Chemoenzymatic Synthesis of Unnatural Nucleotide Sugars for Enzymatic Bioorthogonal Labeling

Article abstract of DOI:10.1021/acscatal.8b02081

In recent years, the development of the enzymatic bioorthogonal labeling strategy has offered exciting possibilities in the probing of structure-defined glycan epitopes. This strategy takes advantage of relaxed donor specificity and strict acceptor specificity of glycosyltransferases to label target glycan epitopes with bioorthogonal reactive groups carried by unnatural nucleotide sugars in vitro. The subsequent covalent conjugation by bioorthogonal chemical reactions with either fluorescent or affinity tags allows further visualization, quantification, or enrichment of target glycan epitopes. However, the application and development of the enzymatic labeling strategy have been hindered due to the limited availability of unnatural nucleotide sugars. Herein, a platform that combines chemical synthesis and enzymatic synthesis for the facile preparation of unnatural nucleotide sugars modified with diverse bioorthogonal reactive groups is described. By this platform, a total of 25 UDP-GlcNAc and UDP-GalNAc derivatives, including the most well explored bioorthogonal functional groups, were successfully synthesized. Furthermore, the potential application of these compounds for use in enzymatic bioorthogonal labeling reactions was also evaluated.

Full text of DOI:10.1021/acscatal.8b02081

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Article
Chemoenzymatic Synthesis of Unnatural Nucleotide
Sugars for Enzymatic Bioorthogonal Labeling
Liuqing Wen, Madhusudhan Reddy Gadi, Yuan Zheng, Christopher Gibbons,
Shukkoor Muhammed Kondengaden, Jiabin Zhang, and Peng George Wang
ACS Catal., Just Accepted Manuscript • Publication Date (Web): 12 Jul 2018
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Chemoenzymatic Synthesis of Unnatural Nucleotide Sugars for En-
zymatic Bioorthogonal Labeling
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Liuqing Wen,* Madhusudhan Reddy Gadi, Yuan Zheng, Christopher Gibbons, Shukkoor Mu-
hammed Kondengaden, Jiabin Zhang, Peng George Wang*
Department of Chemistry, Georgia State University, Atlanta, Georgia, 30303, United States
ABSTRACT: In recent years, the development of enzymatic bioorthogonal labeling strategy offers exciting possibilities in
the probing of structure-defined glycan epitopes. This strategy takes advantage of relaxed donor specificity and strict ac-
ceptor specificity of glycosyltransferases to label target glycan epitopes with bioorthogonal reactive groups carried by un-
natural nucleotide sugars in vitro. The subsequent covalent conjugation by bioorthogonal chemical reactions with either
fluorescent or affinity tags allows further visualization, quantification, or enrichment of target glycan epitopes. However,
the application and development of enzymatic labeling strategy have been hindered due to the limit availability of unnat-
ural nucleotide sugars. Herein, a platform that combines chemical synthesis and enzymatic synthesis for the facile prepa-
ration of unnatural nucleotide sugars modified with diverse bioorthogonal reactive groups is described. By this platform,
total 25 UDP-GlcNAc and UDP-GalNAc derivatives, including the most well-explored bioorthogonal functional groups,
were successfully synthesized. Furthermore, the potential application of these compounds for use in enzymatic
bioorthogonal
labeling
reactions
was
also
evaluated.
