Probing the catalytic promiscuity of a regio- and stereospecific C-glycosyltransferase from Mangifera indica

Article abstract of DOI:10.1002/anie.201506505

The catalytic promiscuity of the novel benzophenone C-glycosyltransferase, MiCGT, which is involved in the biosynthesis of mangiferin from Mangifera indica, was explored. MiCGT exhibited a robust capability to regio- and stereospecific C-glycosylation of 35 structurally diverse druglike scaffolds and simple phenolics with UDP-glucose, and also formed O- and N-glycosides. Moreover, MiCGT was able to generate C-xylosides with UDP-xylose. The OGT-reversibility of MiCGT was also exploited to generate C-glucosides with simple sugar donor. Three aryl-C-glycosides exhibited potent SGLT2 inhibitory activities with IC₅₀ values of 2.6×, 7.6×, and 7.6×10^-7-M, respectively. These findings demonstrate for the first time the significant potential of an enzymatic approach to diversification through C-glycosidation of bioactive natural and unnatural products in drug discovery. C-glycodiversification: MiCGT, as the first benzophenone C-glycosyltransferase (CGT) from Mangifera indica, showed robust regio- and stereospecific C-glycosylation activity for 35 structurally diverse acceptors with UDP-glucose or xylose. The aryl-C-glycoside 1 exhibited potent antidiabetic activity toward SGLT2.

Full text of DOI:10.1002/anie.201506505

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International Edition: DOI: 10.1002/anie.201506505
German Edition: DOI: 10.1002/ange.201506505
Enzyme Catalysis
Probing the Catalytic Promiscuity of a Regio- and Stereospecific
C-Glycosyltransferase from Mangifera indica
Dawei Chen, Ridao Chen, Ruishan Wang, Jianhua Li, Kebo Xie, Chuancai Bian, Lili Sun,
Xiaolin Zhang, Jimei Liu, Lin Yang, Fei Ye, Xiaoming Yu, and Jungui Dai*
Abstract: The catalytic promiscuity of the novel benzophe-
none C-glycosyltransferase, MiCGT, which is involved in the
biosynthesis of mangiferin from Mangifera indica, was
explored. MiCGT exhibited a robust capability to regio- and
stereospecific C-glycosylation of 35 structurally diverse drug-
like scaffolds and simple phenolics with UDP-glucose, and
also formed O- and N-glycosides. Moreover, MiCGT was able
to generate C-xylosides with UDP-xylose. The OGT-reversi-
bility of MiCGT was also exploited to generate C-glucosides
with simple sugar donor. Three aryl-C-glycosides exhibited
potent SGLT2 inhibitory activities with IC50 values of 2.6 ” ,
7.6 ” , and 7.6 ” 10À7 m, respectively. These findings demon-
strate for the first time the significant potential of an enzymatic
approach to diversification through C-glycosidation of bioac-
tive natural and unnatural products in drug discovery.
plant secondary metabolism was rather limited until 2009, and
these CGTs have priority over C-glycosylating flavonoids.[8]
Furthermore, the majority of known CGTs exhibit relatively
narrow substrate spectra, and few CGTs have been applied as
enzymatic tools for the C-glycosylation of various natural and
unnatural compounds. Therefore, mining CGTs with catalytic
promiscuity, but regio- and stereospecificity, from plants will
certainly facilitate the production of a broad range of valuable
products.
Mangifera indica is a traditional Chinese medical herb and
a wide variety of bioactive natural glycosides, including
xanthone and benzophenone C-glycosides, have been isolated
from its leaves and bark.[9] However, little is known about the
enzymes that are responsible for the biosyntheses of these
compounds, particularly in terms of the key step of C-
glycosylation, which inspired us to search for CGTs with
specific catalytic properties. Herein, we report for the first
time, the identification of the novel benzophenone C-
glycosyltransferase, MiCGT, which is involved in the biosyn-
thesis of mangiferin from M. indica. Most importantly, this
communication highlights the catalytic promiscuity and regio-
and stereospecificity of MiCGT.
