 h. 2 mM of quercetin 3-O-β-D-glucoside completely converted into product
258
in 0.5 h. In 1 h, 2.5 mM of quercetin 3-O-β-D-lactoside was produced. The titer of quercetin 3-O-β-D-lactoside
259
increased to 3.9 mM in 3 h. Similarly, the product yield calculated at different time points indicated that the
260
maximum concentration of quercetin 3-O-β-D-lactoside produced was 4.56 mM (2854.6 mg/L) in 4 h (Fig. 4). The
261
result highlights the capacity of one-pot reaction for the high-yield production of the galactosylated product.
262
Biosynthesis of polyphenol lactosides and non-natural saccharides using OPME-CR system
263
Three different sugars (D-glucose, rutinose, or 2-deoxy-D-glucose) conjugated plant secondary metabolites such as
264
flavonol, flavone, isoflavonoid, stilbene, curcuminoid, anthraquinone, and xanthonoid glycosides were used in the
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OPME-CR galactosylation reaction (Fig. 5A). Among the 11 compounds used in the reaction, eight contained D-
266
glucose, two carried rutinose, and one harbored 2-deoxy-D-glucose in their structure. The UHPLC-PDA analysis of
267
the reaction mixtures displayed different turnover rates for each substrate. Analysis of the reaction mixtures using
268
UHPLC-PDA method along with HRQTOF–ESI/MS revealed the conjugation of D-galactose residue in all the
269
compounds tested (Figs. S6-S15). The conversion percentage of seven of the compounds tested reached greater than
270
90% after 120 min (Fig. 5B). The OPME-CR system yielded 1239 mg/L of quercetin 3-O-β-D-lactoside, 1198 mg/L
271
of kaempferol 3-O-β-D-lactoside, 1129 mg/L of aloe emodin 3-O-β-D-lactoside, 1188 mg/L of emodin 3-O-β-D-
272
lactoside, 1544 mg/L of rutin 4′′-O-β-D-galactoside, 1357 mg/L of curcumin 4′-O-β-D-lactoside, and 1422 mg/L of
273
mangostein 3-O-β-D-galactosyl (1-4) β-D-2-deoxyglucoside. The production of other compounds was significantly
274
low (Table S1): 85 mg/L of biochanin A 7-O-β-D-lactoside, 201 mg/L formononetin 7-O-β-D-lactoside, 99 mg/L of
275
resveratrol 3-O-β-D-lactoside, and 123 mg/L of diosmin 4′′-O-β-D-galactoside.
276
DISCUSSION
277
Despite the diverse physiological and pharmacological activities of natural polyphenols,32,33 their use as drugs or
278
food additives has been limited by their low water solubility and absorption. Glycosylation enhances the
279
bioavailability and pharmacological properties of compounds by increasing their solubility and stability.34,35 The
280
sugar moieties of the glycosides often participate in the recognition of their specific biological targets and enhance
281
their efficacy in drug development.1,36 Furthermore, natural product glycosides derived from plants are considered
282
pro-drugs with enhanced absorption and metabolism in the human body.37 Studies have been carried out for the
283
synthesis of D-galactose-conjugated oligosaccharides and flavonoids considering the importance of D-galactose
284
moieties by enzymatic as well as chemo-enzymatic methods.38,39 D-galactose is one of the major building blocks of
285
human milk oligosaccharides (HMOs). Recently, a galactose–aspirin covalent complex pro-drug was synthesized
286
chemically, and galactose-conjugated aspirin was found to enhance the inhibitory activity against the proliferation of
287
cancer cells.40
288
Previously, the OPME reaction was designed for the production of UDP-α-D-glucose and TDP-α-D-2-deoxy-D-
289
glucose.24-26 In this study, we designed an OPME-CR system for the production of an important nucleotide sugar,
290
UDP-α-D-galactose. This system can be used to attach UDP-α-D-glucose or UDP-α-D-galactose to various
291
substrates with a suitable GT enzyme. The GT used in this study, LgtB, is selective for the sole attachment of D-
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galactose from UDP-α-D-galactose.19 Hence, we exploited its specificity for extension of various sugar-containing
293
substrates with D-galactose moiety.
