Synthesis of N-glycan units for assessment of substrate structural requirements of N-acetylglucosaminyltransferase III

Article abstract of DOI:10.1016/j.bmcl.2014.07.074

N-Acetylglucosaminyltransferase (GnT) III is a glycosyltransferase which produces bisected N-glycans by transferring GlcNAc to the 4-position of core mannose. Bisected N-glycans are involved in physiological and pathological processes through the functional regulation of their carrier proteins. An understanding of the biological functions of bisected glycans will be greatly accelerated by use of specific inhibitors of GnT-III. Thus far, however, such inhibitors have not been developed and even the substrate-binding mode of GnT-III is not fully understood. To gain insight into structural features required of the substrate, we systematically synthesized four N-glycan units, the branching parts of the bisected and non-bisected N-glycans. The series of syntheses were achieved from a common core trimannose, giving bisected tetra- and hexasaccharides as well as non-bisected tri- and pentasaccharides. A competitive GnT-III inhibition assay using the synthetic substrates revealed a vital role for the Manβ(1-4)GlcNAc moiety. In keeping with previous reports, GlcNAc at the α1,3-branch is also involved in the interaction. The structural requirements of GnT-III elucidated in this study will provide a basis for rational inhibitor design.

Full text of DOI:10.1016/j.bmcl.2014.07.074

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4533–4537
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of N-glycan units for assessment of substrate structural
requirements of N-acetylglucosaminyltransferase III
Shinya Hanashima a,b, Hiroaki Korekane c, Naoyuki Taniguchi c, Yoshiki Yamaguchi a,
⇑
a Structural Glycobiology Team, Systems Glycobiology Research Group, RIKEN-Max Planck Joint Research Center for Systems Chemical Biology, RIKEN Global Research Cluster,
Wako, Saitama 351-0198, Japan
b Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
c Disease Glycomics Team, Systems Glycobiology Research Group, RIKEN-Max Planck Joint Research Center for Systems Chemical Biology, RIKEN Global Research Cluster, Wako,
Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Acetylglucosaminyltransferase (GnT) III is a glycosyltransferase which produces bisected N-glycans by
transferring GlcNAc to the 4-position of core mannose. Bisected N-glycans are involved in physiological
and pathological processes through the functional regulation of their carrier proteins. An understanding
of the biological functions of bisected glycans will be greatly accelerated by use of specific inhibitors of
GnT-III. Thus far, however, such inhibitors have not been developed and even the substrate-binding mode
of GnT-III is not fully understood. To gain insight into structural features required of the substrate, we
systematically synthesized four N-glycan units, the branching parts of the bisected and non-bisected
N-glycans. The series of syntheses were achieved from a common core trimannose, giving bisected tetra-
and hexasaccharides as well as non-bisected tri- and pentasaccharides. A competitive GnT-III inhibition
assay using the synthetic substrates revealed a vital role for the Manb(1–4)GlcNAc moiety. In keeping
Received 3 June 2014
Revised 22 July 2014
Accepted 29 July 2014
Available online 6 August 2014
Keywords:
Bisecting GlcNAc
N-Glycan
Synthesis
Glycosylation
with previous reports, GlcNAc at the
a1,3-branch is also involved in the interaction. The structural
N-Acetylglucosaminyltransferase III
requirements of GnT-III elucidated in this study will provide a basis for rational inhibitor design.
Ó 2014 Elsevier Ltd. All rights reserved.
Protein N-glycosylation takes place in the endoplasmic reticu-
lum (ER) and influences many of characteristics and functions of
glycoproteins including folding and degradation,1 trafficking,2
and cell adhesion.3 Throughout the glycan processing pathway,
one of the important modifications is the introduction of the
bisecting N-acetylglucosamine (GlcNAc) residue. N-acetylglucos-
aminyltransferase (GnT) III is responsible for inserting bisecting
GlcNAc at O4 of the core b-mannose via a b-linkage, using
UDP-GlcNAc as donor substrate.4 In general, it is thought that the
bisecting GlcNAc is not further glycosylated under physiological
conditions, which is in sharp contrast to the GlcNAc at the mannos-
es on both 1–3 and 1–6 branches.
