Enzymatic glucosylation of unnatural naphthols by a promiscuous glycosyltransferase from Aloe arborescens

Article abstract of DOI:10.1016/j.tetlet.2017.04.041

Enzymatic glucosylation of unnatural products by natural glycosyltransferases (GTs) has great potential in creating novel and bioactive glucosides. A new GT (AaGT3) from Aloe arborescens exhibited catalytic promiscuity and high efficiency to diverse unnatural naphthols. By combing the substrate flexibility and catalytic reversibility of AaGT3, a cost-effective enzymatic approach to novel and bioactive unnatural glucosides was established. These studies indicate the significant potential of promiscuous natural GTs in synthesis of unnatural bioactive glucosides in drug discovery.

Full text of DOI:10.1016/j.tetlet.2017.04.041

Accepted Manuscript
Enzymatic glucosylation of unnatural naphthols by a promiscuous glycosyl-
transferase from Aloe arborescens
Kebo Xie, Yujiao Zhang, Ridao Chen, Dawei Chen, Lin Yang, Xia Liu, Jungui
Dai
PII:
S0040-4039(17)30483-5
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.04.041
TETL 48833
Reference:
Tetrahedron Letters
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Revised Date:
Accepted Date:
17 March 2017
10 April 2017
13 April 2017
Please cite this article as: Xie, K., Zhang, Y., Chen, R., Chen, D., Yang, L., Liu, X., Dai, J., Enzymatic glucosylation
of unnatural naphthols by a promiscuous glycosyltransferase from Aloe arborescens, Tetrahedron Letters (2017),
doi: http://dx.doi.org/10.1016/j.tetlet.2017.04.041
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Enzymatic glucosylation of unnatural naphthols by a promiscuous glycosyltransferase
from Aloe arborescens
Kebo Xiea , Yujiao Zhangb , Ridao Chena , Dawei Chena , Lin Yangb , Xia Liua and Jungui Daia, ∗
aState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking
Union Medical College, 1 Xian Nong Tan Street 1, Beijing 100050, PR China
bCollege of Life and Environmental Sciences, Minzu University of China, Beijing 10081, PR China
ARTICLE INFO
ABSTRACT
Article history:
Received
Enzymatic glucosylation of unnatural products by natural glycosyltransferases (GTs) has great
potential in creating novel and bioactive glucosides. A new GT (AaGT3) from Aloe arborescens
exhibited catalytic promiscuity and high efficiency to diverse unnatural naphthols. By combing
the substrate flexibility and catalytic reversibility of AaGT3, a cost-effective enzymatic
approach to novel and bioactive unnatural glucosides was established. These studies indicate the
significant potential of promiscuous natural GTs in synthesis of unnatural bioactive glucosides
in drug discovery.
Received in revised form
Accepted
Available online
Keywords:
Enzymatic glucosylation
Catalytic promiscuity
Unnatural aglycons
Naphthols
2009 Elsevier Ltd. All rights reserved.
Bioactive glycoside
Glycosylation is widely found in natural products, which can
not only contribute to the diversity of structures but also affect
the physicochemical and biological properties of the
corresponding glycosides.1 In bioactive unnatural products and
even some chemical drugs, sugar moieties are also essential and
their removal oftentimes results in the loss of biological activity2.
However, chemical glycosylation in the synthesis of glycosides
faces challenges in regioselectivity, stereoselectivity, and the
protection and de-protection of functional groups, which limit the
exploitation of diverse unnatural glycosides in drug discovery.3
Glycosyltransferases (GTs) are generally recognized as powerful
synthetic tools for bioactive glycosides, as they can transfer sugar
moieties from donors to acceptors generating various glycosides.4
However, most of the GTs only recognize natural products and
few of them show activity to unnatural products with skeletal
types different from natural products.5 Thus, the discovery of
GTs with catalytic promiscuity and novel specificity to diverse
unnatural products is of necessity in practice.
A. arborescens is a famous medical plant and a wide variety
of structurally diverse glycosides have been isolated from its
leaves.7 The diverse glycosyated secondary metabolites imply the
existence of corresponding GTs, which inspired us to mine GTs
with novel catalytic properties. Thus, to clone novel GTs from A.
arborescens, a degenerate PCR primer for 5′-RACE was
designed based on the conserved PSPG (plant secondary product
glycosyltransferases) motif of five plant GTs which are able to
catalyze unnatural products (Table S1 and Fig. S1 in the
Supplementary Material, SM).8 Combined with 3′-RACE, nine
new A. arborescens GT genes (AaGTs) were successfully cloned
by RT-PCR amplification using the total RNA from A.
arborescens leaves as a template and heterologously expressed in
Escherichia coli as described in SM.
To screen the glucosylation capability of the recombinant
AaGTs in vitro, UDP-glucose (UDP-Glc) along with 2-naphthol
(1), which is a typical and basic structural unit of naphthols were
used in enzymatic assays. The screening reactions (50 mM Tris-
HCl, pH 7.4; 0.5 mM UDP-Glc; 0.25 mM aglycon; 500 µg of
crude AaGTs; 30 °C, 6 h) were analyzed by HPLC-UV/MS
(high-performance liquid chromatography with ultraviolet/mass
spectrometry detection). Of the nine recombinant AaGTs, only
AaGT3 showed glycosylation activity to 2-naphthol (1) with high
conversion rate (Fig. 1). Control reactions lacking either enzyme
or UDP-Glc confirmed that the reactions were dependent upon
both the enzyme and the sugar donor. The glucosylated product
(1a) was isolated from the preparative-scale reaction, and the
Naphthols and their derivatives, as bioactive unnatural
products, are ubiquitous in the field of medicine.6 Glycosylation
of naphthols can not only improve the water solubility but also
affect their pharmacological activity, which might provide lead
compounds for drug discovery. However, to the best of our
knowledge, none of the known GTs is able to glycosylate various
unnatural naphthols. In this work, we reported a new GT from
Aloe arborescens with catalytic promiscuity in glucosylation of
diverse naphthols generating novel and bioactive glucosides.
———
∗ Corresponding author. Tel.: +86-10-63165195; fax: +86-10-63017757; e-mail: jgdai@imm.ac.cn

