Chemo-enzymatic approach to access diastereopure α-substituted GlcNAc derivatives

Article abstract of DOI:10.1080/07328303.2017.1321116

The formation of diastereopure α-substituted GlcNAc derivatives in a simple and straightforward way is a challenging task. Herein, we report the chemical synthesis of diastereomeric α/β-substituted GlcNAc derivatives under non-anhydrous atmosphere using u

Full text of DOI:10.1080/07328303.2017.1321116

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: http://www.tandfonline.com/loi/lcar20
Chemo-enzymatic approach to access
diastereopure α-substituted GlcNAc derivatives
Su-Yan Wang, Pedro Laborda, Ai-Min Lu, Meng Wang, Xu-Chu Duan, Li Liu &
Josef Voglmeir
To cite this article: Su-Yan Wang, Pedro Laborda, Ai-Min Lu, Meng Wang, Xu-Chu Duan, Li Liu &
Josef Voglmeir (2017): Chemo-enzymatic approach to access diastereopure α-substituted GlcNAc
derivatives, Journal of Carbohydrate Chemistry, DOI: 10.1080/07328303.2017.1321116
To link to this article: http://dx.doi.org/10.1080/07328303.2017.1321116
Published online: 05 Jun 2017.
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Date: 07 June 2017, At: 20:22

JOURNAL OF CARBOHYDRATE CHEMISTRY
, VOL. , NO. , –
https://doi.org/./..
Chemo-enzymatic approach to access diastereopure
α-substituted GlcNAc derivatives
a,c
Su-Yan Wanga, Pedro Labordaa, Ai-Min Lub, Meng Wanga, Xu-Chu Duana, Li Liu
and Josef Voglmeir
,
a
aGlycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and
Technology, Nanjing Agricultural University, Nanjing, People’s Republic of China; bCollege of Sciences,
Nanjing Agricultural University, Nanjing, People’s Republic of China; cQlyco Ltd., Nanjing, People’s
Republic of China
ABSTRACT
ARTICLE HISTORY
Received  February 
Accepted  April 
The formation of diastereopure α-substituted GlcNAc derivatives
in a simple and straightforward way is a challenging task.
Herein, we report the chemical synthesis of diastereomeric
α/β-substituted GlcNAc derivatives under non-anhydrous atmo-
sphere using unprotected GlcNAc, followed by a selective enzy-
matic hydrolysis step using a bacterial N-acetyl-hexosaminidase
to provide only α-substituted GlcNAc. This enzyme proved
to selectively hydrolyze the β-anomeric GlcNAc derivatives,
while the α-anomeric GlcNAc derivatives remained unreacted.
The released GlcNAc (and therefore the α/β ratios) could be
quantified using a coupled enzymatic assay consisting of GlcNAc
2-epimerase and N-acetyl- mannosamine dehydrogenase in a
simple and accurate way.
KEYWORDS
Anomeric substitution;
GlcNAc derivatives;
N-acetyl-hexosaminidase;
O-substituted GlcNAc
quantification
GRAPHICAL ABSTRACT
Introduction
Among O-linked glycosides, beta-linked N-acetylglucosaminyl is a commonly
found motif in posttranslational modifications of proteins.[1,2] However, little is
known about the role of α-linked GlcNAc modification of proteins or other aglycons
in nature. Even though some examples have been found in lower eukaryotes,[3,4] no
experimental data on the occurrence of this moiety in higher eukaryotes were found
so far. A recent discovery on human polypeptide-GalNAc transferases suggests that
CONTACT Li Liu
liu.lichen@qlyco.com; Josef Voglmeir
and Technology, Nanjing Agricultural University, No. Weigang, Nanjing , China.
josef.voglmeir@njau.edu.cn
College of Food Science
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lcar.
©  Taylor & Francis Group, LLC