Accumulating evidence suggests that bisecting GlcNAc regu-
lates tumor progression and migration, and Alzheimer’s disease.
Bisecting GlcNAc suppresses tumor progression by an effect on
growth factor signaling5 induced through galectin-mediated cross-
linking.6 Cell adhesion molecules with bisecting GlcNAc on their
N-glycans, particularly with respect to E-cadherin and integrins,
retard tumor migration.7–9 The mechanism of action may be due
to the fact that the bisected N-glycan is a poor acceptor for
GnT-V thereby arresting the formation of migration-related hyper
branched N-glycans.10,11 Bisecting GlcNAc is also involved in
Alzheimer’s disease, altering both the clearance and production
of amyloid-b.12,13 It also plays a pivotal role in suppression of
N-glycan branching, that is, inhibition of b1,6-GlcNAc transfer by
GnT-V and of core fucosylation by Fut8.14–16
Despite the synthetic challenge and because of its importance in
cellular events, the chemical synthesis of N-glycans with bisecting
GlcNAc has now been successfully achieved.17–21 One example of
an answer to overcoming some of the difficulties in the synthesis
has been to couple bisecting GlcNAc before the introduction of
the 1,3 and 1,6-branch mannose to avoid steric hindrance.
‘Designed’ bisected glycans through synthetic approaches are of
great importance for analyses of structure, dynamics and interac-
tions. The synthetic bisected hexasaccharide 1 has been used as a
ligand for mouse dendritic cell inhibitory receptor 2 (DCIR2),
which allowed the ligand recognition mode of DCIR2 to be revealed
by X-ray crystallography and NMR.22 Crystallographic analysis of a
legume lectin PHA-E, which is widely used for detection of bisected
glycans, was assisted using chemically synthesized ligands 1.23,24
The substrate specificity of GnT-III has been studied using trun-
cated substrates at the non-reducing terminus.25 GnT-III requires a
⇑
Corresponding author. Tel.: +81 48 467 9619; fax: +81 48 467 9620.
E-mail address: yyoshiki@riken.jp (Y. Yamaguchi).
b-GlcNAc residue on the
a1,3-Man which is transferred by GnT-I,
http://dx.doi.org/10.1016/j.bmcl.2014.07.074
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

4534
S. Hanashima et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4533–4537
while the other b-GlcNAc residue on the
a
1,6-Man moiety is not
when the enzyme acts on inner glycan residues. Since GnT-III is
essential.25 The addition of further b1,4-galactose units on the
expected to recognize the sugar residues emanating from the
b-mannose, we considered that studying individual parts of the
whole glycan structure could be very rewarding.
a1,6-Man has little effect on the activity of GnT-III while the addi-
tion of galactose onto
a1,3-Man branch inhibits the GnT-III reac-
tion.26 Glycans with tri- or tetra-antenna are also accepted as
substrates. In contrast, there are limited studies of the importance
of the chitobiose unit.
Glycan units 1, 2, 3, and 4 were systematically synthesized
using a parallel approach through common intermediates 12a
and 12b (Schemes 1 and 2).17,27–29 Synthesis of tetrasaccharide 3
was initiated from diallyl protected mannoside 517 (Scheme 1).
To avoid difficulty of the introduction by steric reason, the bisect-
ing GlcNAc was introduced at an early stage of the synthesis. It was
inserted on the O4 of mannoside 5 using the imidate building block
619 via an activation with catalytic amounts of TMSOTf30 to give
disaccharide 7. Then, the allyl protection at the 3,6-positions was
readily removed via a 2-step procedure involving treatment with
Iridium complex and then I2 in H2O to give acceptor 8. Mannosyla-
tion using imidate 9 by activation with TMSOTf in the presence of
MS 4 Å gave core tetrasaccharide 10. This was then treated with
1,2-ethylenediamine in 1-butanol at 100 °C followed by acetyla-
tion of amino and hydroxy groups to quantitatively yield 11.
