Design of N-acetyl-6-sulfo-β-D-glucosaminide-based inhibitors of influenza virus sialidase

Article abstract of DOI:10.1016/j.bmc.2004.01.013

Biological activity of N-acetyl-6-sulfo-β-D-glucosaminides (6-sulfo-GlcNAc 1) having a structural homology to N-acetylneuraminic acid (Neu5Ac 2) and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en 3) was examined in terms of inhibitory activity against influenza virus sialidase (influenza, A/Memphis/1/71 H3N2). pNP 6-Sulfo-GlcNAc 1a was proved to show substantial activity to inhibit the virus sialidase (IC₅₀=2.8 mM), though p-nitrophenyl (pNP) GlcNAc without 6-sulfo group and pNP 6-sulfo-GlcNH₃⁺ 1b without 2-NHAc showed little activity (IC₅₀ >50 mM). The activity was enhanced nearly 100-fold when the pNP group of 1a was converted to p-acetamidophenyl one 5 (IC₅₀=30 μM) or replaced with 1-naphthyl 6 (IC₅₀=10 μM) or n-propyl one 8 (IC₅₀=11 μM).

Full text of DOI:10.1016/j.bmc.2004.01.013

Bioorganic & Medicinal Chemistry 12 (2004) 1367–1375
Design of N-acetyl-6-sulfo-ꢀ-D-glucosaminide-based inhibitors of
influenza virus sialidase
Kenji Sasaki,a,d Yoshihiro Nishida,a,d,* Mikie Kambara,a Hirotaka Uzawa,b
Tadanobu Takahashi,c,d Takashi Suzuki,c,d Yasuo Suzukic,d and Kazukiyo Kobayashia,d,
*
aDepartment of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Chikusa-ku,
Nagoya 464-8603, Japan
bResearch Center of Advanced Bionics, National Institute of Advanced Industrial Science and Technology (AIST), Central 5,
1-1-1 Higashi, Tsukuba 305-8565, Japan
cDepartment of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
dCREST, Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi 332-0012, Japan
Received 4November 2003; revised 10 January 2004; accepted 14January 2004
Abstract—Biological activity of N-acetyl-6-sulfo-b-d-glucosaminides (6-sulfo-GlcNAc 1) having a structural homology to N-acetyl-
neuraminic acid (Neu5Ac 2) and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en 3) was examined in terms of inhibitory
activity against influenza virus sialidase (influenza, A/Memphis/1/71 H3N2). pNP 6-Sulfo-GlcNAc 1a was proved to show sub-
stantial activity to inhibit the virus sialidase (IC50=2.8 mM), though p-nitrophenyl (pNP) GlcNAc without 6-sulfo group and pNP
6-sulfo-GlcNH+3 1b without 2-NHAc showed little activity (IC50 >50 mM). The activity was enhanced nearly 100-fold when the
pNP group of 1a was converted to p-acetamidophenyl one 5 (IC50=30 mM) or replaced with 1-naphthyl 6 (IC50=10 mM) or
n-propyl one 8 (IC50=11 mM).
# 2004Elsevier Ltd. All rights reserved.
1.Introduction
essential roles at the stage of infection and propagation.
For example, a vibrio cholerae sialidase cleaves sialyl
linkages of gangliosides to expose GM1 on the host cells.
This process allows a cholerae toxin to bind and inter-
nalize into the host cells.5a Influenza viruses utilize sia-
lidases to avoid self-aggregation by their hemagglutinins
and to leave from the host cell during budding pro-
cess.5b These microbial events indicate that sialidase
inhibitors can become good candidates of anti-influenza
medicines. Actually, several sialidase inhibitors with a
skeleton of Neu5Ac2en 3 have been developed and
applied for anti-influenza therapy like the well-known
case of Zanamivir 4.6
Sialic acids are highly functional molecules bearing a
carboxylate anion, an acetamide (or N-glycolyl) group,
and an acyclic side chain in the nine-carbon skeleton as
represented by N-acetyl neuraminic acid (Neu5Ac 2,
Fig. 1).1,2 They are linked to the non-reducing terminal
of oligosaccharides in cell surface glycoproteins and
glycolipids. The sialyl oligosaccharides are involved in
various cell–cell recognition events, which include
microbe infection to host cells, fertilization, and meta-
stasis of cancer cells.1,2 Therefore, the mimic design of
sialyl linkages, especially for sialyl LewisX and Lewisa
antigens, is thought to lead to the development of sac-
charide-based medicines and biomaterials.3
Along with our continuous studies on the design of oli-
gosaccharide mimics and their polyvalent models based
on the concept of a carbohydrate module,7 we recently
found that an acrylamido copolymer of N-acetyl-6-
sulfo-b-d-glucosaminide (6-sulfo-GlcNAc) inhibits the
binding interaction between sialyl LewisX and L-selec-
tin.8 The result led us to the speculation that the 6-sulfo-
GlcNAc module might serve as a mimic of sialyl lin-
kages. We have tried to verify this speculation and
Much interest is being directed also to the design of
inhibitors to viral and bacterial sialidases4 which play
Keywords: Sialidases; Inhibitors; Sulfo-sugars; Influenza virus.
* Corresponding authors. Tel.: +81-52-789-2488; fax: +81-52-789-
2528; e-mail addresses: nishida@mol.nagoya-u.ac.jp (Y. Nishida);
kobayash@mol.nagoya-u.ac.jp (K. Kobayashi)
0968-0896/$ - see front matter # 2004Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.01.013

1368
K. Sasaki et al. / Bioorg. Med. Chem. 12 (2004) 1367–1375
showed the experimental evidence in a preceding study9
that p-nitrophenyl (pNP) 6-sulfo-b-d-GlcNAc 1a has
substantial inhibitory activity to vibrio cholerae siali-
dase. In this paper, we describe that the pNP 6-sulfo-b-
d-GlcNAc 1a and its derivatives 5–9 (Schemes 1 and 2)
display inhibitory activity also to influenza virus siali-
dase. The results enable us to confirm that the skeleton
1 provides a new class of sialyl mimics giving a common
basis to the design of sialidase inhibitors.
acyclic side chain in the latter. The difference suggests
that introduction of an appropriate aglycon in 1 may
induce strong activity. It is also of practical meaning
that the difference at the aglycon can be modulated
variably in chemical ways. Such expectation prompted
us to prepare a series of 6-sulfo-GlcNAc derivatives 5–9
with various groups at the aglycon (phenyl, naphthyl,
alkyl, and glycerol) (Schemes 1 and 2) and examine their
biological activities against an influenza virus sialidase.