KEYWORDS: unnatural nucleotide sugars; bioorthogonal chemistry; chemoenzymatic labeling; chemoenzymatic synthesis;
glycan biomarker; glycan epitopes
INTRODUCTION
In recent years, the development bioorthogonal report-
er strategy has emerged as a powerful tool for glycan
analysis.16 Typically, bioorthogonal functional groups car-
ried by unnatural monosaccharides analogues are meta-
bolically incorporated into glycans in vivo, allowing for
further covalent conjugation by bioorthogonal chemical
reactions to tag a specific class of glycans (Figure 1).17,18
The metabolic labeling strategy is based on a covalent
modification of target glycans, and therefore, has much
better sensitivity than traditional methods such as anti-
body and lectin binding. The introduction of high-affinity
tag such as biotin group in second reaction step makes it
Glycans are involved in a variety of physiological and
pathological processes such as protein folding and degra-
dation, immune responses, cell growth and proliferation,
cell-cell
communications,
and
cell-pathogen
interactions.1-3 It is well established that aberrant glyco-
sylation in the glycan structure and site occupancy is a
hallmark of human cancer and many other diseases.4-8
Unlike like DNA transcription and protein translation in
living cells, glycan assembly is not a template-driven pro-
cess. Instead, glycans are installed onto proteins or lipids
through a stepwise, enzyme-catalyzed process. The size
and structural complexity of glycans have posed great
challenges in glycan detection. Traditionally, chemical
modification and affinity binding by antibody or lectin
have been the primary methods used for glycan analysis.9-
possible
to
globally
profile
glycan-attached
glycoproteins.19,20 Currently, the most popular two
bioorthogonal chemistry groups are azide and alkyne. In
addition, many other bioorthogonal groups such as al-
kene, ketone, nitrile, diazirine have also been explored for
use in metabolic labeling.18 These fundamental studies
have made metabolic labeling strategy very popular in
glycan analysis. However, higher order glycans that con-
tain disaccharide or trisaccharide motifs cannot be
uniquely detected by metabolic labeling method. Moreo-
ver, unnatural monosaccharides need to compete with
natural substrates while being incorporated into glycans
through biosynthetic pathways, resulting in lower label-
ing efficiencies.
12
However, chemical modification of glycans is normally
destructive. For example, periodic acid, which is widely
used for sialic acid analysis, can cause permanent damage
to the treated glycans,13 preventing downstream analysis.
Alternatively, antibody and lectin binding based on non-
covalent interaction is a mild and non-destructive meth-
od. To date, large numbers of lectins and antibodies have
been explored as tools for the detection of glycan
epitopes.14,15 Nevertheless, antibodies and lectins suffer
many well-document drawbacks such as low affinity and
cross-reactivity.
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Alternatively, enzymatic bioorthogonal labeling, also
labeling strategy, and enzymatic bioorthogonal labeling
strategy.
known as chemoenzymatic labeling strategy, provides an
in vitro labeling method to detect structure-defined gly-
can epitopes.21 This method takes advantage of the sub-
strate tolerance of recombinant glycosyltransferase to
label target epitope with bioorthogonal groups carried by
unnatural nucleotide sugars (Figure 1).22-26 As the glyco-
syltransferase-mediated reactions and the following
bioorthogonal chemical reactions both proceed with high
efficiency and specificity, enzymatic bioorthogonal label-
ing method provides higher sensitivity and selectivity
than other analytical methods. More importantly, enzy-
matic labeling
doesn’t rely on feeding with metabolic substrates and
therefore could be used to label complex samples such as
tissue extracts in vitro.27
To date, the enzymatic bioorthogonal labeling strategy
has not proven to be as popular as metabolic labeling for
use in glycan analysis. The most important reason is the
limited availability of the unnatural nucleotide sugars
which functionalized with biorthogonal reactive groups.
This has hindered the application and future develop-
ment of enzymatic bioorthogonal labeling strategy. To
address this issue, we describe herein a platform that
combines chemical synthesis and enzymatic synthesis to
produce unnatural nucleotide sugars modified with
bioorthogonal groups. With these unnatural nucleotide
sugars in hand, we also evaluated their potentials for use
in enzymatic bioorthogonal labeling reactions.
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RESULTS AND DISCUSSION
Design of unnatural nucleotide sugars for glycan
analysis by enzymatic bioorthogonal labeling. N-
Acetylgalactosaminyltransferase,
N-
acetylglucosaminyltransferase, and sialyltransferase are
the most popular three classes of enzymes that used in
enzymatic labeling strategy. CMP-Neu5Ac analogues can
be prepared from ManAc or Neu5Ac analogues by using
sialic acid aldolase from E. coli K12 and CMP-sialic acid
synthetase from Neisseria meningitis, both of which have
extremely relaxed substrate specificity.28 However, the
synthesis of UDP-GalNAc and UDP-GlcNAc derivatives
are more difficult. Now only several azido nucleotide sug-
ars are available (2, 3, 17, and 18 in Figure 2) for enzymat-
ic labeling. In this work, we design to synthesize 12 UDP-
GlcNAc analogues and 13 UDP-GalNAc analogues.