Deacetylation of hydroxy groups using NaOMe provided 12a in
82% yield (3 steps). Finally, removal of all benzyl ethers on 12a
using 20% Pd(OH)2/C under a hydrogen atmosphere gave the
desired tetrasaccharide with bisecting GlcNAc 3 in 78% yield. The
trisaccharide counterpart 4 was synthesized according to the pre-
viously reported procedure.31
Here we have investigated the structural requirements on the
acceptor substrate of GnT-III using a series of synthetic glycans.
To achieve the purpose, we designed branching structures 2 and
4 including core b-mannose having C4-OH which is the acceptor
hydroxy group of bisecting GlcNAc, but excluding the chitobiose
moiety at non-reducing termini (Fig. 1). We also synthesized com-
pounds 1 and 3, to investigate the effect of bisecting GlcNAc on
GnT-III binding. The design focuses on the GlcNAc residues sur-
rounding to the core trimannnose unit. The synthesis was achieved
using common building blocks to construct bisectGN-Man3GN2 1,
Man3GN2 2, bisectGN-Man3 3, and Man3 4, respectively. Using
these synthetic glycans, we performed a competitive inhibition
assay to reveal the key sugar units for the acceptor recognition
by GnT-III.
The substrate specificity of glycosyltransferases including GnT-
III has previously been studied using a series of glycans purified
from natural sources. Such approaches, however, often have limita-
tions in identifying minimum structural requirements especially
NHAc
HO
HO
OH
O
O
O
HO
O
H
N
N-glycan; R1
=
HO
HO
Asn
HO
NHAc
HO
R
2 = H or GlcNAc
OH
O
AcHN
O
O
HO
O
R2O
OR1
OH
OH
O
HO
H
GnGnbi-PA; R1
=
N
N
HO
HO
NHAc
HO
O
R2 = H
HO
NHAc
HO
HO
HO
O
O
NHAc
NHAc
HO
HO
HO
HO
HO
O
HO
HO
HO
O
O
O
O
O
HO
HO
HO
HO
OH
O
OH
O
NHAc
O
O
HO
O
HO
HO
O
O
O
HO
OMe
OMe
HO
HO
HO
HO
O
O
HO
NHAc
HO
NHAc
1
2
HO
HO
HO
HO
O
O
HO
O
O
HO
OH
O
OH
O
HO
HO
HO
HO
HO
HO
NHAc
OH
O
OH
O
O
O
HO
HO
HO
HO
O
O
O
O
OMe
OMe
HO
HO
HO
HO
HO
O
OH
O
4
HO
3
OH
Figure 1. Structures of bisected N-glycans and synthetic bisected N-glycan units (bisectGN-Man3GN2) 1 and (bisectGN-Man3) 3, and corresponding bibranched glycan
(Man3GN2) 2 and pauch-mannose (Man3) 4. PA; pyridylamino.

S. Hanashima et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4533–4537
4535
OAc
O
NH
NPhth
BnO
BnO
BnO
NH
CCl3
BnO
O
CCl3
BnO
O
O
BnO
OBn
O
OBn
O
NPhth
O
9
AllO
HO
6
RO
O
BnO
BnO
BnO
AllO
RO
TMSOTf, CH2Cl2
OMe
OMe
TMSOTf, CH2Cl2
5
7; R = All
8
; R = H
OR
O
OAc
O
BnO
BnO
BnO
BnO
BnO
BnO
H2, 10% Pd/C
MeOH
a) ethylenediamine
1-BuOH 100 oC
OBn
O
OBn
O
NHAc
O
NPhth
O
3 (78%)
BnO
BnO
BnO
BnO
BnO
BnO
O
O
O
O
O
O
b) Ac2O, pyridine
OMe
OMe
BnO
BnO
BnO
BnO
O
OR
O
OAc
BnO
BnO
11; R = Ac
10
NaOMe, MeOH
12a; R = H (82%; 3 steps)
Scheme 1. Synthesis of the bisected tetrasaccharide 3.