2.Results and discussion
2.1. Design and synthesis of 6-sulfo-GlcNAc derivatives
5–9 with various aglycon groups
Though sulfonated glycosaminoglycans10 and inorganic
sulfates11 are reported to inhibit several sialidases, little
structural basis has been provided to rationalize the
activity. Our preceding study9 disclosed that pNP
6-sulfo-b-d-GlcNAc 1a shows structure-dependent activ-
ity against the vibrio cholerae sialidase (IC50=2 mM).
Instead of Neu5Ac 2 with a typical 1C4 (chair) con-
formation, the 2-ene compound 3 is thought to simulate
intermediates at the transitional states in sialidase reac-
tions and thus provide a key molecular basis for the
inhibitor design.6 As can be seen in Figure 1, the skele-
ton of 6-sulfo-GlcNAc 1 and 1a (R=pNP) has a struc-
tural homology not only to Neu5Ac 2 but also to
Neu5Ac2en 3 in spatial relationship (distance and heli-
city) between NHAc and anionic groups. The structural
homology implies that the simple GlcNAc may provide
an alternative structural basis for the design of sialidase
inhibitors. Recently, Schworer and Schmidt also cited a
homology between GlcNAc and Neu5Ac 2 for the
design of CMP-sialyltransferase inhibitors showing
the versatility of this idea.12
Scheme 1. Reagents and conditions: (a) (i) Pd(OH)2/C, H2, H2O, rt;
(ii) Ac2O, K2CO3, H2O, rt, 80% (2 steps); (b) 1- or 2- naphthol,
TBAB, 1N NaOH aq/CH2Cl2, rt, 66% (for 12), 65% (for 13); (c)
NaOMe, MeOH, rt, 99% (for 14) and 99% (for 15); (d) Me3NSO3,
DMF, 50 ꢀC, 40% (for 6), 45% (for 7). TBAB=tetrabutylammonium
bromide.
As depicted in Figure 1, a major difference between 1
and 2 (or 3) arises at the aglycon moiety of the former
(pNP group in the case of 1a) that corresponds to an
Scheme 2. Reagents and condiꢀtions: a) Pd(OH)2/C, H2, MeOH, rt,
99%; (b) Me3NSO3, DMF, 4 0 C, 53%; (c) Ac2O, pyr, rt, 98%; (d)
AD-mix, tert-BuOH/H2O, 0 ꢀC 80%; (e) Me2C(OMe)2, PPTS, DMF,
rt, 60%; (f) (i) NaOMe, MeOH, rt; (ii) Me3NSO3, DMF, 40 ꢀC; (iii)
TFA, H2O, MeOH, rt, 34% (3 steps). PPTS=pyridinium p-toluene-
sulfonic acid, TFA=trifluoroacetic acid.
Figure 1. Structure of 6-sulfo-b-d-GlcNAc skeleton 1 having homol-
ogy to Neu5Ac 2. Neu5Ac2en 3 is considered as a putative transition
state-like analogue in sialidase reaction.

K. Sasaki et al. / Bioorg. Med. Chem. 12 (2004) 1367–1375
1369
Compound 5 with a 4-acetamidophenyl group was
derived from 1a in the conventional way of catalytic
hydrogenation in water and N-acetylation with acetic
anhydride. Here, it should be noted that 4-acet-
amidophenol in 5 per se is the analgesic and antipyretic
medicine called acetaminophen.13 Considering the pos-
sible metabolizing pathway of 6-sulfo-GlcNAc deriva-
tives, we expect that the p-acetamidophenyl glycoside
may represent a promising skeleton of O-glycoside-
based medicines. Naphthyl compounds 6 and 7 were
prepared from 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
a-d-glucopyranosyl chloride 1114 in the glycosylation
with either 1- or 2-naphthol under phase-transfer cata-
lytic conditions.15 Direct O-sulfonation using Me3NSO3
in DMF16 successfully gave the corresponding 6-O-sul-
fated GlcNAc derivatives in moderate yields. Thus, 1-O-
aryl glycosides 1a, 5, 6, and 7 were available in a com-
mon way and used as key models to investigate the
effect of aryl groups on sialidase inhibitory activity.
data summarized in Table 1 indicated that the 6-sulfo-
GlcNAc compound 1a showed substantial activity
(IC50=2.8 mM), while neither pNP GlcNAc nor 1b
showed activity. These results, similar to those observed
for the vibrio cholerae sialidase (IC50=2 mM) in our
preceding studty,9 allowed us to confirm that 6-sulfo
and 2-NHAc groups are recognized as key functional
groups also by the influenza virus enzyme. The similar
activity of 1a for both bacterial and viral sialidases may
be ascribed to the conserved topology of their catalytic
domains in the active site.4
The model compounds 5–9 subjected to the same assay
had variable activities depending on the aglycon struc-
tures (Table 2). Interestingly, compound 5 showed ca.
90 times stronger activity (IC50=33 mM) than 1a, in
spite of the fact that there was no diversity between the
two structures except for the p-substituting groups (p-
nitro and p-acetamido). The enhancement should be
ascribable to the higher affinity of the p-acet-
amidophenyl group in 5 to this enzyme than that of the
p-nitrophenyl one in 1a.
n-Propyl glycoside 8 was prepared from allyl N-acetyl-
b-d-glucosaminide 1617 obtained as a major product in
the reaction of GlcNAc and allyl alcohol (60–70 ꢀC) in
the presence of trimethylsilyl chloride (TMSCl). Here, the
TMSCl accelerates the allyl glycosidation by trapping
water molecules in the form of TMSOH. The 1,2-diol
compound 9 was prepared as a mixture of diastereomers
(55: 45, 1H NMR analysis) in the dihydroxylation of tri-
O-acetyl derivative 18 and AD-mix.18 A mixture of the
diastereomers, which could be hardly separated from
each other, was applied to an inhibitory assay to see
how the hydrophilic aglycon in 9 works for the activity
in comparison with the hydrophobic aglycon in 8 and
others.
Compound 7 with a 2-naphthyl group was less active
than 5, while compound 6 with a 1-naphthyl group was
much more active (IC50=10 mM) (Fig. 2). A large
hydrophobic group at the aglycon may change the
morphology of the glycoside to take a supra-molecular
structure in aqueous solution. The assembling structure
Table 1. Inhibition activity of pNP-GlcNAc derivatives against
influenza virus sialidase
Compd
Aglycon
IC50 (mM)a
1a
2.8
ndb
nd
2.2. Inhibitory activities of 6-sulfo-GlcNAc derivatives
against influenza virus sialidase
1b (2-NH+3
)
An influenza virus (A/Memphis/1/71 H3N2) was used
to evaluate the biological potential of the synthetic 6-
sulfo-GlcNAc derivatives as anti-influenza agents. The
assay was conducted with the authentic fluorescent
method19 utilizing 20-(4-methylumbelliferyl)-a-d-N-
acetylneuraminic acid as a substrate. Though the
substrate is non-fluorescent, the enzymic hydrolysis
generates the fluorescent 4-methylumbelliferone detect-
able at Em 450 nm (Ex 365 nm). As reference com-
pounds, pNP GlcNAc (6-OH) without a 6-sulfo group
and pNP 6-sulfo-GlcNH+3 1b (Scheme 3) without
2-NHAc were also applied to the assay to investigate
the role of each of the two functional groups. The assay
pNP GlcNAc (6-OH)
a The mean of duplicate experiments.
b Not detected.