Figure 1. Three common strategies used for glycan analy-
sis: affinity binding strategy, metabolic bioorthogonal
Figure 2. Structures of the designed UDP-GlcNAc and UDP-GalNAc derivatives.
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scale synthesis as GlmU has a much higher expression
level than AGX1 in E.coli system. Preparative-scale syn-
thesis (more than a hundred milligrams) was carried by
one-pot multienzyme systems containing monosaccha-
ride analogues (S4 to S13), NahK and GlmU. Inorganic
pyrophosphatase PPA was added in reaction to hydrolyze
the newly formed inorganic pyrophosphate to improve
the overall yields. Products 4, 8, 9, 12 and 13 were purified
by silver nitrate precipitation method.39,40 Silver nitrate
precipitation is a newly developed method used for the
purification of phosphorylated sugars, by which sugar
adenosines (ATP and ADP) can be cleanly precipitated
out of aqueous solution while sugar phosphates (with five
or more than five carbons) are still in solution.39,40 This
method has widely been used for sugar phosphate purifi-
cation.41-43 We noticed that silver nitrate could also clean-
ly precipitate other phosphorylated nucleotides such as
UTP and UDP in aqueous solution, while sugar nucleo-
tides with one or two internal phosphate groups (UDP-
sugars, GDP-sugars, and CMP-sugars) were not affected.
Therefore, once the above-mentioned reactions finished,
excess silver nitrate was added to precipitate ATP, ADP,
and UTP. Then, sodium chloride was added to precipitate
the redundant silver nitrate. This process can be finished
in less than 10 minutes. Then, the final products can be
obtained after desalting by P-2 column with high purity.
It is worth mentioning 13 is sensitive to light as it con-
tains a diazirine group. The synthetic manipulations re-
quire protection from light. Meanwhile, compounds 5, 6,
7 and 10 can’t be well obtained by silver nitrate precipita-
tion method as they have alkyne group, which can be pre-
cipitated by silver ions to a certain extent. Therefore, for
the purification of compounds 5, 6, 7 and 10, alkaline
phosphatase was added to digest ATP, ADP, and UTP to
facilitate further purification by gel filtration. The struc-
tures of the isolated products were confirmed by NMR
and MS analysis (see supporting information). 4, 5, 9 and
12 were obtained in more than 80% isolated yield (Table
1), while the rest derivatives were obtained in relatively
lower isolated yields, due to limit efficiency of the second
reaction step (a high amount of intermediate phosphory-
lated sugars was observed on TLC). Prolonging the reac-
tion time or adding more enzyme was not shown to im-
prove the reaction yield.
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4
5
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8
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These analogues include most of known bioorthogonal
reporter groups that used in metabolic labeling strategy.
These reactive groups can be probed by well-established
methods such as copper-catalyzed azide-alkyne cycload-
dition (azide and alkyne groups),29 strain promoted al-
kyne-azide cycloaddition (azide group),29 Staudinger-
Bertozzi ligation (ketone group; 9 and 25),30 Pd-catalyzed
bioorthogonal elimination reaction (10, 11, 26, and 27),31
Diels-Alder reaction (alkene group; 8, 11, 24, and 27 ),32
Raman reporter strategy (nitrile group; 12 and 18),33 pho-
toactivatable crosslinking (diazirine group; 13 and 29),34
one-step labeling strategy (biotin group; 14, 15, 30 and
31).35,36
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Chemoenzymatic synthesis of UDP-GlcNAc deriva-
tives functionalized with bioorthogonal reactive
groups.
Monosaccharide
analogues
containing
bioorthogonal reactive groups shown in Figure 1 were
prepared as described in supporting information. Scheme
1 is a common strategy to prepare UDP-sugars. A prereq-
uisite of this strategy is the availability of a kinase that
can phosphorylate monosaccharide to sugar-1-phosphate.
NahK (N-Acetylhexosamine 1-Kinase) from Bifidobacte-
rium is a kinase that used to phosphorylate GlcNAc and
GalNAc and their derivatives.37 To test whether NahK can
recognize our analogues, His-tagged NahK was prepared
using E.coli expression system and purified by Ni-
NTA agarose. Analytical reactions containing monosac-
charide analogues, NahK and ATP, were carried in 20 ul
reaction system. The reaction was analyzed by both HPLC
and TLC. Substrate specificity study showed that all these
analogues can be efficiently accepted by NahK (Table S1).
