Synthesis of selective inhibitors against V. cholerae sialidase and human cytosolic sialidase NEU2

Article abstract of DOI:10.1039/c2ob25335f

Sialidases or neuraminidases catalyze the hydrolysis of terminal sialic acid residues from sialyl oligosaccharides and glycoconjugates. Despite successes in developing potent inhibitors specifically against influenza virus neuraminidases, the progress in designing and synthesizing selective inhibitors against bacterial and human sialidases has been slow. Guided by sialidase substrate specificity studies and sialidase crystal structural analysis, a number of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA or Neu5Ac2en) analogues with modifications at C9 or at both C5 and C9 were synthesized. Inhibition studies of various bacterial sialidases and human cytosolic sialidase NEU2 revealed that Neu5Gc9N₃2en and Neu5AcN₃9N ₃2en are selective inhibitors against V. cholerae sialidase and human NEU2, respectively.

Full text of DOI:10.1039/c2ob25335f

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
This article is part of the
OBC 10th anniversary
themed issue
All articles in this issue will be gathered together
online at
www.rsc.org/OBC10

Dynamic Article Links
Organic &
View Article Online
Biomolecular
Chemistry
Cite this: Org. Biomol. Chem., 2012, 10, 6112
www.rsc.org/obc
PAPER
Synthesis of selective inhibitors against V. cholerae sialidase and human
cytosolic sialidase NEU2†‡
Zahra Khedri,§ Yanhong Li,§ Hongzhi Cao,¶ Jingyao Qu, Hai Yu, Musleh M. Muthana and Xi Chen*
Received 16th February 2012, Accepted 8th May 2012
DOI: 10.1039/c2ob25335f
Sialidases or neuraminidases catalyze the hydrolysis of terminal sialic acid residues from sialyl
oligosaccharides and glycoconjugates. Despite successes in developing potent inhibitors specifically
against influenza virus neuraminidases, the progress in designing and synthesizing selective inhibitors
against bacterial and human sialidases has been slow. Guided by sialidase substrate specificity studies
and sialidase crystal structural analysis, a number of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid
(DANA or Neu5Ac2en) analogues with modifications at C9 or at both C5 and C9 were synthesized.
Inhibition studies of various bacterial sialidases and human cytosolic sialidase NEU2 revealed that
Neu5Gc9N32en and Neu5AcN39N32en are selective inhibitors against V. cholerae sialidase and
human NEU2, respectively.
viral, and human sialidases share a catalytic domain with a
Introduction
common canonical six-bladed β-propeller fold.13
Sialidases or neuraminidases (EC 3.2.1.18) are common exo-
glycosidases6 (EC 3.2.1.18) that catalyze the cleavage of termi-
nal sialic acid residues from sialyl oligosaccharides, glycolipids,
and glycoproteins. They are expressed by various organisms
varying from bacteria and viruses to fungi, protozoa, animals,
and humans.1,2 Endo-sialidases (EC 3.2.1.129) that catalyze the
hydrolysis of internal sialyl glycosidic bonds have also been
reported.3 Based on their protein sequence similarities, all exo-
sialidases known to date are mainly grouped into three glycoside
hydrolase families (GH33, GH34, and GH83) in the Carbo-