2
Tetrahedron
A
B
C
A: 35 °C
B: pH 8.0
C: Mg2+
AaGT3
UDP-Glc
UDP
Figure 1. Catalytic promiscuity of AaGT3 in glucosylation of
unnatural naphthols. (A) Percent conversions of glucosylated
products catalyzed by AaGT3. The library numbers are listed based
on the structural scaffolds shown in part B. (B) The structures of the
aglycons and corresponding glucosylated products.
5
5a
Up to 81% yield
Figure 2. Enzymatic glucosylation of 2-amino-2'-hydroxy-1,1'-
binaphthalene (5) by AaGT3 with UDP-Glc as a sugar donor.
Relative activities of AaGT3 at different tempetures (A), pH buffers
(B) and with divalent cations (C) are shown. ND: not detected. The
yield of glucosylated product 5a was up to 81% when the reaction
was performed at 35 °C and in pH 8.0 buffer with Mg2+.
Table 1 Neuroprotective effects of 2-amino-2'-hydroxy-1,1'-
binaphthalene (5) and its glucoside (5a) on the glutamate-induced
toxicity in SK-N-SH cells
Compounds
Concentration
(µM)
Cell viability
(%)
Increased cell
viability (%)
Control
0
100
0
0
novel compounds, and all anomers are in the β-configuration due
to anomeric protons with large coupling constants (J > 7.0 Hz,
Table S2 in SM). It is important to note that most glucosylated
products exhibited improved water-solubility and the glucoside
form 5a had a more potent neuroprotective effect than the
corresponding aglycon 5 toward the glutamate-induced toxicity
in SK-N-SH cells,9 which might be a potential drug lead for
neurodegenerative disease (Table 1).
L-Glutamic
acid
2.5 × 104
62.08 3.74
Resveratrola
10
10
10
72.40 4.13
63.33 8.27
72.08 6.78
16.69
2.48
5
5a
16.05*
a Resveratrol was used as a positive control.
* P<0.05
In order to improve the yield of bioactive product 5a, the
biochemical characteristics of AaGT3 were investigated with 5 as
an acceptor and UDP-Glc as a sugar donor, respectively. The
optimum temperature and pH of purified AaGT3 were
determined to be 35 °C and pH 8.0, and this enzyme was
independent of metal ions, while Mg2+ could greatly enhance the
activity (Fig. 2). Thus the yield of 5a was up to 81% under the
optimum reaction conditions (50 mM Tris-HCl, pH 8.0; 0.5 mM
UDP-Glc; 0.25 mM aglycon 5; 0.5 mg of purified AaGT3; 5 mM
MgCl2; 35 °C, 6 h). Although the yield of 5a has been improved
to be more than 80%， the cost of this method is still not
effective due to the high price of UDP-Glc.
structure was characterized by MS, H NMR and 13C NMR
spectroscopic data analyses (Figs. S6‒S7 in SM). The observed
large anomeric proton-coupling constant (J = 7.3 Hz, Table S2 in
SM) indicated the formation of the β-anomer and an inverting
mechanism for AaGT3. The cDNA sequence of AaGT3 (1410
bp, GenBank accession number KY662486) contained an ORF
encoding 469 amino acids, and the AaGT3 showed the highest
identity to a predicted UGT88F-like glycosyltransferase from
Phoenix dactylifera. Purification of recombinant His6-AaGT3
was accomplished by His-tag affinity chromatography, and
purified AaGT3 was analyzed by SDS-PAGE (Fig. S2 in SM).
1
In addition to the capability of glycosylating unnatural
products, AaGT3 was also able to catalyze the deglycosylation of
unnatural products such as 4-nitrophenyl-β-D-glucopyranoside
(7), which means the glycosylation catalyzed by AaGT3 was
reversible. As UDP-Glc was an expensive sugar donor, we thus
exploited the reversibility of AaGT3 to provide sugar donors for
generating bioactive glycosides through one-pot reaction (Fig 3).
In the coupled reactions, UDP-Glc was generated by AaGT3-
catalyzed deglycosylation of 7 with UDP, and the sugar moiety
was intermediately transferred to the targeted aglycon acceptor
(5) through the glycosylation catalyzed by AaGT3. The relative
higher concentration of sugar donor (7) drives the one-pot
reaction toward the deglycosylation of 7 and glycosylation of 5.
Thus, the high yield (62%) of 5a occurred in the presence of only
a catalytic amount of UDP (1/100 of 7), which revealed that UDP
was under cyclic utilization throughout the coupled reactions
(Figs. 3A and S5 in SM). To further explore the substrate
spectrum toward the NDP moieties, AaGT3 was subsequently
probed with 5 and four other commercially available NDPs in
one-pot reactions. Interestingly, AaGT3 displayed broad
substrate specificity, recognizing ADP, TDP, CDP and GDP, as
well as their corresponding activated glucoses, albeit with lower
To investigate the catalytic promiscuity of AaGT3 in
recognizing diverse naphthols, a typical naphthols library
including
2,3-dihydroxynaphthalene
(2),
1,5-
dihydroxynaphthalene (3), 1,1′-bi-2,2′-naphthol (4), 2-amino-2'-
hydroxy-1,1'-binaphthalene (5) and 1,1'-methylenedi-2-naphthol
(6) was employed for enzymatic assays (Fig. 1B). From the first-
pass analysis with HPLC-UV/MS, AaGT3 was able to recognize
all the naphthols in the library with high catalytic efficiency (Fig.
1). Of particular note is that all the acceptors are unnatural
products, the structures of which have great differences to the
natural products from A. arborescens. Generally, most of the
reported GTs are capable of glucosylating acceptors with a
typical phenol unit. However, AaGT3 was able to glucosylate
structurally diverse unnatural naphthols, which indicated the
catalytic promiscuity of this plant enzyme. The broad substrate
spectrum and high efficiency make AaGTs being a potential tool
enzyme in enzymatic synthesis of unnatural glycosides. To
confirm the structures and evaluate their pharmacological
activities, glycosylated products of 4, 5 and 6 were prepared from
the scale up reactions and identified by MS and NMR (Figs.
S8‒S15 in SM). All the prepared glucosides 4a, 5a and 6a are

