N-acetyl-6-sulfo-D-glucosamine as a promising mimic of N-acetyl neuraminic acid

Article abstract of DOI:10.1016/S0960-894X(03)00611-5

6-Sulfo-D-GlcNAc with a molecular geometry close to that of N-acetylneuraminic acid (Neu5Ac) was hypothesized to serve as a simple Neu5Ac mimic possessing high potential in biochemical and medicinal applications. The hypothesis was evidenced with a neuraminidase inhibition assay using p-nitrophenyl (pNP) 3-, 4-, and 6-sulfo-β-D-GlcNAc (4, 5 and 2a) and 6-sulfo-β-D-Glc 6, in which only pNP 6-sulfo-β-D-GlcNAc 2a was found to show substantial activity.

Full text of DOI:10.1016/S0960-894X(03)00611-5

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2821–2823
N-Acetyl-6-sulfo-D-glucosamine as a Promising Mimic
of N-Acetyl Neuraminic Acid
Kenji Sasaki,a,c Yoshihiro Nishida, * Hirotaka Uzawa and Kazukiyo Kobayashi *
a,c,
b
a,c,
aDepartment of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Chikusa-ku,
Nagoya 464-8603, Japan
b
National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305-8565, Japan
cCREST, Japan Science and Technology Corporation (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan
Received 21 March 2003; accepted 18 June 2003
Abstract—6-Sulfo-d-GlcNAc with a molecular geometry close to that of N-acetylneuraminic acid (Neu5Ac) was hypothesized to
serve as a simple Neu5Ac mimic possessing high potential in biochemical and medicinal applications. The hypothesis was evidenced
with a neuraminidase inhibition assay using p-nitrophenyl (pNP) 3-, 4-, and 6-sulfo-b-d-GlcNAc (4, 5 and 2a) and 6-sulfo-b-d-Glc
6
#
, in which only pNP 6-sulfo-b-d-GlcNAc 2a was found to show substantial activity.
2003 Elsevier Ltd. All rights reserved.
Sialic acids, bound to exo-terminal b-galactosyl residues
in cell surface glycoproteins and glycolipids, play a key
role in the carbohydrate-protein recognition events
leading to cell adhesion, trafficking of leukocytes, and
performed in such a way as to simulate the key recog-
nition structure of the sialyl linkage exactly. This holds
true for the sialyl linkages potentially useful as ther-
apeutic agents against many types of infectious diseases.
1
infection of influenza viruses to host cells. They are
recognized by many types of sialic acid-binding proteins
such as sialoadhesins (siglecs), selectins, and influenza
hemagglutinins. Therefore, the sialyl linkage particu-
larly of the most abundant N-acetylneuraminic acid
Along with our continuous studies on the design and
application of polyvalent glycoconjugates, we have
investigated practical syntheses of human oligosaccha-
6
rides and their mimics. In our recent effort to establish
a chemical pathway based on the carbohydrate mod-
(
Neu5Ac 1, Fig. 1), possesses high potential in applica-
7
tions to medicinal agents and bio-mimetic materials.
However, the practical application has been hampered
by the property of Neu5Ac-glycoside linkages labile to
chemical and enzymatic degradations as well as by the
difficulty of their large-scaled preparations. Therefore,
many different approaches have been designed for their
mimic synthesis aiming at the development of anti-
ule, we found that an acylamido copolymer carrying
cluster of N-acetyl-6-O-sulfo-b-d-glucosaminide
a
(6-sulfo-b-d-GlcNAc) possesses potent activity to block
X
8
l-selectin/sLe binding (IC =3 mM). The activity is
5
0
much higher than that of a copolymer carrying a cluster
0
X
of 3 -sulfo-Le mimics. This finding, indicating the high
biological potential of 6-sulfo-d-GlcNAc, allows us to
2
3
inflammation drugs and anti-influenza virus agents.
For the anti-inflammation drugs working as selectin
antagonists, the linkage of sialyl LewisX (sLe ) tetra-
saccharide has been replaced with a simple anionic
group such as carboxylate, phosphate, and sulfate
X
4
groups. This approach works effectively when the
receptor proteins recognize the sialyl linkage mainly at
X
the carboxylate group, as in the case of selectin/sLe
5
interactions. Otherwise, the mimic synthesis should be
Figure 1. A homology of molecular structures between Neu5Ac 1 and
6-sulfo-d-GlcNAc 2. The structure of 2 is viewed from a ring frame
along the C2–C3–C4 bonds. The C5–C6 conformation is given in gt
(+60for the dihedral angle of C5–C6/C6–O6).
*
2
Corresponding authors. Tel.: +81-52-789-2488; fax: +81-52-789-
528; e-mail: nishida@mol.nagoya-u.ac.jp (Y. Nishida); kobayash@
mol.nagoya-u.ac.jp (K. Kobayashi)
0
960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00611-5