2
S.-Y. WANG ET AL.
Scheme. ReactionA:traditionalglycosylationreactionusingprotectedGlcNAcasthestartingmate-
rial; Reaction B: glycosylation reactions using unprotected GlcNAc as the starting material.
α-linked GlcNAc to serine or threonine may also be a naturally occurring motif in
humans.[5] In addition, several studies reported that mucosal glycans contain termi-
nal α-GlcNAc moieties,[6,7] which were shown to be of interest as therapeutic agents
to cure Helicobacter pylori infections.[8]
Most reported synthetic schemes to obtain anomerically modified GlcNAc
derivatives are based on the complete protection of non-anomeric hydroxyl
groups.[9–16] Thus, a suitable leaving group, usually a halogen or an acetyl group,
is fixed at the anomeric center of the pyranose ring (Sch. 1, reaction A). After the
successful glycosylation and removal of the protecting groups, the resulting anomer-
ically substituted GlcNAc commonly consists mainly of the β-anomer. To increase
the proportion of the α-anomer, several efforts have been made to improve the
reactivity of the hydroxyl group at the anomeric center of unprotected GlcNAc (1)
(Sch. 1, reaction B).[11,17–20] Typically, unprotected glycosylation reactions are car-
ried out in strongly acidic media or in the presence of a Lewis acid at elevated tem-
peratures thus allowing the synthesis of the GlcNAc derivative as a diastereomeric
α/β-mixture.
Considerable progress in the development of regioselective glycosylation strate-
gies has been achieved in recent years. However, the direct synthesis of anomerically
substituted GlcNAc derivatives from unprotected GlcNAc remains a challenging
task, due to self-condensation reactions and the requirement of strict anhydrous
reaction conditions.[21] In this work, α/β-substituted GlcNAc derivatives are syn-
thesized under non-anhydrous conditions, and β-substituted GlcNAc derivatives
were selectively hydrolyzed allowing the facile isolation of the α-substituted GlcNAc
derivatives. The released GlcNAc could be subsequently used to quantify the α/β
ratios of anomerically substituted GlcNAc.
Results and discussion
Synthesis of anomerically substituted GlcNAc derivatives
Several synthetic routes to anomerically substituted GlcNAc derivatives using
unprotected glycosyl donors have been reported to date. Matsumura and co-workers
reported the glycosylation of 1 with methanol in the presence of 2% HCl;[19]

JOURNAL OF CARBOHYDRATE CHEMISTRY
3
Scheme . Glycosylation of unprotected GlcNAc (1) under non-anhydrous conditions at room tem-
perature. BF·EtO was used as the activator; methanol (a), ethanol (b), -propanol (c), n-butanol (d),
and benzyl alcohol (e) were used as the glycosyl acceptors; acetonitrile (AcCN) was used as solvent.
GlcNAc derivatives were obtained as an α(2a–e)/β(3a–e) mixture.
Ferreires et al. reported the ultrasound-assisted glycosylation of 1 with n-octanol
under anhydrous conditions obtaining predominantly the α-anomer with good
regioselectivity.[22] Wu and co-workers used BF3·Et2O and allyl alcohol obtaining
71% of the α-anomer.[23] Roy et al. glycosylated 1 with 2-chloroethanol in the pres-
ence of acetic acid.[24] Mahal et al. used 2-azidoethanol and azidotriethylene glycol
as acceptors for the direct glycosylation of 1 in the presence of anhydrous Dowex 50
resin to provide only the α anomer.[25] Gao et al. reported glycosylation of 1 with
methanol in presence of Amerlite IR-120 (H).[26] Roy et al. synthesized α-propargyl
GlcNAc employing H2SO4-silica as a catalyst at 65°C.[27] Babic et al. obtained 1
using Fisher glycosylation with benzyl alcohol in the presence of p-toluenesulfonic
acid monohydrate and benzene.[28] Shaikh and co-workers applied ultrasound-
assisted glycosylation between 1 and propargyl alcohol or 2-azidoethanol in the
presence of H2SO4-silica at 40°C.[13] Another approach by Polanki et al. described
the synthesis of α-glycosylation products in high proportion using solvent-free con-
ditions and bismuth nitrate as a catalyst.[18]
Both solvents and the catalysts have been demonstrated to be key elements
in the chemical glycosylation of unprotected glycosyl donors.[22] In order to
avoid self-condensation of unprotected GlcNAc, tetrahydrofuran, 1,4-dioxane,
dichloromethane, and acetonitrile, in which 1 can be slightly dissolved, were
evaluated in preliminary experiments as solvents; glycosylation of 1 under non-
anhydrous conditions could only be detected when acetonitrile was employed
(Sch. 2).
The Lewis acids FeCl3, ZnCl2, and BF3·Et2O were evaluated as glycosylation acti-
vators, with only the latter yielding glycosylation products. This result is in agree-
ment with the results reported by Ferrieres et al, in which ZnCl2 did not yield any
glycosylation products either.[22] The highest yields for the glycosylation of 1 were
observed in the presence of five equivalents of BF3·Et2O. Interestingly, the observed
yields for the glycosylation reaction under non-anhydrous conditions (Table 1) are
similar or higher than the yield reported for the glycosylation of 1 with n-octanol
under strict anhydrous conditions (30%).[22] Furthermore, the reported procedure
required elevated temperatures (60°C) and ultrasound to achieve the glycosylation
of GlcNAc in a dry atmosphere, whereas the glycosylation reaction described herein
was performed at room temperature.
Substituted GlcNAc derivatives (2a–e and 3a–e) were synthetized using methanol
(a), ethanol (b), 2-propanol (c), n-butanol (d), and benzyl alcohol (e) as glycosyl