294
The OPME-CR system regenerates ATP and UDP-α-D-galactose, the latter of which is coupled with LgtB-mediated
295
galactosylation of various secondary metabolite glycosides. In this system, two components, D-galactose and
296
acetylphosphate, are continuously consumed throughout the reaction process, while acetate and Pi are the
297
byproducts. The concentration of UMP and ATP used initially is also very low. Both of these substrates, which are
298
consumed in the reaction, are inexpensive and easily available, resulting in very cost-effective and sustainable
299
outcome. However, the disadvantage of the system is the simultaneous decrease in pH of the reaction because of the
300
continuous production of acetate.24 Thus, the pH should be monitored frequently in a large-scale reaction.
301
The optimal pH of the enzyme was 7.5 to 8 which is within the pH range of majority of glycosyltransferase.14, 17-20
302
Although Tris-HCl buffer has been known to retain the potential to interact with the molecules under the study, it is
303
being used frequently in several biological studies.41 In our study of buffer effect, glycine buffer and phosphate
304
buffer resulted in more than 90% conversion of quercetin 3-O-β-D-glucoside to quercetin 3-O-β-D-lactoside
305
whereas the reaction in Tris-HCl buffer resulted in reduction in product formation. Tris-HCl buffer has also been
306
found to have an inhibitory effect on the activity of glycosyltransferase from Citrus paradisi.42,43
307
The optimized OPME system for the conjugation of D-galactose residue was used to successfully convert eight
308
compounds containing D-glucose, two carrying rutinose disaccharide, and one with 2-deoxy-D-glucose. Some of the
309
substrates were completely converted to products while conversion of other substrates was significantly low. The
310
ability of the enzyme LgtB to contribute D-galactose residue to compounds containing glucoside, rutinoside, or 2-
311
deoxy-D-glucoside may be used to conjugate various oligosaccharide residues and synthesize novel glycoside
312
derivatives. In this experiment, LgtB conjugated D-galactose to various compounds within a short reaction time of 2
313
h. We successfully synthesized kaempferol 3-O-β-D-lactoside, quercetin 3-O-β-D-lactoside, biochanin A 7-O-β-D-
314
lactoside, formononetin 7-O-β-D-lactoside, resveratrol 3-O-β-D-lactoside, curcumin 4′-O-β-D-lactoside, emodin 3-
315
O-β-D-lactoside, aloe emodin 3-O-β-D-lactoside, rutin 4′′-O-β-D galactoside, diosmin 4′′-O-β-D galactoside, and α-
316
mangostein 3-O-β-D-galactosyl (1-4) β-D-2-deoxy-D-glucoside in varying quantities (Table S1). To the best of our
317
knowledge, none of these compounds has been reported from any natural source nor have they been synthesized.
318
The biological activities of these compounds may find application in a number of medicinal, cosmetic, and
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pharmacologically active products. The potential health benefits of these compounds facilitate the development of
320
therapeutic interventions in the future safely, and cost-effectively.
321
Based on the use of engineered system, the final titer of quercetin 3-O-β-D-lactoside was 2854.6 mg/L, where the
322
maximum number of UDP-α-D-galactose regeneration cycles (RCmax) was 18.24 (4.56/0.25) by fed-batch reaction.
323
The LgtB activity was improved by the presence of large amounts of UDP-α-D-galactose. Thus, the one-pot system
324
that produces donor continuously plays a useful role. In the near future, the system will be further expanded for
325
sustainable and cost-effective production of various non-natural NDP-sugars coupled with glycosylation reactions to
326
generate a diverse array of natural product glycosides.
327
ACKNOWLEDGMENT
328
This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant#: PJ013137),
329
Rural Development Administration, Republic of Korea.
330 331
Supporting Information Available
332
Expression of recombinant proteins is shown in Figure S1; Figures (S2-S5) show the NMR of quercetin 3-O-β-D-
333
lactoside; Figures (S6-S15) show the UHPLC-PDA chromatograms of the galactosylation reaction of various
334
substrates with LgtB enzyme.