2822
K. Sasaki et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2821–2823
speculate that the l-selectin may recognize the structure
group, respectively, were also used. These substances
were subjected to an inhibitory assay in the reported
of 6-sulfo-d-GlcNAc as a mimic of the Neu5Ac linkage.
9
12
Recently, Schwo
¨
rer and Schmidt reported a homology
way using a neuraminidase (EC: 3.2.1.18) of Vibrio
cholerae and 2 -(4-methylumbelliferyl)-N-acetylneur-
0
aminic acid. In this assay, the enzyme reaction (K =1.5
mM for the fluorescent substrate and 1.2 mM for sialyl-
in the structure between d-GlcNAc and Neu5Ac and
applied it to the design of sialyltransferase inhibitors. In
this paper, we describe a similar correlation between 6-
sulfo-d-GlcNAc 2 and Neu5Ac 1 and provide experi-
mental evidence that the 6-sulfo-d-GlcNAc 2 serves as a
simple Neu5Ac mimic.
m
1
2
lactose ) liberates a fluorescent 4-methylumbelliferone
from the non-fluorescent substrate. The results sum-
marized in Figure 2 indicate that neither the referential
pNP 6-sulfo-d-Glc 6 nor pNP d-GlcNAc showed inhi-
bition activity at concentrations below 10mM. This
means that the pNP chromophore commonly applied to
the substrates does not affect the florescence assay. The
assay indicated that pNP 6-sulfo-d-GlcNAc 2a shows
notable activity to inhibit the neuraminidase (IC =2
When the structure of 6-sulfo-d-GlcNAc 2 is viewed
from a ring frame along the C2–C3–C4 bonds as depic-
ted in Figure 1, the homology with Neu5Ac 1 becomes
obvious. The structure 2 locates the NHAc, OH, and
anionic functions along the frame close to those of
NeuNAc. A difference exists in the aglycon structure,
which will be, however, modulated by chemical meth-
ods. Though the location of the 6-O-sulfate group in 2
changes by the free rotation of a C5–C6 single bond, the
5
0
mM). The above data indicate that both of the sulfate
and NHAc groups play a key role in the neuraminidase
inhibition activity of 2a. Among the three isomers, the
6-sulfo-isomer 2a showed the highest activity in the
order of 6-sulfo 2a>4-sulfo 5>3-sulfo 4, indicating that
the position of 6-sulfate is another major factor. The
overall biological data support that the neuraminidase
recognizes the absolute structure of pNP 6-sulfo-d-
GlcNAc 2a as a competitor of Neu5Ac.
1
10,11
H NMR analysis
of pNP 6-sulfo-d-GlcNAc 2a
showed that the exocyclic moiety takes the time-aver-
aged population of gg:gt:tg=ca. 60:40:0 (%). The gt-
conformation locates the sulfate group close to the
position of a carboxylate anion in Neu5Ac 1.
To verify our working hypothesis as described above,
three position isomers of pNP sulfo-d-GlcNAc (2a, 4,
and 5) were prepared in a chemical pathway as shown in
Scheme 1. As referential compounds, pNP 6-sulfo-d-Glc
Our hypothesis was supported also by a molecular
modeling of 2a compared with the X-ray crystallo-
graphic data of a Neu5Ac-neuraminidase complex
(Fig. 3). The molecular modeling using an AMBER
force field showed that the 2-NHAc and 6-sulfate
groups in 2a prefer to take a spatial geometry (distance
and helical pattern) matching with that in Neu5Ac in
1
3
6
and pNP d-GlcNAc lacking an NHAc and a sulfate
the enzyme complex rather than free Neu5Ac with a
1
typical C (chair) conformation. In the crystalline data,
4
the Neu5Ac ligand takes a conformation deviated from
1
a C (chair) one and analogous to that of an oxo-car-
4
benium ion at a transitional state. Thus it is probable
Figure 2. Inhibitory activities of pNP sulfo-sugars against a neur-
aminidase of V. cholerae. The degree of inhibition (%, deviationꢀ5%)
was obtained as an averaged value in three experiments using an
equation as follows. Inhibition (%)=100ꢁF
i o i o
/F , where F and F
indicates the strength of fluorescence at em. 450nm (ex. 365 nm) in the
presence and the absence of inhibitors, respectively. A typical assay
protocol: A reaction buffer (pH 4.6) was prepared with 100 mM
sodium acetate, 150mM NaCl, and 4 mM CaCl
2
in pure water (Milli-
0
Scheme 1. Syntheses of pNP sulfo-d-GlcNAc and sulfo-d-Glc. Reac-
Q, Millipore). A mixture of 2 -(4-methylumbelliferyl)-N-acetylneur-
aminic acid (0.1 mM), a sialidase (30 mU) (V. cholerae, Sigma), and an
inhibitor (10–0.1 mM) was dissolved in the reaction buffer (250 mL)
.
N SO
ꢂ
3
(6 equiv), DMF, 40 C, 3 h, Dowex
tion conditions: (a) Me
+
3
(Na form), 52% for 2a, 42% for 6; (b) TBDPSCl, pyridine, DMAP,
rt, 24 h, 95%; (c) Me
.
N SO
ꢂ
ꢂ
3
3
(6 equiv), DMF, 40 C, 10h, Dowex
Na form), 63% (4/5=1.4/1); (d) TBAF, THF, rt, 24 h, 98%.
and incubated in a micro tube at 37 C for 30min. The enzyme reac-
+
(
TBDPSCl=tert-butyldiphenylsilylchloride, DMAP=4-dimethylamino-
pyridine, TBAF= tetrabutylammonium fluoride.
tion was quenched by adding a glycine buffer (133 mM, pH 10.5, 1
mL) containing 60mM NaCl and 83 mM Na CO . The fluorescence
2 3
at 450nm excited at 365 nm was measured for the solution.

