Biocatalytic C-Glucosylation of Coumarins Using an Engineered C-Glycosyltransferase

Article abstract of DOI:10.1021/acs.orglett.8b00378

The enzymatic synthesis of coumarin C-glucosides by an engineered C-glycosyltransferase, MiCGTb-GAGM, was explored in vitro and in vivo. MiCGTb-GAGM exhibited a robust C-glucosylation capability toward structurally diverse coumarin derivatives. The whole-cell bioconversion of MiCGTb-GAGM was exploited for large-scale production of coumarin C-glucosides. Two C-glucosides exhibited potent SGLT2 inhibitory activities with IC₅₀ values at 10^-6 M. These findings provide cost-effective and practical synthetic strategies to generate structurally diverse and novel bioactive coumarin C-glycosides for drug discovery.

Full text of DOI:10.1021/acs.orglett.8b00378

Letter
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Biocatalytic C‑Glucosylation of Coumarins Using an Engineered
C‑Glycosyltransferase
Dawei Chen, Ridao Chen, Kebo Xie, Tian Yue, Xiaolin Zhang, Fei Ye, and Jungui Dai*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, China
*
S Supporting Information
ABSTRACT: The enzymatic synthesis of coumarin C-gluco-
sides by an engineered C-glycosyltransferase, MiCGTb−
GAGM, was explored in vitro and in vivo. MiCGTb−
GAGM exhibited a robust C-glucosylation capability toward
structurally diverse coumarin derivatives. The whole-cell
bioconversion of MiCGTb−GAGM was exploited for large-
scale production of coumarin C-glucosides. Two C-glucosides
exhibited potent SGLT2 inhibitory activities with IC values
5
0
−6
at 10 M. These findings provide cost-effective and practical
synthetic strategies to generate structurally diverse and novel
bioactive coumarin C-glycosides for drug discovery.
mong the naturally occurring C-glycosides, only a few
coumarin C-glycosides are known. Dauroside D (1a,
also known as mulberroside B), a 6-C-glucosyl coumarin from
unprecedented substrate promiscuity by protein engineering.
To investigate the C-glycosylation capacity of these variants
toward coumarins, 5,7-dihydroxycoumarin (1, the aglycon of
dauroside D) was initially used as a sugar acceptor, and uridine
diphosphate α-D-glucose (UDP-Glc) was used as a sugar donor.
The recombinant proteins were overproduced as N-terminal
1
,2
A
Haplophyllum dauricum, exhibited spasmolytic and hypotensive
3
activities, but with mild toxicity. Two 8-C-glucosyl coumarins
(
8-C-glucosyl-6-methoxy-5,7-dihydroxy-3,4-dihydrocoumarin
and 8-C-glucosyl-5-vinyl-6,7-dimethoxy-3,4-dihydrocoumarin)
His -tagged fusions in Escherichia coli BL21 (DE3) and purified
4
6
from Diceratella elliptica showed cytotoxic activity. Given the
11
with Ni-affinity chromatography. The reactions (50 mM
various biological activities of coumarin derivatives, especially
NaH PO −Na HPO , pH 8.0; 0.4 mM UDP-Glc; 0.2 mM 1;
5
2
4
2
4
the effects of the C-glycosyl moiety on the activity, several
1
00 μg of purified enzyme; 30 °C, 12 h) were analyzed by
examples of chemically synthesizing coumarin C-glycosides in
HPLC−UV/MS (high-performance liquid chromatography−
UV absorption/mass spectrometry). Control reactions lacking
either enzyme or UDP-Glc confirmed that the reactions were
dependent upon both the enzyme and UDP-Glc. To our
delight, a quadruple mutant, MiCGTb−GAGM (S60G/
V100A/T104G/I152M), exhibited higher C-glucosylation
activity than the wild type toward 1 (Scheme 1; Figure S1).
which the C-3 or C-4 position contains a glycosyl residue have
6
been described. The chemical synthesis of coumarin 8-C-
7
glucosides has also been reported by Mahling and Schmidt.