335 336
Conflict of interest
337
The authors declare that they have no competing interests.
338
Ethical approval
339
This article does not contain any studies with human participants or animals performed by any of the authors.
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REFERENCES 1.
342 343
bacterial natural products. Chem Soc Rev. 2015, 44, 7591–7697. 2.
344 345
3.
4.
Simmonds, M. S. Flavonoid–insect interactions: recent advances in our knowledge. Phytochemistry. 2003, 64, 21–30.
5.
350 351
Griffith, B. R.; Langenhan, J. M.; Thorson. J. S. ‘Sweetening’natural products via glycorandomization. Curr Opin Biotechnol. 2005, 16, 622–630.
348 349
Weymouth-Wilson, A. C. The role of carbohydrates in biologically active natural products. Nat Prod Rep. 1997, 14, 99–110.
346 347
Elshahawi, S. I.; Shaaban, K. A; Kharel, M. K.; Thorson, J. S. A comprehensive review of glycosylated
Cheynier, V.; Comte, G.; Davies, K. M.; Lattanzio, V.; Martens, S. Plant phenolics: recent advances on their biosynthesis, genetics, and ecophysiology. Plant Physiol Biochem. 2013, 72, 1–20.
6.
Zhang, T.; Sun, L.; Liu, R.; Zhang, D.; Lan, X.; Huang, C.; Xin, W.; Wang, C.; Zhang, D.; Du, G. A novel
352
naturally occurring salicylic acid analogue acts as an anti-inflammatory agent by inhibiting nuclear factor-
353
kappa B activity in RAW264. 7 macrophages. Mol Pharm. 2012, 9, 671–677.
354
7.
Pandey, R. P.; Kim, D. H.; Woo, J.; Song, J.; Jang, S. H.; Kim, J. B.; Cheong, K. M.; Oh, J. S.; Sohng, J.
355
K. Broad-spectrum neutralization of avian influenza viruses by sialylated human milk oligosaccharides: in
356
vivo assessment of 3′-sialyllactose against H9N2 in chickens. Sci Rep. 2018, 8, 2563.
357
8.
358
Yu, R. K.; Tsai, Y. T.; Ariga, T.; Yanagisawa, M. Structures, biosynthesis, and functions of gangliosides—An overview. J Oleo Sci. 2011, 60, 537–544.
359
9.
360
10. Schnaar, R. L. Gangliosides of the vertebrate nervous system. J Mol Biol. 2016,428, 3325–3336.
361
11. Pandey, R. P.; Parajuli, P.; Koffas, M. A.; Sohng, J. K. Microbial production of natural and non-natural
Kolter, T. Ganglioside biochemistry. ISRN Biochem. 2012, 506160.
362
flavonoids: Pathway engineering, directed evolution and systems/synthetic biology. Biotechnol Adv. 2016, 34,
363
634–662.
364
12. Xiao, Z.; Guo, Y.; Liu, Y.; Li, L.; Zhang, Q.; Wen, L.; Wang, X.; Kondengaden, S. M.; Wu, Z.; Zhou, J.; Cao,
365
X. Chemoenzymatic synthesis of a library of human milk oligosaccharides. J Org Chem. 2016, 81, 5851–5865.
366
13. Muthana, S.; Cao, H.; Chen, X. Recent progress in chemical and chemoenzymatic synthesis of
367
carbohydrates. Curr Opin Chem Biol. 2009, 13, 573–581.
15 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 16 of 29
368
14. Lim, E. K.; Ashford, D. A.; Hou, B.; Jackson, R. G.; Bowles, D. J. Arabidopsis glycosyltransferases as
369
biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides. Biotechnol Bioeng.
370
2004, 87, 623-631.
371 372 373 374 375
15. Coutinho, P. M.; Deleury, E.; Davies, G. J.; Henrissat, B. An evolving hierarchical family classification for glycosyltransferases. J Mol Biol. 2003, 328, 307–317. 16. Liang, D. M; Liu, J. H.; Wu, H.; Wang, B. B.; Zhu, H. J.; Qiao, J. J. Glycosyltransferases: mechanisms and applications in natural product development. Chem Soc Rev. 2015, 44, 8350–8374. 17. Wakarchuk, W. W.; Cunningham, A.; Watson, D. C.; Young, N. M. Role of paired basic residues in the
376
expression
377
meningitidis. Protein Eng. 1998, 11, 295–302.