4
S.-Y. WANG ET AL.
Table . Yields and α/β-ratios detected for the anomeric substitution reaction when methanol
(a), ethanol (b), -propanol (c), n-butanol (d), and benzyl alcohol (e) were used as the glycosyl
acceptors. ∗) Low isolated yields of entries  and  were due to the poor separation of the β-anomer
from the α-anomer using silica gel chromatography, allowing that only non-overlapping elution frac-
tions were further used.
Entry
Acceptor
Yield (%)
α/β ratio (colorimetric assay)
α/β ratio (NMR)
Yield (%) α-anomer

a
b
c
d
e




:
:
:
:
:
:
:
:
:
:
%
%




%
%∗)
%∗)

acceptors for the nucleophilic substitution. Entries 1–4 in Table 1 show that yields
were reduced with the increase in length of the glycosyl acceptor (i.e., entry 1, 74%).
When glycosyl acceptors with bulkier aliphatic chains (n-butanol and benzyl
alcohol) were used, the yields decreased to below 30%. In all cases, a mixture of the
α-anomer (2a–e) and the β-anomer (3a–e) was obtained. Although the equatorial
location of the β-anomer is thermodynamically more favored than the axial loca-
tion of the α-anomer (Table 1, entries 1–4), the proportion of α-anomer increases
with the length of the glycosyl acceptor. Using n-butanol as the glycosyl acceptor,
only 13% was obtained with a β-configuration, whereas that proportion increased
to 49% when using methanol as glycosyl acceptor. The obtained results could be
explained by considering the possible approaches of the glycosyl acceptor to the
anomeric carbocation intermediate (Fig. 1). With bulkier substrates, the equatorial
approach forming the β-anomer is hindered by the steric effect of the amidoacetyl
group linked to the C-2 of the pyranose ring and thus, the α-anomer is kinetically
favored. This is in agreement with the reported α/β ratio for the glycosylation of 1
with n-octanol (90:10).[22] Interestingly, the glycosyl acceptor benzyl alcohol led to
an α/β ratio of 63:37, suggesting that the planar aromatic ring did not have relevant
steric effects on the equatorial approach.
Figure . Possible approaches of the glycosyl acceptor to the anomeric carbocation intermediate. The
approach that allows the formation of the β-anomer is hindered by the steric effect produced by the
acetyl group.

JOURNAL OF CARBOHYDRATE CHEMISTRY
5
Enzymatic hydrolysis of β-substituted GlcNAc derivatives
Despite most of the GlcNAc glycosylation strategies provide α and β mixtures, the
isolation of the α-anomer remains to be a challenging task. In this field, Polanki
and coworkers separated the α- and β-anomers after acetylation of the glycosy-
lated GlcNAc derivative by automated flash chromatography equipped with a UV
detector and ELSD using a column containing AgNO3-impregnated silica gel.[18]
Matsumura et al. reported the purification of the α-GlcNAc derivatives using an
anion exchange resin.[19] In this work, the β-anomer was selectively hydrolyzed
enzymatically, allowing the purification of the α-anomer by simple silica gel
chromatography.
For the selective hydrolysis of the β-substituted portion of GlcNAc deriva-
tives, a bacterial β-N-acetyl-hexosaminidase (E.C. 3.2.1.52) derived from
Zobellia galactanivorans (ZgβHexN2854) was used. This enzyme has been recently
employed by our group for the release of GlcNAc from chitinous mushrooms
extracts.[29] Using p-nitrophenyl-β-GlcNAc (3f, Fig. 2A) as a substrate, the
enzymatic activity could be easily observed by measuring the absorbance of p-
nitrophenol, a yellowish chromogenic side product of the reaction (Fig. 2A).
Hydrolytic reactions were performed at 37°C and pH 8.0 using 0.02 U/mL of
recombinant ZgβHexN2854. This enzyme showed high hydrolytic activity towards
3f, while p-nitrophenyl-α-GlcNAc (2f) could not be hydrolyzed by the enzyme
(Fig. 2B).
Figure . A: ZgβHexN-catalyzed hydrolysis of p-nitrophenyl-β-GlcNAc (3f). ZgβHexN
showed no activity towards p-nitrophenyl-α-GlcNAc (2f). B: UV response at  nm of ZgβHexN-
catalyzed hydrolysis reaction when 2f or 3f was used as the substrates. Hydrolytic reactions were
performed at °C and pH . ( mM NaPO/citrate buffer).