However, chemical C-glycosylation remains restricted by such
disadvantages as poor regio- and stereoselectivities and the
8
tedious protection and deprotection of functional groups. The
catalysis of enzymatic C-glycosylation by specific C-glycosyl-
2
MS and MS analysis of 1a displayed the appearance of an ion
transferases (CGTs; EC 2.4) can alleviate these disadvantages,
9
−
making these enzymes powerful tools. In recent years,
peak at m/z 339 [M − H] , which was 162 amu greater than
significant progress has been achieved in the enzymatic C-
glycosylation of natural and unnatural compounds, including
Scheme 1. MiCGTb−GAGM Catalyzes the C-Glucosylation
of 1
1
0
11
12
flavone, benzophenone and xanthone, and anthraquinone.
However, the biocatalytic C-glycosylation of coumarin
derivatives have not been reported. Thus, exploitation of
universally applicable green approaches with catalytically
promiscuous biocatalysts to generate structurally novel and
diverse bioactive coumarin C-glycosides is highly desired.
Herein, we report the in vitro and in vivo synthesis of
structurally diverse bioactive 6- or 8-C-glucosylated coumarins
using an engineered C-glycosyltransferase.
We have recently characterized two benzophenone CGTs
from Mangifera indica. In an effort to expand their substrate
11
Received: February 2, 2018
scope, we generated a panel of variants of the two CGTs with
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b00378
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
that of 1, and peaks corresponding to the characteristic
fragment ions of C-glucoside, found at m/z 249 [M − H −
−
−
11
9
0] and m/z 219 [M − H − 120] (Figure S2). The
product 1a was then prepared on a larger scale for structural
characterization. All the MS and H and C NMR
1
13
spectroscopic data of 1a were in good agreement with those
3
of dauroside D, and a large coupling constant (J = 9.8 Hz) of
the anomeric proton (H-1′) further supported the formation of
the β-anomer. Therefore, MiCGTb−GAGM could regio- and
stereospecifically C-glucosylate 1 in a high yield (82.2% by
HPLC) and was thus selected for further investigations. This
report is the first example of a CGT that catalyzes C-glucosidic
bond formation with coumarin.
The biochemical characteristics of this C-glucosylation
catalyzed by MiCGTb−GAGM were then further investigated
by using 1 and UDP-Glc as acceptor and donor substrates.
MiCGTb−GAGM displayed its maximum activity at pH 8.0
and 40 °C and was divalent cation independent (Figure S3).
Kinetic analysis revealed that MiCGTb−GAGM exhibited Km
values of 647.1 μM and 230.4 μM for 1 and UDP-Glc,
respectively, and that the corresponding k values were 1.6 and
cat
.3 min− (Figure S4).
1
0
To explore the coumarin-substrate spectrum of MiCGTb−
GAGM and investigate substrate structure−enzyme catalytic
activity relationships, an acceptor library of representative
natural and unnatural coumarins with different substituents at
the C-3, C-4, C-5, or/and C-6 positions (Figure 1) was assessed
with UDP-Glc in vitro. An initial hint to the enzyme’s broad
capability for C-glucosylation was provided by HPLC−UV/MS
analysis (Figure 1; Figures S5 and S6), which revealed the
substantial flexibility of MiCGTb−GAGM to C-glucosylate 12
(1−4, 7, 8, 10−13, 16, and 17) of the 20 library members.
Moreover, high C-glucosylation conversion rates (>80%) were
observed with nine substrates (1−4, 7, 8, and 10−12). The
reactions of 10 members (1−3, 7, 8, 11−13, 16, and 17) led to
a single, chromatographically distinct mono-C-glucoside, which
indicates the regiospecificity of MiCGTb−GAGM. In addition,
MiCGTb−GAGM exhibited both C- and O-glucosylation
activity toward three substrates (1, 2, and 13; Table S2).
Hence, the substrate scope and synthetic potential of
MiCGTb−GAGM were further expanded in the field of
enzymatic C-glycosylation, which rendered MiCGTb−GAGM
a promising enzyme for the production of coumarin C-
glycosides that exhibit structural and bioactive diversities. In
addition to UDP-glucose, we have tested the tolerance of
MiCGTb−GAGM toward other five UDP-sugars (such as
UDP-galactose, UDP-glucuronic acid, UDP-rhamnose, UDP-N-
acetylglucosamine and UDP-xylose) with acceptor 1. However,
MiCGTb−GAGM was only able to catalyze UDP-galactose
with low activity (ca. 10%).