Table 2. Inhibition activity of 6-sulfo-GlcNAc derivatives having
various aglycons against influenza virus sialidase
Compd
Aglycon
IC50 (mM)a
0.033
5
6
7
0.010
0.110
8
9b
–CH2CH2CH3
–CH2CH(OH)CH2OH
0.011
0.019
a The mean of duplicate experiments.
b Tested as a racemic mixture.
Scheme 3. Reagents and conditions: (a) Me3NSO3, DMF, 4 0ꢀC, 67%;
(b) 0.1 N NaOH aq, rt, 89%.

1370
K. Sasaki et al. / Bioorg. Med. Chem. 12 (2004) 1367–1375
Figure 2. Inhibitory activities of 6-sulfo-GlcNAc derivatives against
influenza sialidase. The degree of inhibition (%, deviation Æ5%) was
obtained as an averaged value for duplicate experiments using an
equation as follows. Inhibition (%)=100ÂFi/Fo, where Fi and Fo
indicate the strength of fluorescence at Em. 450 nm (Ex. 365 nm) in the
presence and the absence of inhibitors, respectively.
Figure 3. A proposed binding mode of 6-sulfo-GlcNAc derivatives
with influenza virus sialidase referred to X-ray data of Varghese et
al.20 and Kim et al.6c
often induces strong biological activity due to the effect
of a carbohydrate cluster. In the present case, however,
the large difference in the activity between 5 and 7,
having analogous hydrophobic groups to each other, is
apparently ascribed to the discrimination between the 1-
and 2-naphtyl aglycons by this enzyme. It is probable
that the enzyme may give a hydrophobic pocket at the
position accepting the 1-naphthyl preferentially. Com-
pound 8 with a n-propyl group also showed potent
activity close to that of 6, suggesting that the hydro-
phobic pocket admits both 1-naphthyl and the flexible
alkyl groups. The activity was decreased when the
hydrophobic aglycon was replaced with the glycerol in
compound 9 (IC50=19 mM).
aglycon groups are recognized, respectively, by the
arginine triad in a way similar to the carboxylate anion
and the hydrophobic pocket proposed for the alkyl
groups.
The data in Table 2 have also disclosed that every model
compound 5–9 is more active than the pNP derivative
1a. This fact should mean that the p-nitro group is dis-
favored by the hydrophobic and hydrophilic domains to
show seemingly a negative effect on the inhibitory
activity. Consequently, among the synthetic models
examined here, 1-naphthyl and n-propyl glycosides (6
and 8) showed the highest activity, indicating that
introduction of these hydrophobic groups at the agly-
con improves the inhibitory activity significantly.
The above results have indicated that introduction of
hydrophobic groups into the aglycon of 1 is more effec-
tive than that of hydrophilic ones, though natural sialic
acids (2 and 3) bear a 1,2,3-triol function at the corres-
ponding position. However, as judged from the stronger
activity of 9 than that of 5 and 7, it is obvious that this
enzyme provides a hydrophilic domain for the recogni-
tion of the acyclic 1,2,3-triol moiety in 2.
3.Conclusion
We have demonstrated the notable activity of 6-sulfo-
GlcNAc derivatives inhibiting an influenza virus siali-
dase. Though the observed activities are still poorer
than that of Zanamivir 46a,b (IC50=0.3 mM, in our
inhibition assay), it is of significance that the 6-sulfo-d-
GlcNAc skeleton can be used for the development of
sialidase inhibitors. This is because N-acetyl-d-glucos-
amine is one of the most abundant carbohydrate
resources in nature. Moreover, the biological activity
can be modulated in a variable way by changing the
aglycon. It may be also of interest to replace the 6-sulfo
group with a 6-sulfamide group possessing higher bio-
logical and chemical stability.
By X-ray crystalline analysis of a complex of influenza
sialidase and natural Neu5Ac, Varghese et al.20 dis-
closed that the enzyme possesses mainly three binding
sites for sialyl acceptor substrates, that is, an arginine
triad domain for the carboxylate anion, a hydrophobic
pocket for the NHAc group, and a hydrophilic one for
6c
the acyclic side chain. More recently, Kim et al.
reported the X-ray crystalline data of a complex of
influenza sialidase and shikimic acid-based inhibitor.
There, they have shown that the enzyme locates a
hydrophobic pocket in the vicinity of the hydrophilic
domain and turns to use it for artificial substrates bear-
ing alkyl chains at the corresponding position. These
X-ray data well accord with the present experimental
data. A binding mode for the 6-sulfo-GlcNAc may be
thus illustrated with a cartoon model as shown in Fig-
ure 3, where the 6-sulfo group and the hydrophobic
We envision the possibility that the inhibitory activity of
6-sulfo-d-GlcNAc has a certain biological meaning.
This is because the skeleton is involved widely in mam-
malian cell surface oligosaccharides (glycosaminogly-
cans and glycoproteins). The oligosaccharides including
6-sulfo-GlcNAc are associated with GlcNAc-6-O-sulfo-
transferases distributed in human endothelial cells to
give 6-sulfo-d-GlcNAc residues in the 6-sulfo-sialyl

K. Sasaki et al. / Bioorg. Med. Chem. 12 (2004) 1367–1375
1371
Lewisx motif.21 Moreover, mammalian tissues are
known to express sialidases,22 which can potentially
modulate the biological function of these sialyl glyco-
conjugates.23 The behavior of the synthetic 6-sulfo-d-
GlcNAc derivatives against the mammalian sialidases
may provide key insights into the role of the natural 6-
sulfo-d-GlcNAc linkages and the mammalian enzymes.
The study along this line presently in progress will be
reported elsewhere.
10.0 Hz, H-3), 3.61 (dd, 1H, J=9.0, 9.5 Hz, H-4), 2.13,
2.02 (sÂ2, 6H, acetamido groups); HRFAB+MS m/z:
479.0715 (calcd for C16H21N2Na2O10S 479.0712
[M+Na] ). Anal. calcd for C16H21N2NaO10S 2H2O: C,
39.03; H, 5.12; N, 5.69. Found: C, 38.34; H, 5.19; N, 5.34.