6
S.-Y. WANG ET AL.
Scheme . ZgβHexN-catalyzed hydrolysis of 3a–e to obtain 1. 2a–e remained unreacted in the
presence of ZgβHexN. PhGnE catalyzed the epimerization reaction of 1 to ManNAc (4). Sub-
sequently, 4 was oxidized to N-acetyl-D-mannosamino-lactone (5) using N-acetyl-D-mannosamine
dehydrogenase (NAMDH). The NAMDH-catalyzed oxidation of 4 to 5 involved the reduction of NAD+
to NADH, which was subsequently detected photometrically at  nm.
These results encouraged us to carry out the selective hydrolysis of β-
GlcNAc derivatives of the obtained α(2a–e)/β(3a–e) mixtures (Sch. 3, upper part).
ZgβHexN2854 could successfully release the anomerically-linked substituents of
3a, 3b, 3c, 3d within 12 h, while 24 h were needed for the complete hydrolysis of
compound 3e (Fig. S1). As expected, α-GlcNAc derivatives (2a, 2b, 2c, 2d, and 2e)
remained unreacted during the incubation with ZgβHexN2854, and could be easily
purified by silica gel chromatography (Figs. S2–S6).
Detection of β-linked GlcNAc
In this work, an enzyme coupled plate reader-based photometric assay for the
accurate measurement of β-linked GlcNAc was developed. Thus, free GlcNAc
released by the ZgβHexN2854 was interconverted into ManNAc (4) using a bacte-
rial GlcNAc-2-epimerase (PhGn2E).[30] Then, the obtained ManNAc was selectively
oxidized by a bacterial N-acetyl-D-mannosamine dehydrogenase (NAMDH) in the
presence of NAD+, affording N-acetyl-D-mannosamino-lactone (5) and NADH
(Sch. 3, lower part).[31] Although several colorimetric assays for the detection of
reducing sugars have been reported so far,[32] the developed colorimetric assay
allows the specific quantification of β-GlcNAc.
Both the epimerization and oxidation reactions were carried out in microwell
plate format. A linear correlation between the NADH-based absorbance at 340 nm
and the concentration of GlcNAc could be established in the range of 0.25 mM and
4 mM of GlcNAc (Fig. 3) after a 75 min reaction time. The observed concentra-
tion of GlcNAc in the hydrolysis reaction revealed the concentration of β-GlcNAc
derivatives and, consequently, the α/β ratios. A proton NMR-based calculation of
the ratios in the α/β mixtures confirmed the reliability of the coupled photometric
assay (1H NMR spectra are shown in Figs. S7–S11).

JOURNAL OF CARBOHYDRATE CHEMISTRY
7
Figure . Linear regression of the developed colorimetric assay for the quantitative detection of Glc-
NAc. GlcNAc obtained from the ZgβHexN-catalyzed hydrolysis of 3a–e was detected. Reaction
conditions:μLoftheenzymatichydrolysiswereincubatedwithNAD+ (mM), NaPO-citratebuffer
( mM, pH .), PhGnE (. µM), and NAMDH (. µM, final volume:  μL) at °C for  min.
Absorbance was measured at  nm in  s intervals.
Scheme. Quantificationoftheβ-linkedGlcNAcinacoreglycanstructure. AftertheZgβHexN-
catalyzed hydrolysis of the pNP core  glycan, the released GlcNAc (1) was quantified micro spec-
trophotometrically using a coupled epimerization-oxidation assay. The pNP core  glycan by-product
was quantified using UPLC-based analysis.
To further demonstrate the versatility of the applied method, the enzyme-coupled
photometric assay was also applied to quantify a more complex core 2 glycan struc-
ture bearing β-linked GlcNAc (pNP-α-GalNAc[β1,3Gal]β1,6GlcNAc, Sch. 4). The
GlcNAc concentration obtained from the assay (98.7 4.2 µM) was in good agree-
ment with concentration obtained from the UPLC-based assay which detected the
(aquimolar) reaction by-product pNP-α-GalNAcβ1,3Gal (104.7 4.3 µM).
Conclusion
In summary, an alternative approach for the synthesis to anomerically substituted
GlcNAc derivatives using unprotected GlcNAc as the starting material has been
developed. The herein described synthesis method allows the anomeric modifi-
cation of GlcNAc under non-anhydrous conditions, and allowed the formation