Figure 1. Acceptor promiscuity of MiCGTb−GAGM. (A) Percent
conversion of the C-glucosylated coumarins catalyzed by purified
MiCGTb−GAGM. The members are listed based on the structural
scaffolds in part B. (B) Structures of library members and the
corresponding C-glucosylated coumarins. The conversion rates of O-
glucosylated products (1, 2, 5, 6, 9, 13, 14, and 18−20) are shown in
Table S2. *C-glucosylated coumarins were prepared, and their
1
13
structures were confirmed by HR-ESI-MS and H and C NMR
spectroscopic analyses (Figures S9−S33). N.D.: not detected.
13
skeleton. Moreover, MiCGTb−GAGM exhibited C-glucosy-
lation activity on 5,7-dihydroxycoumarins with hydrophobic
substituents at the C-4 position (1−4 vs 5, 6 and 14), linear
substituents at the C-3 position (7 and 8 vs 9 and 14) or cyclic
substituents at the C-3/4 positions (10−13). Furthermore, a
double bond at the C-3/4 positions of coumarins (1, 2, and 4)
had a beneficial effect on the catalytic efficiency of MiCGTb−
GAGM for C-glucosylation, while the conversion rates of 3,4-
dihydrocoumarins (15−17) were not detected or clearly
decreased. These findings facilitate chemoenzymatic syntheses
of additional novel structurally diverse coumarin C-glycosides
with these functional groups, which might be important for
To understand substrate structure−enzyme catalytic activity
relationships, we subsequently performed a detailed structural
comparison of the above 20 coumarins. The fact that 5,7-
dihydroxycoumarins (1−4, 7, 8, 10−13, 16, and 17) were
converted and that neither 7-hydroxycoumarin (18 and 19) nor
2
,14
6,7-dihydroxycoumarin (20) were converted to C-glucosides
various pharmacological properties.
indicates that the 5,7-dihydroxy substituents on the coumarin
framework are of stringent necessity for the C-glycosylation of
MiCGTb−GAGM. Hence, the hydroxy groups at the C-5/7
positions enhanced the electron density around the attacked
carbon atom, which was crucial for the C-glucosylation of
MiCGTb−GAGM and improved the reactivity and regiose-
lectivity of the glucosyl group substitution on the coumarin
While enzymatic reactions are routinely carried out to
characterize the associated enzyme’s biochemical function,
labor-intensive protein purification, the lack of substrate
solubility in aqueous buffer and the cost of UDP-Glc can all
be hurdles to preparative-scale biocatalysis with practical
potential. To overcome these barriers, whole E. coli cells
harboring MiCGTb−GAGM and the inherent UDP-Glc were
15
B
DOI: 10.1021/acs.orglett.8b00378
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
16,17
used as biocatalyst with coumarins in M9 medium.
All
Table 1. Preparative-Scale Coumarin C-Glucosides with
samples were analyzed by HPLC-UV/MS, which revealed that
the substrate scope of MiCGTb−GAGM for C-glucosylation in
vivo was identical to that of purified enzyme with only one
exception (16, Figures 1 and 2). In addition, the conversion
Purified Enzyme or Whole Cells Containing MiCGTb−
GAGM
no.
catalyst
C-glucosylated products
isolated yield (%)
1
2
3
4
7
8
enzyme
1a
80.0
enzyme
2a
85.6
whole cells
whole cells
whole cells
enzyme
3a
95.6
4a/4b
7a
80.4/7.6
68.4
8a
23.8
1
1
1
1
1
0
0
1
2
7
enzyme
10a/10b
10a/10b
11a
26.7/60.2
88.7/5.3
33.4
whole cells
whole cells
whole cells
whole cells
Figure 2. Conversion rates of coumarin C-glucosides using whole cells
containing MiCGTb−GAGM. The conversion of 11 was indicated at
the final concentration of 0.1 mM in 1 mL mixtures, while other
coumarins were indicated at 0.4 mM (Figure S7). The conversion rates
of O-glucosylated products (1, 2, 4, 6−8, and 11−13) are shown in
Table S2. N.D.: not detected.
12a
88.0
17a
7.3
compounds with IC50 values of 2.2 × 10− and 6.3 × 10−6
Table S3), respectively.