Some monosaccharide analogues were found to have
higher relative activities than their natural substrates
GlcNAc or GalNAc (Table S1). Indeed, all the tested mon-
osaccharides can be clearly converted to corresponding
phosphorylated forms after overnight incubation, indicat-
ing the excellent feasibility of NahK for large-scale syn-
thesis of the designed compounds.
Scheme 1. Chemoenzymatic synthesis of UDP-GlcNAc
derivatives 4 to 13
To prepare UDP-GlcNAc derivatives by scheme 1, we
first tested the activity of UDP-N-acetylgalactosamine
pyrophosphorylase from human (AGX1) and pyrophos-
phorylase from Escherichia coli (GlmU).38 Analytical reac-
tion was performed in a 20 ul system containing mono-
saccharide analogues, ATP, UTP, NahK and pyrophos-
phorylase (AGX1 or GlmU). The reaction was analyzed by
both TLC and HPLC. We found AGX1 only tolerate short-
er modification group (4, 5 and 13), while GlmU is active
to all derivatives (4 to 13). We choose GlmU for large-
Scheme 2. Chemoenzymatic synthesis of 10 and 11
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Scheme 2 is an alternative strategy to prepare 10 and 11.
GlmU has been reported as suitable for the preparation of
some UDP-GalNAc derivatives such as 17 and 18,38 it is
unable to recognize our designed structures in Figure 1.
Other pyrophosphorylases from Pyrococcus furiosus DSM
3638 and Bifidobacterium longum44,45 were also tested to
try to synthesize the rest derivatives. However, no activity
was observed. Preparative-scale synthesis (more two hun-
dred milligrams) was carried by one-pot multienzyme
systems containing monosaccharide analogues (S19, S21,
or S28), NahK and AGX1. Once reaction finished, silver
nitrate precipitation was used to purify 19 and 28 as deꢀ
scribed above. 21 was purified by the treatment of the re-
action solution with alkaline phosphatase. After desalting
by P-2 column, products were obtained in high yields
(Table 1). The structures were confirmed by NMR and MS
analysis (see supporting information).
UDP-GlcNTFA was firstly prepared from GlcNTFA by
NahK and AGX1. This step gave excellent yield (92 %).
Then, the treatment of 0.1 M of sodium hydrate gave a
clean conversion of UDP-GlcNTFA to UDP-GlcNH2 in less
than two hours. UDP-GlcNH2 was dissolved in water con-
taining sodium bicarbonate, and then propargyl chlo-
roformate, which selectively reacts with amine group, was
added slowly to give 10 in quantitative yield. After purifi-
cation by P-2 column, 10 was obtained in an overall yield
of 76% with regards to GlcNTFA. Similarly, incubation of
UDP-GlcNH2 with allyl chloroformate gave 11 in total 79%
yield. Although more reaction steps are required by
Scheme 2 for the synthesis of 10 and 11, the total isolated
yields are still higher than the use of Scheme 1 for the
preparation of 10 and 11.
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Scheme 4. Chemoenzymatic synthesis of UDP-GalNAc
derivatives 19, 21, and 28
In addition to the preparation of 19, 21 and 28 by
Scheme 4, 26 and 27 were prepared from GalTFA by
Scheme 5 as the synthesis of 10 and 11 by Scheme 2. 30
and 31 were prepared from 17 and 18 by CuAAC ligation,
respectively (Scheme 6). 17 and 18 were prepared from
GlcNAz and 6-N3-GlcNAc as reported previously.46 The
products 26, 27, 30 and 31 were obtained in high yields
(Table 1) after purification by P-2 column. The structures
were confirmed by NMR and MS analysis (see supporting
information).
Scheme 3. Chemoenzymatic synthesis of 14 and 15
Scheme 3 was used to prepare 14 and 15 from 2 and 3,
respectively. 14 and 15 were conjugated with biotin and
can be used for one-step labeling strategy.36 One-step
labeling, whereby a reporter group such as biotin or fluo-
rescence group is directly conjugated with an activated
sugar donor for enzymatic labeling, provides not only
higher labeling efficiency than the typical two-step label-
ing but also avoids additional chemical reaction steps
needed to introduce a reporter group after the labeling
reaction.35,36 Therefore, it is a more attractive labeling
strategy. 2 and 3 were prepared by scheme 1 using NhaK
and AGX1 first.38 Then, 2 and 33 were dissolved in water
containing 50% of methanol. Ascorbate, TBTA, and CusO4
were added to initiate the CuAAC ligation. The reaction
went to completion in less than 30 minutes with quantita-
tive yield. Product 14 was obtained after P-2 column puri-
fication in 82% yield regarding to 2. Similarly, 15 was pre-
pared from 3 and 33 in 81% yield regarding to 3. The
structures of 14 and 15 were confirmed by NMR analysis
(see supporting information).