8
S.-Y. WANG ET AL.
of α/β mixtures using different alcohols as glycosyl acceptors. A bacterial β-N-
acetyl hexosaminidase catalyzed the selective hydrolysis of the β-anomers, while
α-anomers remained unreacted. Hence, this procedure allowed the easy isolation of
α-diastereoisomers by silica-gel column chromatography, circumventing the exten-
sive chromatographic or crystallographic workup generally required for the sep-
aration of anomers. Furthermore, the applied enzyme coupled photometric assay
could be successfully used to measure the ratios of the chemically obtainedα/β mix-
tures in a fast, easy, and reliable way. This assay might be especially useful in proce-
dures wherein the NMR analysis is unfeasible, such as in high-throughput or sub-mg
syntheses.
Experimental
Materials
Buffers, salts, and other chemicals used in this study were bought at the highest
grade from commercial suppliers without further purification or modification. The
expression and purification of PhGn2E was performed as previously reported.[30]
ZgβHexN2854 hexosaminidase and NAMDH N-acetyl-D-mannosamine dehydro-
genase were obtained from Qlyco Ltd, Nanjing. NMR spectra were registered on a
Bruker AV-400 instrument using the residual proton signal from the solvent as the
internal standard.
Glycosylation reaction
To a solution of 1 (100 mg, 0.45 mmol) in acetonitrile (15 mL) and the correspond-
ing alcohol (20 mmol, methanol (a), ethanol (b), 2-propanol (c), n-butanol (d), or
benzyl alcohol (e)), boron trifluoride diethyl etherate (300 μL, 2.3 mmol) was added.
The reaction mixture was stirred at room temperature overnight. Then, the solvent
was evaporated under reduced pressure. The residue was then subjected to silica-gel
column chromatography. The α/β mixture of the N-acetyl-D-glucosamine deriva-
tive was obtained as a white solid by eluting the compound with an AcOEt/MeOH
9:1 mixture (2a/3a 51:49 mixture: 74% yield, 2b/3b 80:20 mixture: 68% yield, 2c/3c
82:18 mixture: 58% yield, 2d/3d 87:13 mixture: 18% yield, 2e/3e 63:37 mixture: 25%
yield).
Enzymatic assay using chromogenic pNP-GlcNAc as substrate
Reaction mixtures (final volume 50 μL) containing pNP-α-GlcNAc or pNP-
β-GlcNAc (1 mM final concentration), and 0.001 U of ZgβHexN28545
hexosaminidase in Na3PO4/citrate buffer (50 mM, pH 8.0) were
analyzed photo-spectrometrically at 405 nm in 15 s intervals over a time period of
75 min in a multi-well plate reader.

JOURNAL OF CARBOHYDRATE CHEMISTRY
9
Hydrolysis of β-substituted GlcNAc derivatives
The purified α/β mixtures 2a/3a, 2b/3b, 2c/3c, 2d/3d, or 2e/3e were dissolved in
Na3PO4/citrate buffer (50 mM, pH 8.0) and 0.1 U of ZgβHexN2854 hexosaminidase
were added (final volume 5 mL). The reaction mixture was incubated at 37 °С for
12 h in the case of 2a/3a, 2b/3b, 2c/3c, and 2d/3d and 24 h in the case of 2e/3e. Then,
the solvent was evaporated under reduced pressure. The residue was subjected to
silica-gel column chromatography. 2a, 2b, 2c, 2d, and 2e were obtained as white
solids by eluting them with an AcOEt/MeOH 9:1 mixture (Yields: 2a: 32%, 2b: 53%,
2c: 44%, 2d: 9%, 2e: 15%).
NMR signal annotations
2a: 1H NMR (400 MHz, DMSO-d6): δ 7.76 (d, 1H, J = 8 Hz), 5.01 (d, 1H, J = 5.6 Hz),
4.74 (d, 1H, J = 5.6 Hz), 4.54 (t, 1H, J = 6 Hz), 4.52 (d, 1H, J = 3.6 Hz), 3.69–3.61
(m, 2H), 3.51–3.39 (m, 2H), 3.33–3.28 (m, 1H), 3.23 (s, 3H), 3.15–3.08 (m, 1H), 1.82
(s, 3H).
13C NMR (100 MHz, DMSO-d6): δ .169.9, 98.4, 73.2, 71.4, 71.3, 61.4, 54.8, 54.2,
23.2.
1
2b: H NMR (400 MHz, D2O): δ 4.83 (d, 1H, J = 3.6 Hz), 3.89–3.61 (m, 6H),
3.53–3.33 (m, 2H), 2.00 (s, 3H), 1.16 (t, 3H, J = 6.8 Hz).
13C NMR (100 MHz, D2O): δ 174.3, 96.5, 71.6, 71.0, 69.9, 63.8, 60.4, 53.5, 21.7,
13.9.
2c: 1H NMR (400 MHz, D2O): δ 4.93 (d, 1H, J = 3.6 Hz), 3.91–3.66 (m, 6H), 3.42
(m, 1H), 2.00 (s, 3H), 1.17 (d, 1H, J = 6 Hz), 1.09 (d, 1H, J = 6 Hz).
13C NMR (100 MHz, D2O): δ 174.3, 95.0, 71.7, 70.9, 70.8, 70.0, 60.4, 53.6, 22.2,
21.7, 20.3.
1
2d: H NMR (400 MHz, D2O): δ 4.83 (d, 1H, J = 3.6 Hz), 3.88–3.79 (m, 2H),
3.77–3.64 (m, 4H), 3.47–3.40 (m, 2H), 1.99 (s, 3H), 1.61–1.48 (m, 2H), 1.39–1.26
(m, 2H), 0.86 (t, 3H, J = 7.6 Hz).
13C NMR (100 MHz, D2O): δ 174.3, 96.5, 71.6, 70.8, 69.9, 67.8, 60.3, 53.7, 30.5,
21.7, 18.6, 12.9.
2e: 1H NMR (400 MHz, D2O): δ 7.43–7.34 (m, 4H), 7.22–7.18 (m, 1H) 4.90 (d,
1H, J = 3.6 Hz), 4.73 (d, 1H, J = 12 Hz), 4.51 (d, 1H, J = 11.6 Hz), 3.85–3.56 (m,
5H), 3.85–3.56 (m, 1H), 3.45 (M, 1H), 1.91 (s, 3H).
13C NMR (100 MHz, D2O): δ 174.3, 137.0, 128.7, 128.6, 128.4, 95.8, 72.1, 70.8,
70.0, 69.7, 60.5, 53.7, 21.8.
Enzymatic assay using non-chromogenic GlcNAc derivatives as substrate
To an aqueous solution containing the ZgβHexN2854-catalyzed hydrolysis reaction
solution (5 μL), 2 mM NAD+ in 50 mM Na3PO4/citrate buffer (pH 8.0), PhGn2E
(5.8 µM), and NAMDH (3.4 µM) were added (Final volume: 50 μL). The reac-
tion mixture was analyzed using the same continuous plate reader-based method
described by Awad et al.[33] and was recorded for 75 min. The linear relationship