+
.
4.2. Naphthyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-ꢀ-
D-glucopyranosides (12) and (13)
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-glucopyranosyl
chloride 1114 (200 mg, 0.55 mmol), tetrabutyl-
ammonium bromide (176 mg, 0.55 mmol), and 1- or 2-
naphthol (157 mg, 1.09 mmol) in CH2Cl2 (2.5 mL) and
1 M NaOH aq (2.5 mL) were vigorously stirred at
room temperature. After complete consumption of the
chloride (50 min), the reaction mixture was worked up
by adding ethyl acetate. The organic layer was succes-
sively washed with 1M NaOH aq, water and, finally,
with saturated brine. The organic phase was dried over
MgSO4, concentrated, purified by silica gel column
chromatography (hexane: ethyl acetate=1:2!1:3), and
crystallized from EtOH to give respectively 1-naphthyl
derivative 12 (171 mg 66%) and 2-naphthyl derivative
13 (169 mg 65%) as white powder. 12:15a mp 210–
212 ꢀC; [a]D=À63.5ꢀ (c=0.20 in MeOH); IR (KBr)
4.Experimental
Reagents of the highest commercial quality were pur-
chased and used without further purification. Unless
stated otherwise, reactions were performed in well-dried
glassware under an inert atmosphere of N2 and followed
by thin-layer chromatography (TLC) on Merck alumi-
num roll silica gel 60-F254 and/or glass plate RP-18 F254
to visualize with UV light and p-anisaldehyde-sulfuric
acid in EtOH. Merck silica gel 60 (particle size 40–63
mm) and LiChroprep1 RP-18 (particle size 40–63 mm)
were employed for column chromatography. Dowex1
+
(AG-50WÂ4 , Na form) ion-exchange resin was used
for purification on sulfonation steps.
1
1747 and 1232 (O-acetyl), 1619 and 1556 (amide); H
1H NMR (500 MHz and 300 MHz) spectra were recor-
ded at ambient temperature with a Varian Inova 500 (or
300) spectrometer equipped with a Sun workstation.
Optical rotations were determined on a JASCO DIP-
1000 digital polarimeter at ambient temperature. IR
spectra were recorded on a JASCO FT/IR-230 Fourier
transform infrared spectrometer in the form of KBr
disk. Mass spectra were taken on a JEOL JMS700 mass
spectrometer under FAB conditions with an appro-
priate matrix. Elemental analyses were performed with
PE2400 II CHN/O Analyzer. Fluorescence spectra were
recorded on a JASCO FP-777 spectrofluorometer.
NMR (500 MHz, CDCl3) d 8.17 (m, 1H, H-9 of naph-
thyl), 7.79 (m, 1H, H-6 of naphthyl), 7.53 (d, 1H, J=8.0
Hz, H-4of naphthyl), 7.49 (m, 2H, H-7, H-8 of naph-
thyl) 7.38 (t, 1H, J=8.0, 8.0 Hz, H-3 of naphthyl) 7.03
(d, 1H, J=8.0 Hz, H-2 of naphthyl), 5.57 (d, 1H, J=9.5
Hz, amide), 5.31 (dd, 1H, J=10.5, 10.5 Hz, H-3), 5.24
(d, 1H, J=8.5 Hz, H-1), 5.23 (dd, 1H, J=10.5, 10.5 Hz,
H-4), 4.54 (m, 1H, H-2), 4.33 (dd, 1H, J=5.5, 12.5 Hz,
H-6proR), 4.21 (d, 1H, J=2.5, 12.0 Hz, H-6proS), 3.93 (m,
1H, H-5), 2.10, 2.09, 2.07 (sÂ3, 9H, acetyl), 1.95 (s, 3H,
acetamido); FAB+MS: 474 [M+H]+. Anal. calcd for
C24H27NO9: C, 60.88; H, 5.75; N, 2.96. Found: C, 60.80;
H, 5.71; N, 2.81. 13:15a mp 217–219 ꢀC; [a]D=À12.7ꢀ
(c=0.20 in MeOH); IR (KBr) 1749 and 1224 (O-acetyl),
1658 and 1535 (amide); 1H NMR (500 MHz, CD3OD) d
7.79 (d, 2H, J=9.0 Hz, H-4and H-9 of naphthyl), 7.76
(d, 1H, J=8.0 Hz, H-6 of naphthyl), 7.44 (m, 1H, H-7
of naphthyl), 7.41 (d, 1H, J=2.5 Hz, H-1 of naphthyl),
7.36 (m, H-8 of naphthyl), 7.18 (dd, 1H, J=2.5, 9.0 Hz,
H-3 of naphthyl), 5.45 (d, 1H, J=8.0 Hz, H-1), 5.37
(dd, 1H, J=10.5, 10.5 Hz, H-3), 5.09 (dd, 1H, J=10.5,
10.5 Hz, H-4), 4.33 (dd, 1H, J=5.5, 12.5 Hz, H-6proR),
4.16 (m, 2H, H-2 and H-6proS), 4.08 (m, 1H, H-5), 2.04,
2.03, 2.02 (sÂ3, 9H, acetyl), 1.93 (s, 3H, acetamido);
FAB+MS: 474 [M+H]+. Anal. calcd for C24H27NO9:
C, 60.88; H, 5.75; N, 2.96. Found: C, 60.78; H, 5.70; N,
2.81.