Scheme 5. Chemoenzymatic synthesis of 26 and 27
Chemoenzymatic synthesis of UDP-GalNAc deriva-
tives modified with bioorthogonal reactive groups.
To prepare UDP-GalNAc derivatives by scheme 4, we first
tested the activity of AGX1 and GlmU at analytical scales
as mentioned above. However, only the compounds with
short modifications can be prepared by AGX1 (19, 21, and
28). The rest either can’t be prepared by AGX1 at all or
only show trace activity (synthetically useless). Although
Scheme 6. Chemoenzymatic synthesis of 30 and 31
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aCompound structures of 2, 3, 17, and 18 were shown in Fig-
1
2
3
4
5
6
7
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ure 2, and the rest compound structures were shown in Fig-
ure S1. bThe pyrophosphorylase used in second reaction step
c
can be either AGX1 or GlmU. The pyrophosphorylase used
d
in second reaction step can only be GlmU. The pyrophos-
Scheme 7. Chemoenzymatic synthesis of UDP-GalNAc
derivatives 20, 22, 23, 24, 25 and 29
phorylase used in second reaction step can only be AGX1.
eAll products were prepared on a preparative (134 mg-306 mg)
scale.
For nucleotide sugars that cannot be accessed by en-
zymatic methods, chemical synthesis is the only means to
produce. The most difficult step in the chemical synthesis
of nucleotide sugars is to produce sugar phosphate with α
or β configuration selectively, which requires complicated
protection/deprotection manipulations. Since all the
planned monosaccharide analogues can be efficiently
phosphorylated by NahK, which produces sugar-α-1-
phosphate exclusively, we design Scheme 7 to produce
the rest designed sugar nucleotides (20, 22, 23, 24, 25, and
29). Monosaccharide analogues were incubated with
NahK and ATP. Upon reaction completion (no starting
material was observed), the silver nitrate precipitation
method was
employed to purify the phosphorylated sugars (S30 to
S35). After desalting by P-2 column, the obtained phos-
phorylated sugars were confirmed by NMR and MS analy-
sis (see supporting information). In second reaction step,
sugar-1-phosphates (S30 to S35) were incubated with
UMP morpholodate, which is commercially available, and
tetrazole in pyridine solution for 3 to 5 days to produce
UDP-sugars.47 Once the reaction ceased to move forward,
the pyridine was removed under vacuum, and the prod-
ucts were dissolved in sodium chloride solution. After
purification by P-2 column, products were obtained with
yields ranging from 50% to 63% (Table 1). The synthetic
manipulations of 29 require protection from light as it has
a diazirine group. The products were confirmed by NMR
and MS analysis (see supporting information).