S
ugar moieties are often essential for the biological
activities and pharmacological properties of many natural
products.[1] Most of these products are O-glycosylated. How-
ever, N-, C-, and S-glycosides are also found in nature.[2]
Compared with other glycosides, C-glycosides are of partic-
ular pharmaceutical interest because of their pronounced
stability against hydrolysis in vivo.[3] Chemical glycosylation
remains restricted by such disadvantages as poor regio- and
stereoselectivities, and the protection and deprotection of
functional groups, but enzymatic syntheses catalyzed by
specific glycosyltransferases (GTs; EC 2.4) can alleviate
these disadvantages and are thus powerful tools.[4] Significant
progress has been achieved recently in the O-glycodiversifi-
cation of natural and unnatural products by OGTs.[5] How-
ever, GTs which are naturally responsible for forming C-
glycosidic bonds (CGTs) appear to be comparatively sparse
and exhibit stringent specificities which limit their availability
and scope for synthesis.[6]
To clone permissive CGTs from M. indica, a degenerate
PCR primer for 3’-RACE (see Table S1 in the Supporting
Information) was designed based on the conserved plant
secondary product glycosyltransferases (PSPG) motif (see
Figure S1 in the Supporting Information).[8,10] The 3’-termini
of 23 different UGT cDNAs were obtained using the total
RNA from M. indica leaves as a template. Combined with 5’-
RACE, 15 new putative candidates (MiUGTs) for CGTs were
successfully cloned and heterologously expressed in Escher-
ichia coli as described in the Supporting Information. To
identify the C-glycosylation capability of the MiUGTs in
vitro, UDP-glucose and putative biosynthetic precursors of
mangiferin (see Scheme S1 in the Supporting Information),
maclurin (1), and norathyriol (25)[11] were each incubated
with the crude recombinant protein, and analyzed with
HPLC-UV/MS2. Of the 15 recombinant enzymes, only
MiUGT13 was observed to C-glucosylate 1 to 1a with the
appearance of a parent ion peak at m/z 423 [MÀH]À, which
was 162 amu more than that of 1, and the characteristic
fragment ions of C-glucoside, [MÀHÀ90]À and [MÀHÀ120]À
(Figure 1).[8a,12] The product 1a was prepared in a scale-up
reaction, and its structure, including the attached position of
the glucosyl moiety and the anomeric stereochemistry, were
In the past few years, studies on microbial CGTs have
attracted considerable interest and achieved great pro-
gress.[6a,7] Structurally diverse C-glycosides have also been
isolated from plants, and implies the existence of correspond-
ing CGTs.[3a] However, the identification of CGTs involved in
[*] D. Chen, Dr. R. Chen, Dr. R. Wang, Dr. J. Li, K. Xie, C. Bian, L. Sun,
X. Zhang, J. Liu, Prof. Dr. L. Yang, Prof. Dr. F. Ye, Prof. Dr. X. Yu,
Prof. Dr. J. 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
1 Xian Nong Tan Street, Beijing 100050 (China)
E-mail: jgdai@imm.ac.cn
1
elucidated by MS, and H and 13C NMR spectroscopic data
analyses and compared with those of maclurin 3-C-b-d-
glucoside.[13] The large coupling constant (J = 9.8 Hz) of the
anomeric proton (see Figures S59 and S60 in the Supporting
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201506505.
12678
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Figure S6 in the Supporting Information). Taken together,
these results provide the first solid experimental biochemical
evidence suggesting that maclurin 3-C-b-d-glucoside is an
intermediate in the biosynthetic pathway of mangiferin in M.
indica (see Scheme S1).[11]
To explore the catalytic promiscuity and probe the
synthetic utility of this novel CGT in vitro, an acceptor library
of representative natural and unnatural compounds with
structural diversity was assessed with UDP-glucose
(Figure 2). The library members included benzophenones
(1–4, 14–16, 37–39), flavonoids (11, 41–44), benzil (17),
xanthones (18–25), anthraquinone (40), and simple aromatics
with various substituents such as OH, NH2, CH3, OCH3, F, or
Br (10, 26–29, 31–36, 45–54). Additionally, synthetic com-
pounds, including alkaloids (5, 8, 9), thiophene (6), naphtha-
lene (7), benzoates (12, 13), and ethyl phloroglucinol (30),
which closely resemble the natural substrate of 1 featuring
a phloroglucinol moiety, were designed and chemically
synthesized (see the Supporting Information).
An initial hint about the enzymeꢀs unprecedented broad
capability for C-glycosylation was provided by the HPLC-
UV/MS2 analysis (Figure 2; see Figures S7–S27 in the Sup-
porting Information), which revealed that MiCGT was
sufficiently flexible to C-glycosylate 35 of the 54 library
members (65%), including 18 structurally different types.
Such an ability to utilize structurally varied natural and
unnatural aglycons appears to be more common among
OGTs,[5a,14] but is unusual for CGTs. Moreover, MiCGT
resulted in high C-glycosylation conversion rates (> 80%)
with 13 (1–7, 10–12, 17, 26, 27) of the 35 substrates. Notably,
34 members led only to a single, chromatographically distinct
mono-C-glucoside individually, which indicates the regiospe-
cificity of MiCGT. Weak C-glycosylation activity of MiCGT
turned out to be observed with the xanthones (18–24).