NPhth
OH
O
AcO
AcO
AcO
BnO
NPhth
O
BnO
O
AcO
AcO
AcO
BnO
F
BnO
O
OBn
O
O
BnO
O
3.5 eq.
BnO
13
RO
OBn
O
O
O
RO
OMe
BnO
Cp2HfCl2, AgOTf, MS 4A, toluene
O
BnO
O
OMe
BnO
BnO
OH
BnO
O
BnO
NPhth
NHAc
O
BnO
BnO
BnO
12a; R =
AcO
O
AcO
O
NHAc
AcO
BnO
BnO
BnO
(81%)
12b; R = Bn
14a; R =
O
14b; R = Bn (75%)
NHAc
AcO
AcO
O
O
O
AcO
BnO
BnO
BnO
a) ethylenediamine, 1-BuOH
100 oC
OBn
O
O
RO
O
a) NaOMe, MeOH
OMe
1; (65%)
BnO
b) Ac2O, pyridine
2
BnO
; (78%)
O
BnO
NHAc
b) H2, Pd(OH)2/C
MeOH, H2O, AcOH
AcO
AcO
AcO
O
O
NHAc
BnO
BnO
BnO
(82%)
15a; R =
O
15b; R = Bn (80%)
Scheme 2. Parallel syntheses of bisected hexasaccharide 1 and bibranched pentasaccharide 2.
The syntheses of bisecting hexasaccharide 1 and non-bisecting
pentasaccharide 2 are shown in Scheme 2. Synthetic intermediates
12a and 12b were employed as the glycosyl acceptors. The tetra-
saccharide 12a and trisaccharide 12b31 were coupled with fluoride
1332 following activation with Cp2HfCl2 and AgOTf33,34 in toluene
in the presence of MS 4 Å, to give corresponding 14a and 14b in
81% and 75% yield, respectively. Global deprotection gave desired
glycans 1 and 2. Phthalimide groups were removed by treatment
with 1,2-ethylenediamine in 1-butanol at 100 °C, and re-acetyla-
tion provided 15a and 15b in 82% and 80% yield, respectively. Then
hydrogenolysis with 20% Pd(OH)2/C in methanol produced 1 and 2
in 65% and 78% yield, respectively.
Possible interaction of the synthetic glycans 1, 2, 3, and 4 with
GnT-III was studied through the use of a competitive inhibition

4536
S. Hanashima et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4533–4537
GnT-III
(A)
(B)
Synthetic Inhibitors
PA
PA
UDP
UDP
GlcNAc
mannose
100
80
60
40
20
0
4
3
2
1
0
100
200
300
Inhibitor(µM)
400
500
Figure 2. Substrate specificity of GnT-III by observing inhibition with the synthetic substrates 1, 2, 3, and 4. (A) GlcNAc transfer scheme by GnT-III. (B) The relative sugar
transfer rate by GnT-III at each inhibitor concentration. The model substrate GnGnbi-PA was used at 5 M, and synthetic substrates added to 62.5, 125, 250, and 500 M,
respectively. The time-course quantification of substrate and product was performed by HPLC analysis with fluorescence detection.
l
l
assay (Fig. 2). The bi-branched N-glycan carrying a pyridylamino
(PA) group at the reducing terminus (GnGnbi-PA) served as a typ-
ical acceptor substrate and UDP-GlcNAc as a donor substrate.35,36
GnT-III activity and GlcNAc transfer was evaluated by a shift in
HPLC retention time from GnGnbi-PA to bisectGlcNAc-GnGnbi-
PA. Results indicated that GN2Man3 2 has a significant inhibition
activity. The bisected GlcNAc-GN2Man3 1, a product of GnT-III,
showed less but significant competition activity. Mannose-
terminated 3 and 4 were not inhibitory, even in 100-fold excess.