K. Sasaki et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2821–2823
2823
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In conclusion, we have presented experimental evi-
dences supporting the hypothesis that 6-sulfo-d-
GlcNAc may serve as a simple Neu5Ac mimic. Though
the inhibitory activity may not be strong enough for
practical applications, chemical tuning at the aglycon
and C4 positions in the 6-sulfo-d-GlcNAc skeleton as
well as multivalent design are expected to integrate bio-
+
ꢃ
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3
one of the most abundant carbohydrates occurring in
nature, the hypothesis and experimental evidences pre-
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1632.
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1
1
4
300 MHz, D O, room temp) 5.28 (d, 1H, J1,2=8.7 Hz, H-1),
2
studies
have disclosed that GlcNAc-6-O-sulfo-
4
.41 (dd, 1H, JH-5, H-6proS=2.1 Hz, JH-6proS, H-6proR=11.1 Hz,
transferases are expressed widely in human endothelial
tissues. The enzymes recognize an endo-type GlcNAc
residue as the acceptor substrate and contribute possi-
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saccharides. The present study has suggested also that
the sulfotransferases may have a certain role com-
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an exo-terminal b-d-galacto-residue.
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1
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H-6proR=1.7 gg+10.8 gt+4.1 tg; 100=gg+gt+tg.
1
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Acknowledgements
1
The study was supported by grants from the Japan
Society for the Promotion of Science Research Fellow-
ship for young scientists (K.S.) and the 21st Century
COE program ‘Nature-Guided Materials Processing’ of
the Ministry of Education, Culture, Sport, Science and
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