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Table 1. Chemoenzymatic synthesis of UDP-GlcNAc and
UDP-GalNAc derivatives using the strategies shown in
schemes 1 to 7
Evaluation of the use of UDP-GlcNAc and UDP-
GalNAc derivatives in enzymatic bioorthogonal la-
beling reactions. Having these UDP-GlcNAc and UDP-
GalNAc derivatives in hand, we further tested their poten-
tial applications in enzymatic bioorthogonal labeling re-
actions.21 Figure 3 includes several known glycan epitopes
that can be detected by enzymatic bioorthogonal labeling
strategy. B3GNT6 and GCNT1 were used to analyze Tn
antigen and T-antigen with UDP-GlcNAz, respectively.48
BgtA, CgtA, and Bovine GalT (Y289L) were used to label
the Fucα1,2-Gal epitope, Neu5Acα2,3-Gal epitope, and O-
GlcNAc-modified proteins with UDP-GalNAz, respective-
ly.21 Tn antigen and T antigen are famous Mucin-type O-
glycans, which have no expression on normal cells but
highly expressed on many cancer cells.49,50 They have
shown great potential for application in immunotherapy
and clinical diagnosis. Fucα1,2-Gal and Neu5Acα2,3-Gal
are complex glycan epitopes that expressed normally on
the cell surface. However, both expression levels can sig-
nificantly change on many cancer cell lines.51 In addition,
they also play important roles in many other biological
processes such as cell-cell and cell-pathology
interaction.52,53 O-GlcNAc post modification is an im-
portant monosaccharide modification on proteins in mul-
ticellular eukaryotes, involving in protein degradation,
nutrient sensing, gene expression, and other essential cell
processes.54
Entry
Starting
Schemes
Product
Yielde
materiala
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a
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S1
S2
Scheme 1b
Scheme 1b
Scheme 1c
Scheme 1c
Scheme 1c
Scheme 1c
Scheme 1c
Scheme 1c
Scheme 2
Scheme 2
Scheme 1c
Scheme 1b
Scheme 3
Scheme 3
Scheme 4d
Scheme 7
Scheme 4d
Scheme 7
Scheme 7
Scheme 7
Scheme 7
Scheme 5
Scheme 5
Scheme 4d
Scheme 7
Scheme 6
Scheme 6
4
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S3
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S4
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g1
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k
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n
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p
q
r
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S17
S18
S19
S20
S21
S22
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S24
S25
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<1
<1
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<1
<1
<1
<1
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<1
athe activity with natural substrate UDP-GalNAc 1 was
defined as 100. The reaction was carried at 37°C for 30
minutes. See supporting information for experiment de-
tails.
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BgtA
is
a
bacterial
α1,3-N-
acetylgalactosaminyltransferase from Helicobacter muste-
lae, which specifically recognize Fucα1,2-Gal epitope.24
We tested the donor substrate specificity of BgtA with
Fucα1,2-Gal-β1,4-Glc trisaccharide24 and UDP-GalNAc
derivatives (17 to 31). In addition to the known substrates
UDP-GalNAc and UDP-GalNAz, only a few derivatives
can be accepted by BgtA with relatively lower activity
(Table 3). Therefore, we tested another α1,3-N-
acetylgalactosaminyltransferase GTA from humans,56
which also recognizes Fucα1,2-Gal epitope specifically.
Interestingly, we found GTA has a more relaxed donor
specificity than BgtA (Table 3). Many UDP-GalNAc deriv-
atives 17, 19, 20, 21, 24, 28 and 29 can be efficiently ac-
cepted by GTA, indicating its utility for versatile labeling
of Fucα1,2-Gal glycan epitope (further test is undergoing
in our lab). More importantly, GTA has much better
Figure 3. Enzymatic bioorthogonal labeling of Tn antigen, T
antigen, Neu5Acα2,3-Gal, and Fuca1,2-Gal, and O-GlcNAc
with UDP-GlcNAz and UDP-GalNAz.
Figure 4. Donor specificity study of B3GNT6, GCNT1, BgtA,
GTA, CgtA and Bovine GalT with standard oligosaccharides
or monosaccharide.
Table 3. Donor specificity study of BgtA, GTA, CgtA and
GalT with UDP-GalNAc (16) and UDP-GalNAc derivatives
(17 to 31)
Therefore, diverse probes (unnatural nucleotide sugars
functionalized with bioorthogonal reactive groups) are
important tools for the investigation of the detail roles in
a variety of physiological and pathological processes.
Donor
16
BgtA
100a
84
<1
12
7
GTA
100a
120
7
CgtA
100a
120
3
GalT
100a
75
<1
12
8
17
18
Human B3GNT6 and GCNT 1 were expressed in bacu-
lovirus insect cells. To study the donor specificity of hu-
man B3GNT6, GalNAc-O-Bn55 was incubated with UDP-
GlcNAc derivatives (2 to 15) in the presence of Mn2+, Tris-
HCl, and B3GNT6 (Figure 4). The reaction was analyzed
by HPLC. As shown in Table 2, we found some derivatives
such as 2, 4, and 5 even have better activity than its natu-
ral substrate UDP-GlcNAc 1, indicating their potential use
in the diverse labeling of Tn antigen. Similarly, donor
specificity of GCNT1 was tested with Galβ1,3-GalNAc-O-
Bn55 (Figure 4) and UDP-GlcNAc derivatives (2 to 15). The
results indicated that GCNT1 less tolerant of modifica-
tions like B3GNT6 (Table 2). Nevertheless, some new sub-
strates such as 3, 4, 5, and 12 can still be accepted by
GCNT1.