378 379
of
active
recombinant
galactosyltransferases
from
the
bacterial
pathogen
Neisseria
18. Park, J. E.; Lee, K. Y.; Do, S. I.; Lee, S. S. Expression and characterization of β-1, 4-galactosyltransferase from Neisseria meningitidis and Neisseria gonorrhoeae. J Biochem Mol Biol. 2002, 35, 330–336.
380
19. Blixt, O.; Brown, J.; Schur, M. J; Wakarchuk, W.; Paulson, J. C.; Efficient preparation of natural and synthetic
381
galactosides with a recombinant β-1, 4-galactosyltransferase-/UDP-4′-gal epimerase fusion protein. J Org
382
Chem. 2001, 66, 2442–2448.
383 384
20. Oh, T.; Kim, D. H.; Kang, S. Y.; Yamaguchi, T.; Sohng, J. K. Enzymatic synthesis of vancomycin derivatives using galactosyltransferase and sialyltransferase. J Antibiot. 2011, 64, 103–109.
385
21. Yu, H.; Li, Y.; Zeng, J.; Thon, V; Nguyen, D. M.; Ly, T.; Kuang, H. Y.; Ngo, A.; Chen, X. Sequential one-pot
386
multienzyme chemoenzymatic synthesis of glycosphingolipid glycans. J Org Chem. 2016, 8, 10809–10824.
387
22. Parajuli, P.; Pandey, R. P.; Gurung, R. B.; Shin, J. Y.; Jung, H. J.; Kim, D. H.; Sohng, J. K. Enzymatic
388 389 390
synthesis of lactosylated and sialylated derivatives of epothilone A. Glycoconj J. 2016, 33, 137–146. 23. Darsandhari, S.; Bae, J. Y.; Shrestha, B.; Yamaguchi, T.; Jung, H. J.; Han, J. M.; Rha, C. S.; Pandey, R. P.; Sohng, J. K. Enzymatic synthesis of novel quercetin sialyllactoside derivatives. Nat Prod Res. 2018, 1-9.
391
24. Le, T. T.; Pandey, R. P.; Gurung, R. B.; Dhakal, D.; Sohng, J. K. Efficient enzymatic systems for synthesis of
392
novel α-mangostin glycosides exhibiting antibacterial activity against Gram-positive bacteria. Appl Microbiol
393
Biotechnol. 2014, 98, 8527–8538.
394 395
25. Shin, J. Y.; Pandey, R. P.; Jung, H. Y.; Chu, L. L.; Park, Y. I.; Sohng, J. K. In vitro single-vessel enzymatic synthesis of novel Resvera-A glucosides. Carbohydr Res. 2016, 424, 8–14.
16 ACS Paragon Plus Environment
Page 17 of 29
Journal of Agricultural and Food Chemistry
396
26. Gurung, R. B.; Gong, S. Y.; Dhakal, D.; Le, T. T.; Jung, N. R.; Jung, H. J.; Oh, T. J.; Sohng, J. K. Synthesis of
397
curcumin glycosides with enhanced anti-cancer properties using one-pot multi-enzyme glycosylation
398
technique. J Microbiol Biotechnol. 2017, 27, 1639-48.