Evidently, the bibranched structure 2 is a substrate of GnT-III.
This finding is consistent with a previous study using synthetic
glycan with a b-O-octyl group at the anomeric position of the
core-mannose.37 The relative catalytic efficiencies can now be
understood. Compound 1 is significantly inhibitory, indicating that
GnT-III is sensitive to product inhibition. Furthermore, the GlcNAc
of the Manb(1–4)GlcNAc moiety is highly recognized by GnT-III.
Clearly, GnGnbi-PA is a good substrate for GnT-III and can be use-
recognizes sugar residues between GlcNAc residues at the
1,3-Man branch and Manb(1–4)GlcNAc. Unfortunately, 3D
a
structural information on GnT-III is not available and further
details of the role of the key residues must await atomic structures.
The inhibition constant (Ki) of the synthetic compounds 1 and 2
were determined by kinetic assay (Table 1). The substrates of
GnT-III, either GnGnbi-PA or UDP-GlcNAc is used in a single con-
centration, and the concentrations of the other substrate and the
synthetic compound were changed. The apparent Ki values in the
presence of a fixed concentration (5 lM) of the acceptor GnGnbi-
PA are similar in hexasaccharide 1 (1.5 mM) and pentasaccharide
2 (1.4 mM). In contrast, the apparent Ki values in the presence of
a fixed concentration (20 mM) of the donor UDP-GlcNAc are
14 mM in hexasaccahride 1 and 1.2 mM in pentasaccharide 2,
which causes the higher inhibition efficiency of 2. One-order differ-
ence of Ki in 1 and 2 in the presence of 20 mM UDP-GlcNAc is
explained by the differences in competition efficiency toward the
preferential substrate, GnGnbi-PA.
In summary, we have investigated the substrate structural
requirements of GnT-III using a series of putative synthetic sub-
strates and products. Syntheses of the individual branching parts
of N-glycans were achieved through systematic coupling of core
tri/tetrasaccharide acceptors and GlcNAc donor. A competitive
inhibition assay suggests that GlcNAc residues connecting the
core-trimannose play a vital role in recognition by GnT-III. An
understanding of the substrate structural requirements of GnT-III
will assist in uncovering how N-glycan biosynthesis is controlled
and help design specific glycosyltransferase inhibitors.38
fully assayed at a concentration of 5
and 2 were moderately competitive at rather high concentrations
(62.5–500 M). Structural differences between substrates
lM. The synthetic glycans 1
l
GnGnbi-PA and 2 are the sugar units at the reducing termini, the
GlcNAc residues on Manb(1–4)GlcNAc and the PA tag. The
penultimate GlcNAc residue moiety is important for recognition
by GnT-III. Additionally, previous data showed that b-alkyl linked
pentasaccharides are a substrate of GnT-III.37 Our data, taken
together with the previous studies, suggests that GnT-III likely
Table 1
Acknowledgments
Apparent Ki values of the synthetic compounds 1 and 2 in the inhibition of GnT-III
Compound
Ki (mM)
We thank Dr. Yukishige Ito (RIKEN/ERATO) for support in the
syntheses, and Prof. Yoshitaka Ikeda (Saga University) for valuable
comments on this manuscript. This work was supported in part by
Grant-in-Aid for Young Scientists (B) (24710257) and Scientific
Research (C) (25460054) from Japan Society of the Promotion of
Science (JSPS), by Encouraged Research Grant from Tokyo
Biochemical Research Foundation, and by RIKEN Fund for Seeds
of Collaborative Research.
UDP-GlcNAca
GnGnbi-PAb
1
2
1.5
1.4
14
1.2
a
GnGnbi-PA (5 lM), UDP-GlcNAc (0.5, 1, 2, 4 mM), and 1 or 2 (0, 125, 250,
500
l
M).
GnGnbi-PA (2.5, 5, 10
M).
b
l
M), UDP-GlcNAc (20 mM), and 1 or 2 (0, 125, 250,
500
l

S. Hanashima et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4533–4537
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