hydrate Active enZyme (CAZy) database (http://www.cazy.org).4
Bacterial, mammalian, and protozoan sialidases as well as trans-
sialidases (EC 2.4.1.-) are grouped into the GH33 family. Neura-
minidases from influenza A and B viruses belonging to GH34
and other viral neuraminidases are grouped into GH83. In
addition, a mouse klotho in GH1 has been found to have siali-
dase activity.5,6 Several sialyltransferases in CAZy database,
such as glycosyltransferase families GT427 and GT808–10 have
also been shown to possess sialidase and/or trans-sialidase activi-
ties.11,12 Despite their primary sequence differences, bacterial,
Inhibitors specifically against neuraminidases of influenza
viruses have been developed and applied efficiently for the treat-
ment of flu.14 However, the emergence of drug-resistant viral
strains and the increasing threat of viral pandemics15 urge the
discovery of new neuraminidase inhibitors. Due to their impor-
tant nutritional and pathogenic roles, bacterial sialidases, includ-
ing those from Vibrio cholerae (causes cholera),16 Streptococcus
pneumoniae (causes Otitis media in children),17 gram positive
anaerobic bacteria Clostridium perfringens (causes gas gangrene
disease), and Salmonella typhimurium LT2 (causes gastroenteri-
tis)18,19 are being considered as potential drug targets. Human
sialidases, including lysosomal sialidase NEU1, cytosolic siali-
dase NEU2, and membrane-associated sialidase NEU3 and
NEU420 have been shown to be related to a number of diseases
such as cancer and type I and II sialidosis.21,22 They are cur-
rently being studied as important targets for inhibitor design.
The strategy of protein crystal structure-based rational design
of sialidase inhibitors has been validated by the success of
designing influenza virus neuraminidase inhibitors23–25 as clini-
cally effective anti-influenza drugs [e.g., Zanamivir (Relenza)14
and Oseltamivir (Tamiflu)26]. The development of effective and
selective inhibitors against bacterial and human sialidases has
been less successful despite the fact that an increasing number
of bacterial sialidase crystal structures are becoming available.
A number of derivatives of the common sialidase transition
state analogue 2-deoxy-2,3-dehydro-N-acetylneuraminic acid
(Neu5Ac2en or DANA, 1) have been synthesized as potential
inhibitors against human NEU1,27 NEU2,28 and NEU3,20,29,30
as well as bacterial (e.g. V. cholerae31,32 and C. perfringens32)
sialidases. However, the inhibitory efficiency of the obtained
Department of Chemistry, University of California, One Shields Avenue,
Davis, California 95616, USA. E-mail: chen@chem.ucdavis.edu;
Fax: +1-5307528995; Tel: +1-5307546037
†This article is part of the Organic & Biomolecular Chemistry 10th
Anniversary issue.
‡Electronic supplementary information (ESI) available: 1H and 13C
NMR spectra for compound 4–8. See DOI: 10.1039/c2ob25335f
§These authors contributed equally.
¶Current address: National Glycoengineering Research Center, Shan-
dong University, Jinan, Shandong 250012, China.
6112 | Org. Biomol. Chem., 2012, 10, 6112–6120
This journal is © The Royal Society of Chemistry 2012