399 400 401 402 403 404 405 406 407 408
27. Pandey, R. P.; Parajuli, P.; Koirala, N.; Lee, J. H.; Park, Y. I.; Sohng, J. K. Glucosylation of isoflavonoids in engineered Escherichia coli. Mol Cells. 2014. 37, 172. 28. Ghimire, G. P.; Koirala, N.; Pandey, R. P.; Jung, H. J; Sohng, J. K. Modification of emodin and aloe-emodin by glycosylation in engineered Escherihia coli. World J Microbiol Biotechnol. 2015. 31, 611-619. 29. Lee, J. H.; Chung, S. W.; Lee, H. J.; Jang, K. S.; Lee, S. G.; Kim, B. G. Optimization of the enzymatic one pot reaction for the synthesis of uridine 5′-diphosphogalactose. Bioprocess Biosyst Eng. 2010, 33, 71-78. 30. Lee, H. C.; Lee, S. D.; Sohng, J. K.; Liou, K. One-pot enzymatic synthesis of UDP-D-glucose from UMP and glucose-1-phosphate using an ATP regeneration system. J Biochem Mol Biol. 2004, 37, 503–506. 31. Parajuli, P.; Pandey, R. P; Sohng, J. K. Regiospecific biosynthesis of tamarixetin derivatives in Escherichia coli. J Biol Eng. 2018, 133, 113-121.
409
32. Daglia, M. Polyphenols as antimicrobial agents. Curr Opin Biotechnol. 2012, 23, 174–181.
410
33. Quideau, S.; Deffieux, D.; Douat‐Casassus, C.; Pouysegu, L. Plant Polyphenols: Chemical Properties,
411 412 413 414 415 416 417 418 419
Biological Activities, and Synthesis. Angew Chem Int Ed Engl. 2011, 50, 586–621. 34. Shimoda, K.; Otsuka, T.; Morimoto, Y.; Hamada, H.; Hamada, H. Glycosylation and malonylation of quercetin, epicatechin, and catechin by cultured plant cells. Chem Lett. 2007, 36, 1292–1293. 35. Pandey, R. P.; Sohng, J. K. Glycosyltransferase-mediated exchange of rare microbial sugars with natural products. Front Microbiol. 2016, 7, 1849. 36. Křen, V.; Řezanka, T. Sweet antibiotics–the role of glycosidic residues in antibiotic and antitumor activity and their randomization. FEMS Microbiol Rev. 2008, 32, 858–889. 37. Crespy, V.; Morand, C.; Besson, C.; Manach, C.; Démigné, C.; Rémésy, C. Comparison of the intestinal absorption of quercetin, phloretin and their glucosides in rats. J Nutr. 2001, 131, 2109–2114.
420
38. Schocker, N. S.; Portillo, S.; Brito, C. R.; Marques, A. F.; Almeida, I. C.; Michael, K. Synthesis of Galα (1, 3)
421
Galβ (1, 4) GlcNAcα-, Galβ (1, 4) GlcNAcα-and GlcNAc-containing neoglycoproteins and their
422
immunological evaluation in the context of Chagas disease. Glycobiology. 2015, 26, 39–50.
17 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
423 424 425 426 427 428 429 430
39. Pei, J.; Chen, A.; Zhao, L.; Cao, F.; Ding, G.; Xiao, W. One-pot synthesis of hyperoside by a three-enzyme cascade using a UDP-galactose regeneration system. J Agric Food Chem. 2017, 65, 6042-6048. 40. Huang, G. Synthesis and biological activities of galactose–aspirin conjugate prodrug designed for ADEPT and PMT. Med Chem Res. 2017 (https://doi.org/10.1007/s00044-017-2094-4) 41. Chen, C. C.; Guo, W. J. Isselbacher, K. J. Rat intestinal trehalase. Studies of the active site. Biochem. J. 1987, 247, 715-724. 42. Owens, D. K.; McIntosh, C. A. Identification, recombinant expression, and biochemical characterization of a flavonol 3-O-glucosyltransferase clone from Citrus paradisi. Phytochemistry. 2009, 70, 1382-1391.
431
43. Devaiah, S. P.; Owens, D. K.; Sibhatu, M. B.; Sarkar, T. R.; Strong, C. L.; Mallampalli, V. K.; Asiago, J.;
432
Cooke, J.; Kiser, S.; Lin, Z.; Wamucho, A. Identification, recombinant expression, and biochemical analysis of
433
putative secondary product glucosyltransferases from Citrus paradisi. J Agric Food Chem. 2016, 64, 1957-
434
1969.