4.1. 4-Acetamidophenyl 2-acetamido-2-deoxy-6-O-sulfo-
nate-ꢀ-D-glucopyranoside (5)
To a H2O (2 mL) solution of pNP 6-sulfo-b-d-GlcNAc
1a (50 mg, 0.11 mmol) was added a catalytic amount of
Pd(OH)2/C and the mixture was stirred under H2
atmosphere. After stirring at room temperature for 2 h,
the mixture was filtered through Celite pad and con-
centrated in vacuo. The residue was dissolved in H2O (2
mL) again. K2CO3 (46 mg, 0.33 mmol) was added to the
solution and the mixture was stirred at 0 ꢀC. Ac2O (32
mL, 0.33 mmol) was added dropwise to the cool solution
and stirred at 0 ꢀC for 3 h. The reaction mixture was
concentrated and purified by reverse phase column
chromatography (H2O). After treating the fraction with
ion-exchange resin, the residue was lyophilized to afford
5 (41 mg 80%) as a white solid. [a]D=À78.9ꢀ (c=0.11
in H2O); IR (KBr) 3299 (OH), 1660 and 1548 (amide),
4.3. 1-Naphthyl 2-acetamido-2-deoxy-ꢀ-D-glucopyranoside
(14)
1226 (OSO3); H NMR (500 MHz, D2O, 30 ꢀC) d 7.33
To a MeOH solution (10 mL) of 12 (140 mg, 0.30
mmol) was added a catalytic amount of NaOMe. After
stirring at room temperature for 1 h, the mixture was
neutralized with ion exchange resin and filtered and
then the filtrate was concentrated in vacuo to give a
white powder 1415a (100 mg, 99%). Mp 269–271 ꢀC;
1
(d, 2H, J=9.0 Hz, Hmetha of phenyl group), 7.06 (d, 2H,
J=9.0 Hz, Hortho of phenyl group), 5.12 (d, 1H, J=8.5
Hz, H-1), 4.37 (dd, 1H, J=2.0, 11.5 Hz, H-6proS), 4.23
(dd, 1H, J=5.5, 11.5 Hz, H-6proR), 3.97 (dd, 1H, J=8.0,
10.5 Hz, H-2), 3.84(m, 1H, H-5), 3.65 (dd, 1H, J=10.0,

1372
K. Sasaki et al. / Bioorg. Med. Chem. 12 (2004) 1367–1375
[a]D=À68.6ꢀ (c=0.1 in MeOH); IR (KBr) 3299 (OH),
1619 and 1556 (amide); 1H NMR (500 MHz, CD3OD) d
8.17 (m, 1H, H-9 of naphthyl), 7.83 (m, 1H, H-6 of
naphthyl), 7.50 (d, 1H, J=8.0 Hz, H-4of naphthyl),
7.44 (m, 2H, H-7, H-8 of naphthyl) 7.38 (t, 1H, J=8.0,
8.0 Hz, H-3 of naphthyl) 7.18 (d, 1H, J=8.0 Hz, H-2 of
naphthyl), 5.11 (d, 1H, J=8.5 Hz, H-1), 4.18 (dd, 1H,
J=8.5, 10.0 Hz, H-2), 3.97 (dd, 1H, J=3.0, 12.3 Hz, H-
6proS), 3.75 (dd, 1H, J=5.5, 12.3 Hz, H-6proR), 3.57 (dd,
1H, J=10.5, 10.5 Hz, H-4), 3.53 (m, 1H, H-5), 3.48
(dd, 1H, J=10.0, 10.5 Hz, H-3), 1.95 (s, 3H, acetam-
ido); FAB+MS: 348 [M+H]+. Anal. calcd for
60.67; H, 6.22; N, 3.93. Found: C, 60.99; H, 5.99; N,
3.78.
4.6. 2-Naphthyl 2-acetamido-2-deoxy-6-O-sulfonate-ꢀ-D-
glucopyranoside sodium salt (7)
To a DMF (4mL) solution of 15 (123 mg, 0.35 mmol)
was added Me3NSO3 complex (246 mg, 1.77 mmol) in
DMF (8 mL). After stirring at 50 ꢀC for 3 h, the mixture
was quenched with MeOH (5 mL) and stirred for 12 h.
The residue was concentrated and purified by reverse
phase silica gel column chromatography (H2O to H2O:
MeOH=3:1). The fraction was treated with ion-
exchange resin and lyophilized to afford 7 (72 mg 45%)
as a white amorphous. [a]D=-80.6ꢀ (c=0.12 in H2O);
IR (KBr) 3396 (OH), 1656 and 1548 (amide), 1253
.
C18H21NO6 H2O: C, 59.17; H, 6.34; N, 3.83. Found: C,
59.33; H, 6.18; N, 3.70.
4.4. 1-Naphthyl 2-acetamido-2-deoxy-6-O-sulfonate-ꢀ-D-
glucopyranoside sodium salt (6)
(OSO3); H NMR (300 MHz, D2O, 30 ꢀC) d 7.64(m,
1
3H, H-4, H-9 and H-6 of naphthyl), 7.34 (m, 1H, H-7 of
naphthyl), 7.27 (m, 2H, H-8 and H-1 of naphthyl), 7.03
(dd, 1H, J=2.4, 9.0 Hz, H-3 of naphthyl), 5.12 (d, 1H,
J=8.7 Hz, H-1), 4.23 (dd, 1H, J=2.1, 11.5 Hz, H-
6proS), 4.07 (dd, 1H, J=5.7, 11.5 Hz, H-6proR), 3.89 (dd,
1H, J=8.4, 9.9 Hz, H-2), 3.67 (m, 1H, H-5), 3.56 (dd, 1H,
J=9.9, 9.6 Hz, H-3), 3.44 (dd, 1H, J=9.6, 9.6 Hz, H-4),
1.87 (s, 3H, acetamido); HRFAB+MS m/z: 472.0658
(calcd for C18H20NNa2O9S 472.0654 [M+Na]+). Anal.
To the DMF (4mL) solution of 14 (100 mg, 0.34mmol)
was added Me3NSO3 complex (236 mg, 1.75 mmol) in
DMF (8 mL). After stirring at 50 ꢀC for 3 h, the mixture
was quenched with MeOH (5 mL) and stirred for 12 h.
The residue was concentrated and purified by reverse
phase silica gel column chromatography (H2O to H2O:
MeOH=3:1). The fraction was treated with ion-
exchange resin and lyophilized to afford 6 (60 mg 40%)
as a white solid. [a]D=À89.6ꢀ (c=0.10 in H2O); IR
(KBr) 3292 (OH), 1652 and 1552 (amide), 1236 (OSO3);
1H NMR (500 MHz, D2O, 30 ꢀC) d 8.08 (m, 1H, H-9 of
naphthyl), 7.93 (m, 1H, H-6 of naphthyl), 7.65 (d, 1H,
J=8.5 Hz, H-4of naphthyl), 7.58 (m, 2H, H-7, H-8 of
naphthyl) 7.49 (t, 1H, J=8.0, 8.0 Hz, H-3 of naphthyl)
7.26 (dd, 1H, J=0.7, 7.7 Hz, H-2 of naphthyl), 5.27 (d,
1H, J=8.5 Hz, H-1), 4.44 (d, 1H, J=2.0, 11.5 Hz, H-
6proS), 4.27 (dd, 1H, J=5.5, 11.5 Hz, H-6proR), 4.20 (dd,
1H, J=8.0, 10.5 Hz, H-2), 3.96 (m, 1H, H-5), 3.70 (dd,
1H, J=10.5, 9.5 Hz, H-3), 3.65 (dd, 1H, J=9.5, 9.5 Hz,
H-4), 1.93 (s, 3H, acetamido); HRFAB+MS m/z:
472.0661 (calcd for C18H20NNa2O9S 472.0654
.
calcd for C18H20NNaO9S 0.5H2O: C, 47.16; H, 4.62; N,
3.06. Found: C, 47.48; H, 4.74; N, 3.04.
4.7. Allyl 2-acetamido-2-deoxy-ꢀ-D-glucopyranoside (16)
To an allylalcohol (30 mL) solution of N-acetyl-d-glu-
cosamine (2.0 g, 7.04mmol) was added TMSCl (0.01 eq
for GlcNAc) and the reaction mixture was stirred at 60–
80 ꢀC for 30 min. After cooling to room temperature,
the reaction mixture was neutralized with Et3N and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (CHCl3:MeOH=10:1) and
crystallized from EtOH to afford 1617 (704mg, 35%) as a
colorless crystal. Mp 160–163 ꢀC; [a]D=À28.4ꢀ (c=0.42
in MeOH); IR (KBr) 3284(OH), 1652 and 1564
+
.