19
103
78
53
19
4
38
22
29
21
1
20
21
10
7
14
10
4
22
23
<1
4
24
85
14
9
8
5
25
6
4
15
3
26
86
59
43
1
<1
<1
15
2
27
11
74
39
3
3
28
40
2
29
30
2
<1
<1
Table 2. Donor specificity study of B3GNT6 and GCNT1
with UDP-GlcNAc (1) and UDP-GlcNAc derivatives (2 to
15)
31
1
1
<1
<1
athe activity with natural substrate UDP-GalNAc was de-
fined as 100. The reaction was carried at 37°C for 30
minutes (BgtA, GTA and CgtA tests). For GalT test, the
reaction was carried at 20°C for 2 hours. See supporting
information for experiment details.
Donor
B3GNT6 GCNT1 Donor
B3GNT6 GCNT1
1
2
3
100a
216
3
100a
9
2
<1
25
10
11
17
8
<1
<1
26
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expression level than BgtA in E.coli system (~50 mg pro-
tein can be obtained from 1 L culture, while only ~5 mg of
BgtA can be obtained as the same expression condition).
Experimental details, Supporting Figures, Supporting
Table, 1H NMR and 13C NMR spectra,
AUTHOR INFORMATION
Corresponding Authors
CgtA
is
a
β1,4-N-acetylgalactosaminyltransferase
jejuni, which recognizes
from Campylobacter
Neu5Acα2,3-Gal epitope.57 We found it accept UDP-
GalNAz as substrate and developed a chemoenzymatic
labeling tool to detect cell surface Neu5Acα2,3-Gal gly-
cans.22 CgtA donor specificity was tested by Neu5Acα2,3-
Gal-β1,4-Glc trisaccharide22 and UDP-GalNAc derivatives
17 to 31. The results indicated that UDP-GalNAz is the
best substrate of CgtA, while other substrates such as 19,
21, 26, 27, and 28 can also be well recognized by CgtA,
also indicating their potentials for versatile labeling of
Neu5Acα2,3-Gal glycan epitope. Bovine GalT Y289L is a
β1,4-galactosyltransferase, which catalyzes the transfer of
galactose from UDP-Gal to GlcNAc.58 It can also tolerate
substitutions at C-2 position such as UDP-GalNAc and
UDP-GalNAz for use in O-GlcNAc protein labeling.26,27
We tested donor specificity of Bovine GalT Y289L using
GlcNAc and UDP-GalNAc derivatives 17 to 31. However,
in addition to UDP-GalNAz, only compound 28 can be
efficiently recognized by Bovine GalT, indicating its strict
donor specificity with UDP-GalNAc derivatives. In addi-
tion, we found neither N-acetylglucosaminyltransferase
nor N-acetylgalactosaminyltransferase studied in this
work can efficiently recognize probes 14, 15, 30, and 31,
which can be used in one-step labeling strategies. This is
because the triazole-linked biotin group is too large to be
accepted by these enzymes. Protein engineering to allow
for a larger donor may be a potential solution, one that
may well be worth pursuing given the benefits of one-step
labeling in glycan analysis.
*(P.G.W) Email: pwang11@gsu.edu.
*(L.W) Email: lwen2@gsu.edu.
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Peng George Wang: 0000ꢀ0003ꢀ3335ꢀ6794
Liuqing Wen: 0000ꢀ0001ꢀ9187ꢀ7999
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank the National Institutes of Health (U01GM116263)
for financial support of this work. We thank Dr. Donald L.
Jarvis from University of Wyoming for providing the bacu-
lovirus vector used to produce B3GNT6 and GCNT1 for this
study. This vector was produced as part of the glycoreposito-
ry project, which was supported by NIH P41GM103390.
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In summary, we have developed a platform for the effi-
cient preparation of unnatural nucleotide sugars func-
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combing of chemical and enzymatic synthesis. A total
number of 25 UDP-GlcNAc and UDP-GalNAc derivatives
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ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge on
the at DOI
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