Enzymatic N-Glycosylation of Diverse Arylamine Aglycones by a Promiscuous Glycosyltransferase from Carthamus tinctorius

Article abstract of DOI:10.1002/adsc.201601128

A new glycosyltransferase (UGT71E5) from Carthamus tinctorius exhibited a robust promiscuity towards 30 structurally diverse drug-like aromatic amine scaffolds, making it the first reported glycosyltransferase capable of catalyzing N-glycosylation with multiple diverse nitrogen-heterocyclic aromatic compounds. The catalytic promiscuity and reversibility of UGT71E5 was exploited to generate the rare N-glycoside from the abundant O-glycoside with high efficiency. These findings demonstrate the significant potential of UGT71E5 in the enzymatic synthesis of diverse bioactive N-glycosides. (Figure presented.).

Full text of DOI:10.1002/adsc.201601128

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DOI: 10.1002/adsc.201601128
Enzymatic N-Glycosylation of Diverse Arylamine Aglycones by
a Promiscuous Glycosyltransferase from Carthamus tinctorius
Kebo Xie,a Ridao Chen,a Dawei Chen,a Jianhua Li,a Ruishan Wang,a Lin Yang,b
and Jungui Daia,*
a
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, Peopleꢀs
Republic of China
Fax : (+86)-10-6301-7757; phone: (+86)-10-6316-5195; e-mail: jgdai@imm.ac.cn
College of Life and Environmental Sciences, Minzu University of China, 27 Zhong Guan Cun Southern Street, Beijing
b
100081, Peopleꢀs Republic of China
Received: October 12, 2016; Revised: November 30, 2016; Published online: && &&, 0000
Supporting information for this article can be found under: http://dx.doi.org/10.1002/adsc.201601128.
Abstract: A new glycosyltransferase (UGT71E5)
from Carthamus tinctorius exhibited a robust pro-
miscuity towards 30 structurally diverse drug-like
aromatic amine scaffolds, making it the first report-
ed glycosyltransferase capable of catalyzing N-gly-
cosylation with multiple diverse nitrogen-heterocy-
clic aromatic compounds. The catalytic promiscuity
and reversibility of UGT71E5 was exploited to gen-
erate the rare N-glycoside from the abundant O-
glycoside with high efficiency. These findings dem-
onstrate the significant potential of UGT71E5 in
the enzymatic synthesis of diverse bioactive N-gly-
cosides.
active glycosylated products, as they can transfer
sugar moieties from donors to acceptors generating
O-, N-, S-, and C-glycosidic bonds.[3a,4] Significant
progress has been made in enzymatic O-glycosylation
of natural and unnatural products by GTs.[5] However,
enzymatic N-glycosylation still remains restricted by
enzyme specificity and the availability of suitable
GTs. In the past few years, several GTs with N-glyco-
sylation activity have been reported, such as an engi-
neered microbial GT OleD that exhibits N-glycosyla-
tion activity to some aromatic amines, hydrazines and
hydrazides.[5a] In addition, some GTs from plants also
possess N-glycosylation activity.[5f–h,6] However, the
majority of the known GTs exhibit relatively narrow
substrate spectra and few of them have been utilized
as tools in diverse N-glycosylations. Thus, mining for
novel GTs with catalytically promiscuous N-glycosyla-
tion capability is of significance, as it will allow the
construction of enzymatic methods for the synthesis
of bioactive N-glycosides. Here, we report a new GT
(UGT71E5) from Carthamus tinctorius that can toler-
Keywords: Carthamus tinctorius; catalytic promiscu-
ity; enzyme catalysis; glycosylation; glycosyltrans-
ferases; N-glycosylation
Sugar moieties are commonly found attached to bio- ate a number of structurally different drug-like aro-
logically active natural products and clinical drugs, matic amines to generate diverse N-glycosidic bonds.
which not only can contribute to the diversity of The catalytic reversibility of UGT71E5 in O-glycosy-
structures but also affect the physicochemical and bio- lation allows a one-pot reaction generating rare N-
logical properties of these glycosides.[1] The majority glycosides from abundantly available O-glycosides.