[M+Na] ). Anal. calcd for C18H20NNaO4S 4.5H2O: C,
40.75; H, 5.51; N, 2.64. Found: C, 40.47; H, 4.95; N,
2.36.
1
(amide); H NMR (500 MHz, CD3OD) d 5.88 (m, 1H,
¼CH in allyl group), 5.25 (ddd, 1H, J=1.5, 3.0, 17.0
Hz, ¼CHcisHtrans in allyl group), 5.15 (ddd, 1H, J=1.5,
3.0, 10.5 Hz, ¼CHcisHtrans in allyl group), 4.42 (d, 1H,
J=8.5 Hz, H-1), 4.32 (m, 1H, OCH2 in allyl group),
4.06 (m, 1H, OCH2 in allyl group), 3.86 (dd, 1H, J=2.5,
12.3 Hz, H-6proS), 3.65 (dd, 1H, J=4.0, 12.3 Hz, H-
6proR), 3.32 (dd, 1H, J=8.5, 11.0 Hz, H-2), 3.26 (m, 2H,
H-3 and H-4), 3.23 (ddd, J=2.5, 5.0, 10.5 Hz, H-5),
1.96 (s, 3H, acetamido); HRFAB+MS m/z: 262.1299
(calcd for C11H20NO6 262.1291 [M+Na]+). Anal. calcd
for C11H21NO6: C, 50.18; H, 8.04; N, 5.32. Found: C,
49.01; H, 8.13; N, 5.05.
4.5. 2-Naphthyl 2-acetamido-2-deoxy-ꢀ-D-glucopyrano-
side (15)
To a MeOH solution (10 mL) of 13 (150 mg, 0.31
mmol) was added a catalytic amount of NaOMe. After
stirring at room temperature for 1 h, the mixture was
neutralized with ion exchange resin and filtered and
then the filtrate was concentrated in vacuo to give a
white powder of 1515a (110 mg, 99%). Mp 245–248 ꢀC;
[a]D=+16.4ꢀ (c=0.20 in MeOH); IR (KBr) 3386 (OH),
1658 and 1535 (amide); 1H NMR (500 MHz, CD3OD) d
7.76 (m, 3H, H-4, H-9 and H-6 of naphthyl), 7.42 (m,
2H, H-7 and H-1 of naphthyl), 7.34(m, 1H, H-8 of
naphthyl), 7.18 (dd, 1H, J=2.5, 9.0 Hz, H-3 of naph-
thyl), 5.19 (d, 1H, J=8.5 Hz, H-1), 3.97 (dd, 1H, J=8.5,
10.5 Hz, H-2), 3.95 (dd, 1H, J=2.5, 12.3 Hz, H-6proS),
3.74(dd, 1H, J=6.0, 12.3 Hz, H-6proR), 3.60 (dd, 1H,
J=10.5, 10.5 Hz, H-3), 3.53 (m, 1H, H-5), 3.44 (dd, 1H,
4.8. n-Propyl 2-acetamido-2-deoxy-ꢀ-D-glucopyranoside
(17)
To a MeOH (20 mL) solution of 16 (500 mg, 1.90
mmol) was added a catalytic amount of Pd(OH)2/C.
After vigorously stirring under H2 atmosphere for 1.5 h,
the mixture was filtered through Celite pad and con-
centrated in vacuo. The residue was crystallized from
J=10.3, 10.0 Hz, H-4), 1.99 (s, 3H, acetamido); FAB+MS:
+
.
348 [M+H] . Anal. calcd for C18H21NO6 0.5H2O: C,

K. Sasaki et al. / Bioorg. Med. Chem. 12 (2004) 1367–1375
1373
EtOH to afford 1724 (495 mg, >99%) as a white crystal.
Mp 183–184 ꢀC; [a]D=À28.9ꢀ (c=0.64in MeOH). IR
(KBr) 3270 (OH), 1654and 1556 (amide); 1H NMR
(500 MHz, D2O, 30 ꢀC) d 4.44 (d, 1H, J=8.5 Hz, H-1),
3.83 (dd, 1H, J=1.5, 12.5 Hz, H-6proS), 3.77 (m, 1H,
OCH2 in propyl group), 3.66 (m, 1H, H-6proR), 3.60 (dd,
1H, J=8.5, 10.5 Hz H-2), 3.46–3.36 (m, 4H, H-5, H-3,
1.99, 1.97 (sÂ3, 9H, acetyl), 1.89 (s, 3H, acetamido).
Anal. calcd for C17H25NO9: C, 52.71; H, 6.50; N, 3.62.
Found: C, 52.77; H, 6.50; N, 3.49
4.11. 1 (and 3)-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
ꢀ-D-glycopyranosyl)-sn-glycerol (19)
H-4and OCH in propyl group), 1.96 (s, 3H, acetam-
2
AD-mix-a (5.41 g) was dissolved in tert-BuOH (5 mL)
and H2O (10 mL). Stirring at room temperature pro-
duced two clear phases. The mixture was cooled to 0 ꢀC,
and 18 (1.5 g, 3.87 mmol) dissolved in tert-BuOH (5
mL) was added dropwise to the solution and vigorously
stirred at 0 ꢀC for 12 h. While the mixture was stirred at
0 ꢀC, sodium sulfite (5.7 g) was added and the mixture
was allowed to warm to room temperature and stirred
for 30 min. n-BuOH was added to the reaction mixture,
and after separation of the layers, the aqueous phase
was further extracted with n-BuOH. The combined
organic layers were dried over MgSO4. The residue was
purified by silica gel column chromatography (CHCl3:
MeOH=20:1) to afford the diol 19 (1.3 g, 3.09 mmol) in
80% yield as diastereo mixture (55:45). IR (KBr) 3318
(OH), 1745 and 1232 (acetyl), 1652 and 1546 (amide);
1H NMR (500 MHz, CD3OD) d 5.28 (ddÂ2, 2H,
J=10.5, 10.5 Hz, H-3), 5.06 (ddÂ2, 2H, J=10.5, 10.5
Hz, H-4), 4.63 (dÂ2, J=8.5 Hz, H-1), 4.26 (ddÂ2, 1H,
J=5.0, 12.5 Hz, H-6proR), 4.06 (ddÂ2, 1H, J=2.5, 12.5
Hz, H-6proS), 3.85 (ddÂ2, 1H, J=8.4, 10.2 Hz H-2),
3.79 (m, 1H, H-5), 3.73–3.46 (m, 5H, glycerol), 2.04,
1.99, 1.96 (sÂ3, 9H, acetyl), 1.90 (s, 3H, acetamido);
HRFAB+MS m/z: 422.1656 (calcd for C17H28NO11
422.1662 [M+H]+).