In summary, the potential of a uniquely permissive
6
M
(
engineered C-glycosyltransferase MiCGTb−GAGM to func-
tion as a catalyst for the C-glucosylation of coumarins was
highlighted in vitro and in vivo. MiCGTb−GAGM exhibited a
robust C-glucosylation capacity toward a series of coumarin
derivatives with different substitutions at the C-3 or/and C-4
positions in vitro. Moreover, MiCGTb−GAGM was exploited
as a whole-cell biocatalyst to generate several coumarin C-
glucosides. Two coumarin C-glucosides (10a and 11a)
displayed potent inhibitory effects against SGLT2. These
findings demonstrated the opportunities of performing the
biocatalytic C-glycosylation of structurally diverse coumarins
with endogenous NDP-sugars of host strains. The present
study collectively demonstrates for the first time the significant
potential of a CGT as a powerful biocatalyst for the in vitro and
in vivo synthesis of additional novel bioactive natural and
unnatural coumarin C-glycosides as drug leads and will enhance
ongoing efforts to develop the combinatorial biosynthesis of
NDP-sugars for in vivo C-glycorandomization and the synthetic
biology of bioactive coumarin C-glycosides.
rates of 1a, 2a and 8a by MiCGTb−GAGM in vivo were
significantly lower than that of the purified enzyme, while the
yields of their respective O-glucoside were increased (Table
S2). Interestingly, the ratio between 10a and 10b yields was
changed from 41:59 to 93:7 when the MiCGTb−GAGM was
used as a whole-cell biocatalyst in place of purified enzyme
(
Figures 1 and 2). These observed different yields or
regioselectivity from purified enzymes and whole cells may be
due to the different reaction microenvironment around proteins
between in vitro and in vivo. High C-glucosylation conversion
rates (>80%) were achieved with six substrates (3, 4, 7, and
1
0−12) with the endogenous UDP-Glc of the host strains.
High yields of 7a were obtained from 0.5 to 8 h with different
concentrations of 7 (Figure S8). Therefore, the success of these
C-glucosylation reactions through whole-cell biotransformation
establishes a cost-effective and applicable green approach for
the convenient synthesis of structurally diverse and novel
18
bioactive coumarin C-glucosides.
To further confirm the catalytic properties of MiCGTb−
GAGM and biologically assay the coumarin C-glucosides,
preparative-scale reactions were performed with a purified
enzyme or whole cells based on their respective yields of
products. In total, we obtained 12 C-glucosylated coumarins
ASSOCIATED CONTENT
Supporting Information
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b00378.
(
Table 1), 11 (2a−4a, 4b, 7a, 8a, 10a−12a, 10b, and 17a) of
which were novel compounds. The isolated yields of six C-
Experimental procedures, including protein expression,
whole cells bioconversion, reactions analysis and product
purification, HPLC/MS, HR-ESI-MS, and NMR charac-
terization data and spectra of products (PDF)
glucosides (1a−4a, 10a, and 12a) were greater than 80%. Their
1
13
structures were identified with HR-ESI-MS and H and
C
NMR spectroscopic data (Figures S9−S33). Most of the C-
glucosylated products (1a−4a, 7a, 8a, 10a−12a, and 17a) were
coumarin 6-C-glucosides, and the sugar moieties were attached
at the C-8 position of the coumarins (4b and 10b). All anomers
of coumarin C-glucosides were in the β-configuration, which
was deduced from the large coupling constants (J = 9.7−9.9
Hz) of the anomeric protons. The resulting 12 C-glucosylated
coumarins were biologically evaluated for potential effects on
type-2 diabetes, which was assayed using human sodium-
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: jgdai@imm.ac.cn.
ORCID
Jungui Dai: 0000-0003-2989-9016
Notes
19
glucose cotransporter 2 (SGLT2) inhibitory activity in vitro;
this assay revealed that 10a and 11a exhibited potential as lead
The authors declare no competing financial interest.
C
DOI: 10.1021/acs.orglett.8b00378
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
burg, B.; Oja, T.; Klika, K. D.; Metsa-
̈
Ketela,
̈
M. ACS Chem. Biol. 2013,
ACKNOWLEDGMENTS
■
8
, 2377−2382. (c) Li, L.; Wang, P.; Tang, Y. J. Antibiot. 2014, 67, 65−
This work was supported by the National Science Foundation
of China (Grant Nos. 81703369 and 21572277) and CAMS
Innovation Fund for Medical Sciences (CIFMS-2016-I2M-3-
7
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Okkels, F. T.; Mortensen, U. H.; Lindberg Møller, B.; Frandsen, R. J.
N.; Kannangara, R. ChemBioChem 2017, 18, 1893−1897. (e) Salem, S.
M.; Weidenbach, S.; Rohr, J. ACS Chem. Biol. 2017, 12, 2529−2534.
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