of the glycosides involve O-glycosidic bonds. By con-
In our previous work, we found a promiscuous GT,
trast, the N-glycosylation is relatively rare and exhib- UGT73AE1 from C. tinctorius, that demonstrates
its outstanding pharmacological activity especially in a wide substrate spectrum for O-glycosylation.[5f] In
some chemical drugs that have attracted considerable this work, we aimed to screen for a GT with catalytic
attention.[2] However, chemical N-glycosylation faces promiscuity in rare N-glycosylation to expand the ap-
challenges in regioselectivity, stereoselectivity, and the plication of GTs in diverse enzymatic glycosylations.
protection and de-protection of functional groups, To clone permissive GTs from C. tinctorius, a modified
which limit the exploitation of these rare glycosides in degenerate PCR primer for 5’ RACE was designed
drug discovery.[3] Glycosyltransferases (GTs) are gen- based on the conserved PSPG (plant secondary prod-
erally recognized as powerful synthetic tools for bio- uct glycosyltransferases) motif of six permissive plant
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GTs as shown in the Supporing Information, Fig- reactions lacking either enzyme or UDP-Glu con-
ure S1. Combined with 3’ RACE, 16 new different C. firmed that the reactions were dependent upon both
tinctorius glycosyltransferase genes (CtGTs) were suc- the enzyme and UDP-Glu. The glycosylated products
cessfully cloned by RT-PCR amplification using the (1a, 4a, 34a and 35a) were prepared from scaled-up
total RNA from florets of C. tinctorius as a template reactions and their structures, including anomeric ste-
and heterologously expressed in Escherichia coli.
reochemistry, were characterized by MS, 1H NMR,
For screening the N-glycosylation activity of and 13C NMR spectroscopic analyses (Supporting In-
CtGTs, the small molecules, 7-amino-4-methylcou- formation, Figures S42–S45 and S62–S65). The large
marin (1) and 3,4-dichloroaniline (4) were used as ac- anomeric proton-coupling constants (J=7.9, 8.4, 9.8
ceptors and uridine diphosphate glucose (UDP-Glu) and 9.6 Hz for 1a, 4a, 34a and 35a, respectively) indi-
was used as a sugar donor. The screening reactions cate the formation of the b-anomers and an inverting
(50 mM Tris-HCl, pH 7.4; 0.5 mM UDP-Glu; mechanism for CtGT22 (Supporting Information,
0.25 mM aglycone; 500 mg of crude CtGTs; 308C, 6 h) Table S4).
were analyzed by HPLC-UV/MS (high-performance
The cDNA sequence of CtGT22 (1443 bp, Gen-
liquid chromatography-UV absorption/mass spectrom- Bank accession number KX610759) contained an
etry). Of the 16 recombinant CtGTs, only CtGT22 ORF encoding 480 amino acids and showed the high-
showed glycosylation activity to both 1 and 4 with est identity (67%) to UGT71E1, a flavonoid GT from
high conversion rates (>90%) (Figure 1). Moreover, Stevia rebaudiana.[7] CtGT22 was further named
CtGT22 also showed high catalytic efficiency in S-gly- UGT71E5 according to the UGT Naming Commit-
cosylation with small molecules (34 and 35) and O- tee.[8] Recombinant His6-UGT71E5 was purified using
glycosylation with flavonoids (36–39) (see Figure 1 His-tag affinity chromatography and was analyzed
and the Supporting Information, S35–S38). Control with SDS-PAGE (Supporting Information, Figure S2).
Figure 1. High catalytic efficiency of UGT71E5 in N-, S- and O-glycosylation. N-Glycosylation: Structures of N-glycosylated
products and HPLC analysis of N-glycosylation catalyzed by UGT71E5. S-Glycosylation: Structures of S-glycosylated prod-
ucts and HPLC analysis of S-glycosylation catalyzed by UGT71E5. O-Glycosylation: Structures of flavonoids recognized by
UGT71E5. Percent conversions of acceptors are shown underneath the structures. N- and S-glycosylated products (1a, 4a,
34a and 35a) were isolated and confirmed by MS and 1H, 13C NMR.