435
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Figure Legends
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Scheme 1. Schematic diagram for one-pot multi-enzymes cofactors recycling (OPME-CR) galactosylation reaction
438
of diverse polyphenolic glycosides. Black box shows substrates transformed to galactose conjugated derivatives by
439
LgtB enzyme. Purple box shows cofactors recycling system. In cyan box-substrates and green box-products are
440
shown.
441
Figure 1. UHPLC-PDA and HR-QTOF ESI/MS spectra of one-pot multi-enzymes reactions of quercetin 3-O-β-D-
442
glucoside. A) UHPLC-PDA chromatograms of in vitro one-pot multienzymes reactions of quercetin 3-O-β-D-
443
glucoside to produce quercetin 3-O-β-D-lactoside at different time points: (i) 0 min, (ii) 30 min, and (iii) 60 min. B)
444
UV spectra of (i) quercetin 3-O-β-D-glucoside and (ii) the product. C) HR-QTOF ESI/MS analysis showing
445
production of quercetin 3-O-β-D-lactoside in positive ionization mode along with its mass fragmentation. D) The
446
possible structures of major fragments observed in positive ion mode.
447
Figure 2. Regular galactosylation reaction at different concentration of UDP-α-D-galactose and quercetin 3-O-β-D-
448
glucoside. 2mM of quercetin 3-O-β-D-glucoside was used with the increasing concentration of UDP-α-D-galactose.
449
Likewise 10mM of UDP-α-D-galactose was used for increasing concentration of quercetin 3-O-β-D-glucoside.
450
Figure 3. Optimized reaction conditions for OPME reaction for conversion of quercetin 3-O-β-D-glucoside to
451
quercetin 3-O-β-D-lactoside. A) Temperature, B) divalent metal ions, and C) pH.
452
Figure 4. Preparative scale OPME-CR galactosylation reaction with quercetin 3-O-β-D-glucoside for synthesis of
453
quercetin 3-O-β-D-lactoside. One millimolar quercetin 3-O-β-D-glucoside was added to the reaction mixture at 0.5,
454
1.5, and 2.5 h.
455
Figure 5. A) Structures of different polyphenolic glycosides modified in this study. B) Conversion percentage of
456
each substrate to respective D-galactose conjugated derivative at different time points.
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Tables
Table 1. 1H-NMR of quercetin 3-O-β-D-glucoside, 14 and quercetin 3-O-β-D-lactoside
Position
Quercetin 3-O-β-D-glucoside 14
6 8 2′ 5′ 6′ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′
6.20 (d, J=2.0 Hz, 1H) 6.39 ( d, J=2.0 Hz, 1H ) 7.70 ( d, J=2.0 Hz, 1H ) 6.86 ( d, J=8.5 Hz, 1H ) 7.58 ( dd, J=2.0, 8.5Hz, 1H ) 5.24 ( d, J= 8.0 Hz, 1H ) 3.40-3.70 (4H, m)
1′′′ 2′′′ 3′′′ 4′′′ 5′′′ 6′′′
3.92 (1H, dd, J=12.0, 2.0 Hz) 3.56 (1H, dd, J=12.0, 5.0 Hz) -
Quercetin 3-O-β-Dlactoside 6.14 (s) 6.34 (s) 7.56 (m) 6.84 (d, J=8.26 Hz) 7.56 (m) 5.49(d, J=7.69 Hz) 3.0-4.0
4.21 (d, J=7.2Hz) 3.0-4.0
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Table 2. 13C-NMR of quercetin 3-O-β-D-glucoside,31 and quercetin 3-O-β-D-lactoside Position 2 4 3 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′ 1′′′ 2′′′ 3′′′ 4′′′ 5′′′ 6′′′
Quercetin 3-Oβ-D-glucoside 156.58 177.9 133.77 161.70 99.12 164.61 93.97 156.63 104.43 122.06 115.67 148.92 145.27 116.66 121.63 101.31 74.55 76.96
Quercetin 3-Oβ-D-lactoside 156.94 177.51 133.56 161.58 99.64 166.39 94.26 156.41 103.72 121.41 115.70 149.20 145.37 116.47 121.99 101.22 74.28 76
70.39 78.03 61.43
80.94 75.77 60.86 104.29 73.68 75.22 68.61 70.97 60.86
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