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HO
NO2
Deglycosylation
7a
7
B
Figure 3. One-pot reaction catalyzed by AaGT3. (A) The bioactive
glucoside (5a) was generated from a simple sugar donor (7) with a
catalytic amount of UDP. (B) Besides UDP, other NDPs (TDP,
GDP, ADP and CDP) can also be used in the one-pot reaction.
conversion rates than that of UDP (Fig. 3B). This feature allows
AaGT3 to be applied in producing bioactive glucosides from a
simple sugar donor in vivo with different NDPs of host strains.10
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glucosides without adding activated sugars establishes a cost-
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glucosides.
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In summary, enzymatic glucosylation of unnatural naphthols
for generating novel and bioactive unnatural glucosides was
achieved by a natural GT AaGT3 from A. arborescens. AaGT3
exhibited robust glucosylation activity toward simple basic units
and structurally diverse derivatives of naphthols. Moreover, the
catalytic reversibility of AaGT3 coupled with its catalytic
promiscuity was exploited as a powerful biocatalyst for the
enzymatic synthesis of novel and bioactive unnatural glucosides.
This study not only demonstrates the application prospect of
natural GTs in synthesis of target unnatural glycosides with
pharmacological activities but also provides a potential tool in
glycorandomization of diverse unnatural products.
Supplementary Material
Supplementary material (experimental operations, including
gene cloning, expression, reaction analysis and products
purification protocols, LC/MS, HRESIMS and NMR
characterization data and spectra of glucosylated products)
associated with the article can be found, in the online version, at
http:xxx
Acknowledgments
This work is financially supported by the National Natural
Science Foundation of China (Grant No. 81602999) and CAMS
Innovation Fund for Medical Sciences (CIFMS-2016-I2M-3-012).
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4
Tetrahedron
Graphical Abstract
Leave this area blank for abstract info.
Enzymatic glucosylation of unnatural
naphthols by a promiscuous
glycosyltransferase from Aloe arborescens
Kebo Xie, Yujiao Zhang, Ridao Chen, Dawei Chen, Lin Yang, Xia Liu, Jungui Dai*

Highlights
A new GT from Aloe arborescens exhibited
catalytic promiscuity and high efficiency to diverse
unnatural naphthols.
A cost-effective enzymatic approach to novel and
bioactive unnatural glycosides was established.
This work indicates the significant potential of
natural GTs in synthesis of unnatural bioactive
glycosides.