10
S.-Y. WANG ET AL.
between absorbance values and GlcNAc concentration was established by testing
various GlcNAc standard solutions (between 0.25 mM and 4 mM).
The determination of the β-GlcNAc contents of the pNP-core 2 glycan was per-
formed using a micro spectrophotometer (Nanodrop 1000, Thermo), which allowed
to reduce assay volumes from 50 μL to 10 μL. Approximately, 0.5 mg of the pNP-
core 2 glycan (Carbosynth Ltd., UK) was dissolved in 707 μL of dH2O to obtain a
1 mM stock solution. One microliter of this solution was then mixed with 1 μL of
a 1 mM pNP-β-gal standard solution (used as an inert internal standard for latter
HPLC quantification) and 0.02 U of ZgβHexN2854 hexosaminidase in MES buffer
(pH 6.8, 50 mM, final volume 6 μL), and incubated overnight at 37°C. To this mix-
ture 1 µL NAD+ (20 mM), 1 µL Na3PO4/citrate buffer (pH 8.0, 1 M), 1 µL of PhGn2E
(1.16 µM), and 1 µL NAMDH (0.68 µM) were added. After 75 min of incubation
at 37°C, the sample was first analyzed with a micro spectrophotometer at 340 nm
and subsequently subject to UPLC analysis using a reversed phase column (Phe-
nomenex Kinetex C18, bead size 1.7 µm, dimensions 150 × 2.10 mm) with ammo-
nium formate (50 mM, pH 4.5, 0.2 mL/min) mixed with increasing concentrations
of acetonitrile as eluent (from 15% to 40% in the first 6 min). The eluents (pNP-β-
gal and pNP-α-GalNAcβ1, 3Gal) were detected with an UV wavelength of 300 nm
and quantified based on their relative peak areas.
Funding
This work was supported in part by the Natural Science Foundation of China (grant numbers
31471703, A0201300537, and 31671854 to J.V. and L.L.), and the 100 Foreign Talents Plan (grant
number JSB2014012 to J.V.). The authors would like to thank F. J. Gimenez-Warren for language
editing of this manuscript.
ORCID
Li Liu http://orcid.org/0000-0002-2178-9237
Josef Voglmeir http://orcid.org/0000-0002-4096-4926
References
1. Dwek, R.A. Glycobiology: toward understanding the function of sugars. Chem. Rev. 1996,
96, 683–720.
2. Varki, A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology
1993, 3, 97–130.
3. Jung, E.; Gooley, A.A.; Packer, N.H.; Karuso, P.; Williams, K.L. Rules for the addition of
O-linked N-acetylglucosamine to secreted proteins in Dictyostelium discoideum. Eur. J.
Biochem. 1998, 253, 517–524.
4. Previato, J.O.; Sola-Penna, M.; Agrellos, O.A.; Jones, C.; Oeltmann, T.; Travassos, L.R.;
Mendonça-Previato, L. Biosynthesis of O-N-acetylglucosamine-linked glycans in Try-
panosoma cruzi characterization of the novel uridine diphospho-N-acetylglucosamine:
polypeptide N-acetylglucosamine α1→O-threonine. J. Biol. Chem. 1998, 273,
14982–14988.