Interestingly, MiCGT was able to both C- and O-glycosylate
16 substrates (14–16, 18–24, 31–36; see Table S3 in the
Supporting Information). MiCGT also exhibited N-glycosy-
lation activity toward 3,4-dichloroaniline (54; see Figure S28
in the Supporting Information). MiCGT was also observed to
be able to form C-xylosides (1b, 10b, 27b, 28b) with UDP-
xylose (see Figures S29–S32 in the Supporting Information).
Based on the above results, MiCGT exhibited unusual
substrate (acceptor and donor) promiscuity, which rendered
MiCGT a promising enzyme for the construction of C-
glycoside libraries that exhibit structural and bioactive
diversities.
Given that an understanding of the determinants in the
acceptor molecules that control the mode of glycosylation is
critical for the generation of novel C-glycosyl derivatives,
a detailed structural comparison was performed. It demon-
strated that MiCGT appeared to generate only C-glycosides
with 2,4,6-tri-hydroxy acceptors at the A ring (1–13), both C-
and O- glycosides with 2,4-di-hydroxyl acceptors at the A ring
(14–16), and only O-glycosides with 2- or 4-mono-hydroxy
acceptors at the A ring (38, 39). The same results were
observed with simple phenolics (26–36). Hence, the electron
density around the carbon atom which undergoes attack,
enhanced by electron-donating hydroxy groups, might play an
important role in C-glycosylation, and strongly supports the
Figure 1. C-glycosylation of 1 catalyzed by recombinant MiCGT. A) The
reaction catalyzed by the crude recombinant enzyme. B) HPLC chro-
matograms of 1 and the enzyme product 1a. C,D) Typical negative-ion
MS and MS2 spectra for 1a. The HPLC conditions are provided in
Table S2 in the Supporting Information.
Information) indicated the formation of the b-anomer and an
inversion mechanism for MiUGT13. The above results
unequivocally establish MiUGT13 as a CGT (designated
MiCGT, Mangifera indica C-glycosyltransferase, GenBank
accession No. KT200208). A phylogenetic tree was con-
structed (see Figure S2 in the Supporting Information) and
revealed that MiCGT and other plant CGTs were grouped
into a single clade with the highest identity (50%) with
UGT708D1 from Glycine max.[8e]
Recombinant His6-MiCGT was purified with His-tag
affinity chromatography and analyzed with SDS-PAGE (see
Figure S3 in the Supporting Information) for the biochemical
characteristics. MiCGT displayed the maximum activity at
pH 9.0 and 458C, and was divalent-cation-independent (see
Figure S4 in the Supporting Information). MiCGT exhibited
Km values of 47.0 mm and 159.2 mm for 1 and 25 (for structures
see Figure 2), respectively, and the corresponding kcat values
of 1.6 sÀ1 and 0.8 sÀ1 (see Figure S5 in the Supporting
Information). Generally, the C-glucosylation in the mangi-
ferin biosynthesis is hypothesized to occur at the benzophe-
none (1) or the xanthone (25) stage (see Scheme S1).[11] In our
experiments, we observed that MiCGT could C-glycosylate
1 to 1a, but only O-glycosylate 25. MiCGT exhibits a kcat
/
Km value of 3.4 ” 104 mÀ1 sÀ1 to 1, which was roughly sevenfold
that to that for 25 (5.2 ” 103 mÀ1 sÀ1). Cumulatively, MiCGT
can be definitively validated as the first benzophenone CGT.
Furthermore, maclurin 3-C-b-d-glucoside (1a) was enzymati-
cally converted into mangiferin (73% yield for 2 h) when
incubated with the crude enzyme from M. indica leaves (see
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Figure 2. Exploring the catalytic promiscuity of MiCGT. A) Percent conversions of the C-glycosylated products catalyzed by MiCGT. The members
are listed based on the structural scaffolds shown in part B. The black and grey columns represent the conversion of C-glucosides and C-
xylosides, respectively. B) The structures of the library members and corresponding C-glycosylated products. Compounds 5, 7–9, 12, 13, and 30
are designed novel scaffolds. The O- or N-glucosylated yields of the compounds (14–16, 18–25, 31–54) are shown in the Supporting Information.
*=C-glycosylated products were prepared and confirmed by MS, and 1H and 13C NMR spectroscopy. N.D.=not detected, D.=detected in MS.
occurrence of aryl-C-glycosylation by a direct Friedel–Crafts-
like reaction mechanism.[2a,3b,6,7] Additionally, the facts that
neither 45 nor various acetophenone derivatives with either
electron-donating groups, such as NH2, CH3, and OCH3 (48,
49, 51, 52), or electron-withdrawing groups, such as F and Br
(46, 47, 50, 53), were accepted indicates that the 2,4-di-
hydroxy substituents on the aromatic ring are of stringent
necessity for MiCGT to C-glycosylate. Thus, the number and
position of the electron-donating hydroxy group at an
aromatic ring are the critical determinants which mediate
the C- or O-glycosylation capacity of MiCGT.