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UGT71E5 displayed the maximum catalytic activity rings were substituted by nitrogen, the conversion
at pH 9.0 and 308C and was divalent cation independ- rates were lower. Introduction of nitrogen atoms into
ent (Supporting Information, Figure S3). UGT71E5 the aromatic ring may affect, but not completely de-
exhibited Km values of 274.8 mM, 76.6 mM and stroy, the aromatic properties of the acceptors.[10] Ad-
181.4 mM for 1 (N-glycosylation), 34 (S-glycosylation) ditionally, the heterocyclic ring might form a p–p in-
and 40 (O-glycosylation), respectively (Supporting In- teraction with an enzyme amino acid residue around
formation, Figure S4). Given the cDNA sequence ho- the active site and help to allocate the substrate in an
mology, the catalytic activity to flavonoids and the ex- appropriate orientation.[6a] Hence, UGT71E5 glycosy-
istence of various flavonoid glycosides in C. tinctorius, lated these N-heteroarylamine compounds, utilizing
UGT71E5 was tentatively hypothesized as a flavonoid its robust catalytic promiscuity. In total, 30 structural-
GT.
ly diverse unnatural compounds, including 14 (18–23,
To systematically explore the catalytic promiscuity 25–31 and 33) derivatives of nitrogen heterocycles,
and probe the synthetic utility of UGT71E5 in N-gly- were shown as acceptors of UGT71E5 to generate N-
cosylation, a series of structurally diverse representa- glycosides. This remarkable catalytic promiscuity for
tive drugs and drug-like compounds containing N- nitrogen heterocycles is uncommon for a plant GT.
based nucleophiles (amino/imino groups) was collect- To the best of our knowledge, this is the first reported
ed. Therefore, a compound library including deriva- GT capable of catalyzing N-glycosidic bond formation
tives of aniline (1–16), aminopyridine (18–21), amino- with such diverse nitrogen heterocyclic aromatic com-
pyridazine (22–24), aminopyrimidine (25–29), amino- pounds. This finding will expand the applications of
pyrazine (30), aminotriazine (31–33) and an aliphatic GTs in the field of enzymatic glycosylation.
amine (17) was constructed (Figure 2B and C). Nota-
In addition to N-glycosylation, UGT71E5 also ex-
bly, most of the library members are clinical anticarci- hibited S-glycosylation activity toward the SH func-
nogen (Lenakidomide, 2), antileprotic (diaminodi- tion of an nitrogen heterocyclic derivative (2-mercap-
phenyl sulfone, 5), anesthetic (Benzocaine, 9), choli- to-4-methylpyridine, 41), which has not been reported
nesterase inhibitor (Tacrine, 20), hypotensor (Prazo- for other GTs (Supporting Information, Figures S29
sin, 28), antibacterial synergist (Trimethoprim, 29) and S33). Moreover, a different pattern of S-glycosy-
and bioactive natural or un-natural products with sig- lation was observed when 2,4-dichlorobenzyl thiol
nificant pharmacological activity but poor solubility (42), which only possesses an aliphatic thiol, was used
and negative side effects. For example, 9-amino-camp- as an acceptor (Supporting Information, Figures S29
tothecin (7) is an inhibitor of topoisomerase I with and S34).
potent antitumor activity against a wide spectrum of
To further confirm the catalytic properties of
human tumors. However, its poor water solubility and UGT71E5 and the structures of the glycosylated
hematologic toxicity have limited its clinical applica- products, eight N-glucosylated (1a, 4a-8a, 10a and
tion and development.[9] Above all, 33 members with 20a) and three S-glucosylated (34a, 35a and 42a)
diverse structures were employed in the N-glycosylat- products were isolated from the preparative-scale re-
ed modification.