View Article Online
Fig. 1 Structures of Neu5Ac2en 1 and Neu5AcN32en 2.
inhibitors was usually similar to or even lower than Neu5Ac2en
(1) (Fig. 1).
Fig. 2 Overlay of sialidase-bound Neu5Ac2en structures in the
reported crystal structures of sialidases11,36–41 from S. pneumonia
(NanB, carbons are shown in cyan), V. cholerae (carbons are shown in
light pink), C. perfringens (NanI, carbons are shown in orange), S. typhi-
murium (carbons are shown in magenta), and human NEU2 (carbons are
shown in green). Nitrogen atoms are shown in blue and oxygen atoms
are shown in red. Neu5Ac2en structures are manually overlaid in
PyMOL to allow the maximum overlap of the ring and C1 carboxyl of
Neu5Ac2en.
Recently, conformational flexible loops close to the catalytic
site of sialidases were used as potential targets for inhibitor
design.13,33,34 Based on information gained from mechanism-
based labelling, a potent inhibitor selective against V. cholerae
sialidase with a Ki of 73 nM was identified by screening of a
focused library.35
We have shown previously that results from sialidase substrate
specificity studies can be used to guide the design of selective
sialidase inhibitors.11 In this strategy, α2–3- and α2–6-linked
sialyl galactosides containing different sialic acid forms or ana-
logues are synthesized and used as sialidase substrates.
The structural features of sialic acid forms in substrates selec-
tively cleaved by certain sialidases can be introduced to
Neu5Ac2en to obtain inhibitors that are selective against target
sialidases. Indeed, sialidase substrate specificity studies using
α2–3- and α2–6-linked sialyl galactosides with C5-modified
sialic acid residues11,36 helped to identify Neu5AcN32en (2)
(a C5-derivative of Neu5Ac2en) (Fig. 1) as a selective
inhibitor against human NEU2.11 Herein, we explore the appli-
cation of the strategy in designing additional selective sialidase
inhibitors.
Fig. 3 Structures of Neu5Ac9N32en 3, Neu5Gc9N32en 4,
Neu5AcN39N32en 5, Neu5Ac9Me2en 6, and Neu5Ac9NPro2en 7.
C. perfringens, S. typhimurium, and S. pneumoniae sialidases.
Therefore, we hypothesized that Neu5Ac9N32en (3) would be
a similarly good inhibitor as Neu5Ac2en against C. perfringens,
V. cholerae, S. typhimurium, and S. pneumoniae bacterial siali-
dases but will be a weaker inhibitor, compared to Neu5Ac2en,
for human NEU2. In addition, Neu5Gc9N32en (4) would be a
similarly good inhibitor as Neu5Ac2en against human
NEU2 and V. cholerae sialidase, but a worse inhibitor against
C. perfringens, S. typhimurium, and S. pneumoniae sialidases.
Furthermore, as we demonstrated before,11 azido modification at
C5-NHAc group on Neu5Ac2en provided an improved and
selective inhibitor against human NEU2. Therefore, we predicted
that combining the features of azido modifications at both
C5-NHAc group and C9 of Neu5Ac2en will result in a selective
inhibitor (Neu5AcN39N32en, 5) against human NEU2. Also,
Neu5Ac9Me2en (6) with a methoxy at C9 of Neu5Ac2en may
be a better or a similarly good inhibitor compared to Neu5Ac2en
against S. typhimurium or S. pneumoniae sialidase based on our
substrate specificity tests. To test these hypotheses, we syn-
thesized a library of Neu5Ac2en analogues with modifications at
C9 and both C5 and C9, including Neu5Ac9N32en (3),
Neu5Gc9N32en (4), Neu5AcN39N32en (5), and Neu5Ac9Me2en
(6) (Fig. 3). In addition, a Neu5Ac2en derivative with a C9-pro-
pylamine modification (Neu5Ac9NPro2en, 7) was synthesized to
test the effect of a larger C9-group in sialidase inhibitory
activities.
Results and discussion
Designing of sialidase inhibitors
To identify the logical positions for systematic modifications, the
structures of sialidase-bound inhibitor Neu5Ac2en (a common
transition-state analogue) in the reported crystal structures of bac-
terial and human sialidases were compared. Fig. 2 shows that in
addition to the N-acetyl group at C5, two significant differences
of sialidase-bound Neu5Ac2en are the positions of C9 and the
orientations of the hydroxyl groups attached to C9
(Fig. 2).13,37–42 Therefore, modifications at C9 or at both C9 and
C5 will most likely provide selectivity for Neu5Ac2en-based
sialidase inhibitors.
Our recent substrate specificity studies of bacterial sialidases
from C. perfringens, V. cholerae, S. typhimurium, and S. pneu-
moniae as well as human NEU2 using thirty α2–3/6-linked sia-
losides containing C9-modified sialic acid43 indicate that α2–
3-linked sialoside containing Neu5Ac9N3 (Neu5Ac9N3α2–
3GalβpNP) is a similarly good substrate as the sialoside contain-
ing non-modified Neu5Ac (Neu5Acα2–3GalβpNP) for bacterial
sialidases. In contrast, α2–3-sialoside containing Neu5Gc9N3
(Neu5Gc9N3α2–3GalβpNP) is a similarly good or better sub-
strate than sialoside containing non-modified Neu5Ac
(Neu5Acα2–3GalβpNP) for human NEU2 and V. cholerae siali-
dase, but not for other bacterial sialidases tested including
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 6112–6120 | 6113