ido), 1.47 (m, 2H, CCH2 in propyl group), 0.79 (t, 1H,
J=7.5, 7.5 Hz, CCH3 in propyl group); FAB+MS 286
+
.
[M+Na] . Anal. calcd for C11H21NO6 0.1H2O: C,
49.84; H, 8.06; N, 5.28. Found: C, 49.69; H, 7.47; N,
5.09.
4.9. n-Propyl 2-acetamido-2-deoxy-6-O-sulfonate-ꢀ-D-
glucopyranoside sodium salt (8)
To a DMF (15 mL) solution of 17 (100 mg, 0.38 mmol)
was added Me3NSO3 complex (318 mg, 2.28 mmol).
After stirring at 40 ꢀC for 6 h, the reaction was quen-
ched with MeOH (10 mL). The mixture was stirred at
room temperature for 12 h and concentrated in vacuo.
The residue was purified by reverse phase silica gel col-
umn chromatography (H2O). Treating the mixture with
ion-exchange resin, the residue was lyophilized to afford
8 (74mg, 53%) as a white solid. [ a]D=À62.5ꢀ (c=0.17
in H2O); IR (KBr) 3295 (OH), 1656 and 1554(amide),
1251 (OSO3); H NMR (500 MHz, D2O, 30 ꢀC) d 4.39
1
(d, 1H, J=8.5 Hz, H-1), 4.20 (dd, 1H, J=1.5, 12.3 Hz,
H-6proS), 4.07 (dd, 1H, J=5.5, 12.3 Hz, H-6proR), 3.70
(ddd, 1H, J=10.0, 10.0, 10.0, OCH2 in propyl group),
3.55 (dd, 1H, J=8.5, 10.5 Hz H-2), 3.50 (m, 1H, H-5),
3.42 (m, 2H, H-3, H-4 and OCH2 in propyl group),
1.89 (s, 3H, acetamido), 1.41 (m, 2H, CCH2 in
propyl group), 0.72 (t, 1H, J=7.5, 7.5 Hz, CCH3 in propyl
group); HRFAB+MS m/z: 388.0653 (calcd for
C11H20NNa2O9S 388.0654[M+Na] +). Anal. calcd for
4.12. 1,2 (and 2,3)-O-Isopropylidene-3 (and 1)-O-(2-acet-
amido-2-deoxy-ꢀ-D-glucopyranosyl)-sn-glycerol (20)
To a DMF (10 mL) solution of 19 (670 mg, 1.59 mmol)
was added Me2C(OMe)2 (390 mL, 3.18 mmol) and pyr-
idinium p-toluenesulfonic acid (PPTS) (40 mg 0.16
mmol). After stirring under N2 atmosphere at room
temperature for 2.5 h, the mixture was neutralized with
Et3N. The residue was eluted with EtOAc and the
organic layer was washed with brine and H2O. The
solution was dried over MgSO4. The residue was con-
centrated in vacuo and purified by silica gel column
chromatography (CHCl3:MeOH=10:1) to afford col-
orless syrup 20 (421 mg, 60%) as diastereo mixture
.
C11H20NNaO9S 2H2O: C, 32.92; H, 6.03; N, 3.49.
Found: C, 32.91; H, 5.94; N, 3.44.
4.10. Allyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-ꢀ-D-
glucopyranoside (18)
To a pyridine (20 mL) solution of 16 (700 mg, 2.44
mmol) was added acetic anhydride (2 mL) at 0 ꢀC and
the mixture was stirred overnight. After concentration
of the reaction mixture, the residue was diluted with
ethyl acetate and the solution was washed with 1 N HCl
aq, satd NaHCO3 and brine. The organic layer was
dried over MgSO4 and concentrated in vacuo. The resi-
due was purified by silica gel column chromatography
(toluene: ethyl acetate=1:1) to give 18 (940 mg, 98%) as
1
(55:45). H NMR (500 MHz, CDCl3) d 6.12 (dÂ2, 1H,
J=8.5 Hz, acetamide), 5.28 (ddÂ2, 1H, J=10.5, 10.5
Hz, H-3), 5.06 (ddÂ2, 1H, J=10.5, 10.5 Hz, H-4), 4.77
(dÂ2, 1H, J=8.0 Hz, H-1), 4.26 (m, 2H, H-6proR and
glycerol), 4.06 (dd, 1H, J=2.5, 11.4Hz, H-6 proS), 4.02
(m, 1H, glycerol), 3.89 (m, 1H, glycerol), 3.79 (ddÂ2,
1H, J=8.4, 10.2 Hz H-2), 3.73 (m, 1H, H-5), 3.63
(ddÂ2, 1H, J=6.0, 10.5 Hz, glycerol), 2.09, 2.03, 2.02
(sÂ3, 9H, acetyl), 1.95 (s, 3H, acetamido), 1.42, 1.34
(sÂ2, 6H, isopropylidene); HRFAB+MS m/z: 462.1985
(calcd for C20H32NO11 462.1975 [M+H]+).
a white powder. [a]D=À24.1ꢀ (c=1.00 in MeOH); H
1
NMR (500 MHz, CD3OD) d 5.88 (m, 1H, ¼CH in allyl
group), 5.25 (ddd, 1H, J=1.5, 3.0, 17.0 Hz,
¼CHcisHtrans in allyl group), 5.20 (dd, 1H, J=10.5, 10.5
Hz, H-3), 5.15 (ddd, 1H, J=1.5, 3.0, 10.5 Hz,
¼CHcisHtrans in allyl group), 4.97 (dd, 1H, J=10.5, 10.5
Hz, H-4), 4.65 (d, 1H, J=8.5 Hz, H-1), 4.30 (m, 1H,
OCH2 in allyl group), 4.27 (dd, 1H, J=5.0, 12.3 Hz, H-
6proR), 4.10 (dd, 1H, J=2.5, 12.3 Hz, H-6proS), 4.05 (m,
1H, OCH2 in allyl group), 3.86 (dd, 1H, J=8.5, 11.0
Hz, H-2), 3.76 (ddd, J=2.5, 5.0, 10.5 Hz, H-5), 2.05,
Compound 20 was dissolved in MeOH (10 mL) and a
catalytic amount of NaOMe was added in the solution.