actions, four (6a, 7a, 10a and 20a) of which were
The unprecedented catalytic promiscuity of novel compounds (Figures 2 and the Supporting Infor-
UGT71E5 in N-glycosylation was observed from mation, S29). The structures were identified by mass
a first-pass analysis with HPLC-UV/MS, which re- and NMR spectroscopic analyses (Suppoorting Infor-
vealed that UGT71E5 was flexible enough to N-gly- mation, Figures S42–S67). For example, 1H and
cosylate sixteen (1–16) structurally different library 13C NMR, HSQC and HMBC were employed to con-
1
members (Figure 2A and B). Of particular note, most firm the structure of 7a. In the H NMR spectrum,
of the acceptors have large structural differences, in- the presence of the glucosyl moiety was suggested by
cluding varied skeletons. As N-heteroarylamine com- the characteristic signals for H-Glc1’ (dH =4.55, 1H)
pounds are common drug scaffolds, exploring and ex- and H-Glc2’-6’ (dH =3.16–3.72, 6H). The location of
panding the utility of UGT71E5 in the glycosylation the glucosyl moiety was indicated by the correlation
of heteroarylamines is significant. Thus, by focusing between H-Glc1’ and C-9 (dC =143.5) in the HMBC
on heteroarylamines, another 16 library members spectrum and the unchanged hydroxy proton signal of
1
(18–33), including derivatives of N1-, N2-, and N3-het- OH-20 (dH =6.52, 1H) in the H NMR spectrum. The
erocycles, were employed. UGT71E5 was sufficiently large coupling constant (J=8.0 Hz) of H-Glc1’ indicat-
flexible to N-glycosylate all the derivatives of amino- ed the b configuration of the glucosidic linkage. Thus,
pyridine (18–21) (Figure 2A and C). To our surprise, the glycosylated product of 9-amino-camptothecin (7)
derivatives of aminopyridazine (22, 23), aminopyrimi- was identified as 9-amino-camptothecin 9-N-b-d-glu-
dine (25–29), aminopyrazine (30), and aminotriazine coside (7a). The water solubility of the glycosylated
(31, 33) were also recognized by UGT71E5. The im- products was greatly increased while the pharmaco-
portance of aromatic properties is evidenced by the logical activity needs to be further investigated. For
fact that when more carbon atoms in the aromatic N-glycoside 7a, the water solubility increased nearly
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Figure 2. Catalytic promiscuity of UGT71E5 in N-glycosylation. (A) Percent conversions of N-glycosylated products cata-
lyzed by UGT71E5. The library numbers are listed based on the structural scaffolds shown in parts B and C. The black and
grey columns represent conversions of glucosides and galactosides, respectively. *=glycosylated products were isolated and
confirmed by MS and NMR; D=detected in MS; N=not detected. (B, C) The structures of the library members and corre-
sponding N-glycosylated products.
39-fold compared to the aglycone (Supporting Infor- but, however, has not been achieved due to the lack
mation, Table S3). The anomers of all the isolated of suitable GTs. Thus, based on the tri-functional fea-
products were in the b configuration, as evidenced by tures of UGT71E5 in N-, S-, and O- glycosylation in-
anomeric protons with large coupling constants (J> cluding the catalytic reversibility in O-glycosyla-
7.0 Hz) (Supporting Information, Table S4).
tion,[11,5g] model reactions were performed with 4-ni-
Given the current low diversity of N- and S-glyco- trophenyl b-d-glucopyranoside (43) as a sugar donor
sides, exploitation of universally applicable green and 7-amino-4-methylcoumarin (1) or 7-mercapto-4-
chemical approaches to generate rare N- and S-glyco- methylcoumarin (34) as an acceptor under the cataly-
sides from abundant O-glycosides is highly desired, sis of UGT71E5 (Figure 3). The desired N- and S-glu-
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form S-glycosidic bonds (Supporting Information, Fig-
ures S29, S31 and S32). The broad acceptor and
donor spectra make UGT71E5 a powerful enzymatic
tool in the synthesis of glycosides and combinatorial
application for the future drug discovery.
In summary, the astonishing catalytic diversity of
UGT71E5 in N-glycosylation was highlighted. As
a plant GT, UGT71E5 exhibited robust N-glycosyla-
tion to a series of drug and drug-like compounds. In
particular, the surprising glycosylation activity to
–NH2 and –NH– functions of nitrogen heterocyclic
derivatives makes UGT71E5 the first reported GT ca-
pable of catalyzing N-glycosylation with multiple di-
verse nitrogen heterocyclic compounds. In addition to
the acceptor diversity, UGT71E5 also showed an in-
teresting tolerance to sugar donors in N- and S-glyco-
sylation, which had not yet been reported in plant
GTs. Moreover, the remarkable enzymatic conversion
of O- into N- and S-glycosidic bonds may create new
opportunities for the synthesis of rare bioactive N-
Figure 3. Exploiting O-GT reversibility of UGT71E5 to gen-
erate N- and S-glucosides from a simple O-glucoside (43).