Accordingly, the minimal recognition unit of MiCGT for
C-glycosylation was identified as resorcinol (36), while that of
the previously characterized plant CGTs is 2’,4’,6’-trihy-
droxyacetophenone (28).[8a–c] Most importantly, MiCGT was
able to catalyze similar scaffolds (26–36) to the corresponding
C-glycosides, which could serve as common building blocks
for the chemical syntheses of structurally varied bioactive C-
glycosyl natural products and drugs.
Based on the dual specificity for C- and O-glycosylation of
MiCGT and the reversibility of OGTs,[5b,f, 14,15] we attempted
to synthesize C-glycosides with a simple sugar donor by
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of a benzophenone CGT may provide more genetic informa-
tion for the future verification of additional novel CGTs for
the C-glycodiversification of bioactive natural and unnatural
products. Given the dual selectivity for both C- and O-
glycosylation, MiCGT is probably a good model for future
kinetic/mechanistic and structural studies that aim to eluci-
date the catalytic mechanism and substrate promiscuity, and
to address how the enzyme governs the C- and O-glycosyla-
tions. The MiCGT reported here hints at more exciting and
novel CGTs hiding in plants as enzymatic tools for C-
glycosylation in the search for drug leads, and would also
facilitate further enzyme engineering for the development of
novel biocatalysts in combinatorial biosynthesis.
Figure 3. Exploiting the OGT reversibility of MiCGT to generate
C-glucoside with a simple sugar donor (the HPLC analysis is shown in
Figure S33 in the Supporting Information).
coupled reactions. As shown in Figure 3, the OGT-reversi-
bility of MiCGT was exemplified in the coupling reactions, in
which the C-glucosylated product 1a was obtained in a high
yield (77%) with 4-nitrophenyl-b-d-glucopyranoside (55) and
only a catalytic amount of UDP (see Figure S33 in the
Supporting Information). Without adding relatively costly
UDP-glucose, this study provides an economic and environ-
mentally general approach to the C-glycodiversification of
bioactive molecules.
Acknowledgements
The work was financially supported by the National Natural
Science Foundation of China (Grant No. 81273405), and the
National Science & Technology Major Project “Key New
Drug Creation and Manufacturing”, China (Grant No.
2012X09301002-001-005).
To further confirm the catalytic properties of MiCGT and
biologically assay the C-glycosides, we obtained 16 C-
glycosylated products by preparative-scale reactions, 12 (3a,
5a–10a, 12a, 14a, 16a, 18a, 28b) of which were novel C-
glycosides. Their structures were identified with HRESIMS,
and 1H and 13C NMR spectroscopic data. The sugar moieties
were observed to be attached at the C3-position of the A ring,
a carbon atom in the ortho/para position to the hydroxy
groups, and is similar to the observation of C-glycosylations
by all known CGTs.[6–8] The b-glycosidic linkages were
deduced from the large coupling constants (J = 9.7–9.9 Hz)
of the anomeric protons (see Figures S59–S94 in the Support-
ing Information). Therefore, MiCGT is a regio- and stereo-
specific C-glycosyltransferase. The resulting 16 C-glycosy-
lated compounds were biologically evaluated for potential
effects on type-2 diabetes using human sodium-glucose co-
transporter 2 (SGLT2) inhibitory activity in vitro,[16] thus
revealing that 10a–12a exhibited potential as lead com-
pounds with the IC50 values of 2.6 ” , 7.6 ” , and 7.6 ” 10À7 m,
respectively.
In summary, the surprising catalytic promiscuity of
MiCGT, which is a novel benzophenone CGT involved in
the biosynthesis of mangiferin from M. indica, was high-
lighted. MiCGT exhibited robust regio- and stereospecific C-
glycosylation activity toward numerous natural and unnatural
druglike compounds and simple phenolics with either UDP-
glucose or xylose and the abilities to form C-, O-, and N-
glycosides. The OGT reversibility of MiCGT was also
exploited to economically generate C-glucosides with simple
sugar donors without the need for using the expensive UDP-
sugar moiety. Three aryl-C-glycosides (10a–12a) displayed
strong inhibitory effects against SGLT2. The present studies
have successfully demonstrated, for the first time, the
significant potential of an enzymatic approach not only to
the chemoenzymatic formation of structurally diverse bioac-
tive aryl-C-glycosides for lead compound discovery, but also
for the formation of key and common building blocks for the
bioactive compound syntheses. Moreover, the identification
Keywords: carbohydrates · enzyme catalysis · glycosylation ·
regioselectivity · transferases
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Received: July 14, 2015
Published online: September 1, 2015
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