JOURNAL OF CARBOHYDRATE CHEMISTRY
11
5. Both, P.; Green, A.; Gray, C.; Šardzík, R.; Voglmeir, J.; Fontana, C.; Austeri, M.; Rejzek, M.;
Richardson, D.; Field, R. Discrimination of epimeric glycans and glycopeptides using IM-MS
and its potential for carbohydrate sequencing. Nat. Chem. 2014, 6, 65–74.
6. André, S.; Kaltner, H.; Kayser, K.; Murphy, P.V.; Gabius, H.-J. Merging carbohydrate chem-
istry with lectin histochemistry to study inhibition of lectin binding by glycoclusters in the
natural tissue context. Histochem. Cell Biol. 2016, 145, 185–199.
7. Nakayama, J.; Yeh, J.-C.; Misra, A.K.; Ito, S.; Katsuyama, T.; Fukuda, M. Expression cloning
of a human α1, 4-N-acetylglucosaminyltransferase that forms GlcNAc α1→ 4Galβ→ R, a
glycan specifically expressed in the gastric gland mucous cell-type mucin. P. Natl. Acad. Sci.
USA. 1999, 96, 8991–8996.
8. Kawakubo, M.; Ito, Y.; Okimura, Y.; Kobayashi, M.; Sakura, K.; Kasama, S.; Fukuda, M.N.;
Fukuda, M.; Katsuyama, T.; Nakayama, J. Natural antibiotic function of a human gastric
mucin against Helicobacter pylori infection. Science 2004, 305, 1003–1006.
9. Arihara, R.; Nakamura, S.; Hashimoto, S. Direct and stereoselective synthesis of 2-
acetamido-2-deoxy-β-D-glycopyranosides by using the phosphite method. Angew. Chem.
Int. Edit. 2005, 44, 2245–2249.
10. Haddoub, R.; Laurent, N.; Meloni, M.M.; Flitsch, S.L. Straightforward Synthesis of 2-
acetamido-2-deoxy-β-D-glucopyranosyl esters under microwave conditions. Synlett 2009,
20, 3328–3332.
11. Hanessian, S.; Lou, B. Stereocontrolled glycosyl transfer reactions with unprotected glycosyl
donors. Chem. Rev. 2000, 100, 4443–4464.
12. Šardzík, R.; Noble, G.T.; Weissenborn, M.J.; Martin, A.; Webb, S.J.; Flitsch, S.L. Prepa-
ration of aminoethyl glycosides for glycoconjugation. Beilstein J. Org. Chem. 2010, 6,
699–703.
13. Shaikh, N.; Russo, L.; Cipolla, L.; Nicotra, F. Ultrasonic assisted Fischer glycosylation: gen-
erating diversity for glycochemistry. Mol. Divers. 2011, 15, 341–345.
14. Campo, V.L.; Martins, M.B.; da Silva, C.H.; Carvalho, I. Novel and facile solution-phase syn-
thesis of 2, 5-diketopiperazines and O-glycosylated analogs. Tetrahedron 2009, 65, 5343–
5349.
15. Seibel, J.; Hillringhaus, L.; Moraru, R. Microwave-assisted glycosylation for the synthesis of
glycopeptides. Carbohyd. Res. 2005, 340, 507–511.
16. Carvalho, I.; Scheuerl, S.L.; Kartha, K.R.; Field, R.A. Practical synthesis of the 2-acetamido-
3, 4, 6-tri-O-acetyl-2-deoxy-β-D-glucosides of Fmoc-serine and Fmoc-threonine and their
benzyl esters. Carbohyd. Res. 2003, 338, 1039–1043.
17. Bernardes, G.J.; Gamblin, D.P.; Davis, B.G. The direct formation of glycosyl thiols from
reducing sugars allows one-pot protein glycoconjugation. Angew. Chem. Int. Edit. 2006, 45,
4007–4011.
18. Polanki, I.K.; Kurma, S.H.; Bhattacharya, A.K. Direct glycosylation of unprotected and unac-
tivated sugars using bismuth nitrate pentahydrate. J. Carbohyd. Chem. 2015, 34, 196–205.
19. Matsumura, S.; Kawamura, Y.; Yoshikawa, S.; Kawada, K.; Uchibori, T. Surface activities,
biodegradability and antimicrobial properties of glucosamine derivatives containing alkyl
chains. J. Am. oil Chem. Soc. 1993, 70, 17–22.
20. Zhu, Y.; Pan, Q.; Thibaudeau, C.; Zhao, S.; Carmichael, I.; Serianni, A.S. [13C, 15N] 2-
Acetamido-2-deoxy-d-aldohexoses and their methyl glycosides: synthesis and NMR inves-
tigations of J-couplings involving 1H, 13C, and 15N. J. Org. Chem. 2006, 71, 466–479.
21. Banoub, J.; Boullanger, P.; Lafont, D. Synthesis of oligosaccharides of 2-amino-2-deoxy sug-
ars. Chem. Rev. 1992, 92, 1167–1195.
22. Ferrières, V.; Bertho, J.-N.; Plusquellec, D. A new synthesis of O-glycosides from totally O-
unprotected glycosyl donors. Tetrahedron Lett. 1995, 36, 2749–2752.