Conversions of 1a and 34a were 96% and 100%, respective-
ly. The HPLC analysis is shown in the Supporting Informa-
tion, Figures S39 and S40.
cosylated products (1a and 34a) were obtained with and S-glycosides. So far, GTs with N-glycosylation ac-
conversions of 96% and 100%, respectively. These tivity have not been well studied due to the lack of
high conversions occurred in the presence of only suitable models.[13] UGT71E5 might be an OGT in
a catalytic amount of UDP (1/10 of 43), which re- vivo while possessing potent N-glycosylation catalytic
vealed that UDP was under cyclic utilization through- activity in vitro, that is a good model for deciphering
out the coupled reactions. Therefore, O-deglycosyla- the promiscuity of plant GTs in vitro. Cumulatively,
tion and N- or S-glycosylation were successfully cou- the present study demonstrated, for the first time, the
pled together with UDP via UGT71E5. To investigate significant potential of a plant enzyme as a powerful
the substrate spectrum toward the NDP moieties, biocatalyst for the synthesis of rare bioactive natural
UGT71E5 was subsequently probed with 1 and four and unnatural glycosides.
other commercially available NDPs in one-pot reac-
tions. Interestingly, UGT71E5 displayed broad sub-
strate specificity, recognizing dTDP, ADP, CDP and Experimental Section
GDP, as well as their corresponding activated gluco-
ses. In those reactions, glucose was transferred from Materials
dTDP-, ADP-, CDP- and GDP-Glu donors at 50%
Chemicals and reagents were purchased from Sigma–Al-
that of UDP-Glu (Supporting Information, Fig-
drich (St. Louis, MO, USA), J&K Scientific Ltd. (Beijing,
ure S41). This feature allows UGT71E5 to be applied
China), InnoChem Science & Technology Co., Ltd. (Beijing,
in N-glycodiversification from O-glucosides in vivo
with different NDPs of host strains.[12] Above all, this
one-pot reaction mediated by UGT71E5 generating
N- and S-glycosides from abundant O-glycosides with-
out adding activated sugars establishes a cost-effective
enzymatic method for synthesizing diverse bioactive
N- and S-glycosides. To be noticed, UGT71E5, as
a tri-functional N-, S- and O-GT with catalytic pro-
China) and BioBioPha (Kunming, China).
Methods
UGT71E5 was amplified by PCR, and the gene fragment
was then inserted into plasmid pET-28a. The resultant plas-
mid was then transformed into E. coli (TransGen Biotech,
miscuity and high efficiency, can be applied to glyco- China) for heterologous expression. Recombinant
UGT71E5 was purified using His-tag affinity chromatogra-
phy. The analyses of enzymatic products were performed on
an Agilent 1200 series HPLC system (Agilent Technologies,
Germany) coupled with an LCQ Fleet ion trap mass spec-
trometer (Thermo Electron Corp., USA) equipped with an
electrospray ionization (ESI) source. HR-ESI-MS data were
measured with an Agilent Technologies 6520 Accurate Mass
Q-TOF LC/MS spectrometer. Compounds were character-
ized by 1H NMR at 400 or 600 MHz and 13C NMR at
150 MHz on Mercury-400 and Bruker AVIIIHD-600 spec-
diversification.
UGT71E5 also showed tolerance to sugar donors.
The commercially available sugar donors UDP-Glu,
UDP-galactose (UDP-Gal) and UDP-glucuronic acid
(UDP-GA) were employed to investigate the promis-
cuity of sugar donors. UGT71E5 was able to accept
UDP-Glu and UDP-Gal, generating N-glycosidic
bonds when 3,4-dichloroaniline (4) was used as an ac-
ceptor. For 3,4-dichlorobenzenethiol (35), UGT71E5
can recognize UDP-Glu, UDP-Gal, and UDP-GA to trometers.
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The detailed experimental procedures, including CtGTs
cloning, expression, reaction analysis and products purifica-
tion protocols, LC/MS, HRESIMS and NMR characteriza-
tion data and spectra of glucosylated products can be found
in the Supporting Information.
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Acknowledgements
This work was supported by the National Science Foundation
of China (Grant Nos. 21572277 and 81573317), CAMS Inno-
vation Fund for Medical Sciences (CIFMS-2016-I2M-3-012)
and Fundamental Research Funds for the Central Universi-
ties (2016ZX350040).
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COMMUNICATIONS
Enzymatic N-Glycosylation of Diverse Arylamine
Aglycones by a Promiscuous Glycosyltransferase from
Carthamus tinctorius
7
Adv. Synth. Catal. 2017, 359, 1 – 7
Kebo Xie, Ridao Chen, Dawei Chen, Jianhua Li,
Ruishan Wang, Lin Yang, Jungui Dai*
Adv. Synth. Catal. 0000, 000, 0 – 0
7
ꢁ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!