After stirring at room temperature for 1 h, the mixture
was neutralized with ion exchange resin and filtered and

1374
K. Sasaki et al. / Bioorg. Med. Chem. 12 (2004) 1367–1375
then the filtrate was concentrated in vacuo to give dea-
cetylated one as a white powder [320 mg, 90%, dia-
stereo mixture (55:45)]. This compound was used to the
next reaction without further purification.
(500 MHz, D2O, 30 ꢀC) d 8.21 (d, 2H, J=9.0 Hz, Hmetha
of phenyl group), 7.21 (d, 2H, J=9.0 Hz, Hortho of
phenyl group), 5.20 (d, 1H, J=8.5 Hz, H-1), 4.32 (d,
1H, J=2.0, 11.5 Hz, H-6proS), 4.17 (dd, 1H, J=6.0, 11.5
Hz, H-6proR), 3.88 (m, 1H, H-5), 3.52 (m, 2H, H-3 and
H-4), 3.03 (m, 1H, H-2); HRFAB+MS m/z: 381.0602
(calcd for C12H17N2O10S 381.0604[M+H] +). Anal.
4.13. 1 (and 3)-O-(2-acetamide-2-deoxy-6-O-sulfonate-ꢀ-
D-glucopyranosyl)-sn-glycerol sodium salt (9)
.
calcd for C12H16N2O10S 2H2O: C, 34.62; H, 4.84; N,
6.73. Found: C, 34.36; H, 4.90; N, 6.46.
To a DMF (10 mL) solution of the deacetylated com-
pound (244 mg, 0.73 mmol) was added Me3NSO3 complex
(303 mg, 2.19 mmol). After stirring at 40 ꢀC for 2 h, the
mixture was quenched with MeOH (8 mL) and stirred
for 12 h. The residue was concentrated and purified by
silica gel column chromatography (CHCl3:MeOH=3:1)
to afford sulfated compound (100 mg) as a syrup. The
syrup was dissolved in TFA:H2O:MeOH=1:12:12 (5
mL) and the mixture stirred at room temperature for 1 h
and concentrated. The residue was dissolved in H2O,
treated with ion-exchange resin and lyophilized to
afford 9 (100 mg, 34%) as diastereo mixture (55:45). IR
(KBr) 3399 (OH), 1656 and 1558 (amide), 1218 (OSO3);
1H NMR (500 MHz, D2O, 30 ꢀC) d 4.39 (dÂ2, 1H,
J=8.5 Hz H-1), 4.19 (dd, 1H, J=2.0, 11.4Hz, H-6 proS),
4.08 (dd, 1H, J=5.5, 11.4Hz, H-6 proR), 3.70 (m,
2H), 3.30–3.58 (m, 7H), 1.89 (s, 3H, acetamido);
HRFAB+MS m/z: 420.0535 (calcd for C11H20NNa2O11S
420.0552 [M+Na]+).
4.16. Sialidase inhibition assay using influenza virus
The inhibition assay was carried out with 100 mM
sodium acetate buffer (pH 5.0) in a micro tube (the
number of replicates=2). A buffer solution (5 mL, 10
mg/mL based on protein volume) of influenza virus
(A/Memphis/1/71 H3N2)26 was incubated with 4mM
20-(4-methylumbelliferyl)-a-d-N-acetylneuraminic acid
(SIGMA, M8639) (5 mL) and inhibitor (5 mL) prepared
at appropriate concentration at 37 ꢀC for 30 min. The
reaction was stopped with 1 mL of cold 0.1 M sodium
carbonate (pH 11.5). The fluorescence of released
4-methylumbelliferone was detected on a spectro-
fluorometer (Jasco FP-777) with excitation at 365 nm
and emission at 450 nm.
Acknowledgements
4.14. p-Nitrophenyl 2-deoxy-6-O-sulfonate-sulfo-2-tri-
fluoroacetamido-ꢀ-D-glucopyranoside sodium salt (22)
This study has been supported by CREST of JST
(Japan Science and Technology Agency) and the 21st
Century COE program ‘Nature-Guided Materials Pro-
cessing’ of the Ministry of Education, Culture, Sport,
Science and Technology of Japanese Government. The
author (K.S.) thanks for grants from Research Fellow-
ships of the Japan Society for the Promotion of Science
for Young Scientists. We also thank Mr. Kin-ichi
Oyama, Chemical Instrument Center, Nagoya Uni-
versity, for the mass spectral measurements.
To a DMF (6 mL) solution of p-nitrophenyl 2-deoxy-2-
trifluoroacetamido-b-d-glucopyranoside 2125 (200 mg,
0.50 mmol) was added Me3NSO3 complex (210 mg, 1.50
mmol) in DMF (4mL). After stirring at 40 ꢀC for 3 h,
the mixture was quenched with MeOH (4mL) and stir-
red at room temperature for 12 h. The residue was con-
centrated and purified by reverse phase silica gel column
chromatography (H2O). After treating the fraction with
ion-exchange resin, the residue was lyophilized to afford
22 (166 mg 67%) as a white solid. [a]D= À21.2ꢀ
(c=0.07 in H2O); IR (KBr) 3448 (OH), 1716 and 1600
(amide), 1245 (OSO3); 1H NMR (500 MHz, D2O, 30 ꢀC)
d 8.18 (d, 2H, J=9.0 Hz, Hmetha of phenyl group), 7.13
(d, 2H, J=9.0 Hz, Hortho of phenyl group), 5.34(d, 1H,
J=8.5 Hz, H-1), 4.34 (d, 1H, J=2.0, 11.5 Hz, H-6proS),
4.19 (dd, 1H, J=6.0, 11.5 Hz, H-6proR), 4.08 (dd, 1H,
J=8.0, 10.5 Hz, H-2), 3.90 (m, 1H, H-5), 3.73 (dd, 1H,
J=10.5, 9.0 Hz, H-3), 3.58 (dd, 1H, J=9.0, 10.0 Hz, H-4);
HRFAB+MS m/z: 521.0073 (calcd for C14H14F3N2Na2-
O11S 521.0066 [M+Na]+).
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