12
S.-Y. WANG ET AL.
23. Wu, B.; Yang, J.; Robinson, D.; Hofstadler, S.; Griffey, R.; Swayze, E.E.; He, Y. Synthesis of
linked carbohydrates and evaluation of their binding for 16S RNA by mass spectrometry.
Bioorg. Med. Chem. Lett. 2003, 13, 3915–3918.
24. Roy, R.; Kim, J.M. Cu (II)-self-assembling bipyridyl-glycoclusters and dendrimers bearing
the Tn-antigen cancer marker: syntheses and lectin binding properties. Tetrahedron 2003,
59, 3881–3893.
25. Sanki, A.K.; Mahal, L.K. A one-step synthesis of azide-tagged carbohydrates: versatile inter-
mediates for glycotechnology. Synlett 2006, 2006, 0455–0459.
26. Gao, F.; Yan, X.; Shakya, T.; Baettig, O.M.; Ait-Mohand-Brunet, S.; Berghuis, A.M.; Wright,
G.D.; Auclair, K. Synthesis and structure-activity relationships of truncated bisubstrate
inhibitors of aminoglycoside 6ꢀ-N-acetyltransferases. J. Med. Chem. 2006, 49, 5273–5281.
27. Roy, B.; Mukhopadhyay, B. Sulfuric acid immobilized on silica: an excellent catalyst for Fis-
cher type glycosylation. Tetrahedron Lett. 2007, 48, 3783–3787.
ˇ
ˇ
28. Babic, A.; Pecar, S. Synthesis of protected hydroxyethylamine transition-state analogs of N-
Ac-muramyl-L-alanine. Synthetic Commun. 2008, 38, 3052–3061.
29. Lv, Y.; Laborda, P.; Huang, K.; Cai, Z.; Wang, M.; Lu, A.; Doherty, C.; Liu, L.; Flitsch, S.;
Voglmeir, J. Highly efficient and selective biocatalytic production of glucosamine from chitin.
Green Chem. 2017.
30. Wang, S.-Y.; Laborda, P.; Lu, A.-M.; Duan, X.-C.; Ma, H.-Y.; Liu, L.; Voglmeir, J. N-
acetylglucosamine 2-epimerase from Pedobacter heparinus: first experimental evidence of
a deprotonation/reprotonation mechanism. Catalysts 2016, 6, 212.
31. Yamamoto-Otake, H.; Koyama, Y.; Horiuchi, T.; Nakano, E. Cloning, sequencing, and expres-
sion of the N-acyl-D-mannosamine dehydrogenase gene from Flavobacterium sp. strain
141–8 in Escherichia coli. Appl. Environ. Microb. 1991, 57, 1418–1422.
32. Marques, T.A.; Rampazo, E.M.; Zilliani, R.R.; Marques, P.A.A.; Benincasa, F. Automated
sugar analysis. Food Sci. Technol. 2016, 36, 70–75.
33. Awad, F.N.; Kulinich, A.; Yao, M.J.; Duan, X.C.; Cai, Z.P.; Gu, B.; Liu, L.; Voglmeir, J. Enzy-
matic glycosylation of indoxyglycosides catalyzed by a novel maltose phosphorylase from
Emticicia oligotrophica. J. Carbohyd. Chem. 2016, 35, 301–314.