View Article Online
Synthesis of sialidase inhibitors
Inhibition studies of bacterial sialidases and human NEU2
Compounds 9-azido-9-deoxy-Neu5Ac2en (Neu5Ac9N32en, 3),
9-azido-9-deoxy-Neu5Gc2en (Neu5Gc9N32en, 4), 9-azido-5-
(2-azidoacetamido)-5,9-dideoxy-Neu5Ac2en (Neu5AcN39N32en,
5), 9-O-methyl-Neu5Ac2en (Neu5Ac9OMe2en, 6), and 9-amino-
propyl-5,9-dideoxy-Neu5Ac2en (Neu5Ac9NPro2en, 7) were
synthesized as shown in Scheme 1. Compound Neu5Ac9N32en
(3) was generated using a reported method with optimiza-
tion.27,29 Neu5Gc9N32en (4) was synthesized from compound 8
by treating it with Boc2O44 to produce N-acetyl-N-Boc protected
product 11, which was deacetyled under Zemplén condition to
form 12. Selective tosylation of the primary hydroxyl group of
12 followed by azidation provided intermediate 13. After
removal of the Boc group of 13 with TFA in water, the obtained
amine was treated with acetoxyacetyl chloride in the presence of
base DIPEA and coupling reagents HOBT and EDCI in CH2Cl2
followed by deprotection produced the desired compound
Neu5Gc9N32en (4) in a high yield. To synthesize Neu5AcN39-
N32en (5), compound 13 was acetylated and the Boc protecting
group was removed by treatment with TFA in CH2Cl2. Coupling
of the obtained amine with N3CH2COOH in the presence of
EDCI–HOBT afforded compound 16 in a good yield. Deprotec-
tion of 16 followed by hydrolysis in aqueous NaOH afforded the
desired product Neu5AcN39N32en (5). Compound Neu5A-
c9OMe2en (6) was obtained by base (NaOH in H2O)-catalyzed
hydrolysis of compound 17 which was formed from intermediate
9 by gradual addition of a solution of NaOMe in MeOH
followed by neutralization with Dowex (H+) resin. To prepare
Neu5AcN39NPro2en (7), compound 10 was hydrogenated under
atmospheric pressure to give amine 18. Reductive amination of
18 with propanal and subsequent demethylation provided the
desired product 7 in a moderate yield.
The IC50 values of the C9- as well as both C9- and C5-modified
Neu5Ac2en derivatives 3–7 synthesized above were determined
using human NEU2 and four commercially available bacterial
sialidases from Salmonella typhimurium (Prozyme), Clostridium
perfringens (Sigma), Vibrio cholerae (Sigma), and Streptococcus
pneumoniae (NanB, Prozyme). Inhibition studies were con-
ducted using Neu5Acα2–3GalβpNP or Neu5Acα2–6GalβpNP as
sialidase substrate. As shown in Table 1, Neu5Ac9N32en (3)
was a similarly effective inhibitor as Neu5Ac2en (1) against
α2–3-specific S. typhimurium and S. pneumoniae sialidases
as predicted. However, unlike the prediction, Neu5Ac9N32en
(3) was 3-fold less efficient than Neu5Ac2en (1) against
C. perfringens sialidase when either Neu5Acα2–3GalβpNP or
Neu5Acα2–6GalβpNP was used as the substrate. For V. cholerae
sialidase and human NEU2, Neu5Ac9N32en (3) was 2.3-fold or
2.4-fold less efficient than Neu5Ac2en (1) when Neu5Acα2–
3GalβpNP was used as the substrate, while the IC50 values of
these two inhibitors were similar when Neu5Acα2–6GalβpNP
was used as the substrate. Neu5Ac9N32en (3) along with a series
of C9-triazole derivatives of Neu5Ac2en have been tested pre-
viously as inhibitors against human NEU3.29 Neu5Ac9N32en
(3) showed less inhibition activity against NEU3 than
Neu5Ac2en (1). Nevertheless, slightly improved activity was
observed using phenyl-, hexyl-, and phenoxymethyl-C9-triazole
Neu5Ac2en derivatives (IC50 values were 20, 23, and 45 μM,
respectively).29
As predicted from substrate specificity studies,43 Neu5Gc9-
N32en (4) was a weaker inhibitor than Neu5Ac2en (1) against
C. perfringens, S. typhimurium, and S. pneumoniae sialidases.
However, in disagreement with the prediction, Neu5Gc9N32en
(4) was also a weaker inhibitor than Neu5Ac2en (1) against
Scheme 1 Synthesis of Neu5Ac2en derivatives.
6114 | Org. Biomol. Chem., 2012, 10, 6112–6120
This journal is © The Royal Society of Chemistry 2012

View Article Online
Table 1 IC50 values of potential inhibitors against bacterial and human sialidases using either Neu5Acα2–3GalβpNP or Neu5Acα2–6GalβpNP as
sialidase substrate
IC50 (μM) with Neu5Acα2–3GalβpNP as sialidase substrate
Sialidases
Neu5Ac2en (1)
Neu5Ac9N32en (3) Neu5Gc9N32en (4) Neu5AcN39N32en (5) Neu5Ac9Me2en (6) Neu5Ac9NPro2en (7)
C. perfringens 20
1
59
20
5
1
(1.9 0.4) × 103
(1.6 0.2) × 103
(1.4 0.1) × 102
(1.6 0.4) × 104
>1 × 103
(4.6 0.6) × 102
(1.3 0.3) × 102
(1.3 0.2) × 102
(3.0 0.6) × 103
(1.1 0.1) × 103
>1 × 103
V. cholerae
8.6 1.0a
18
3
>1 × 103
S. typhimurium (2.0 0.1) × 102a (2.1 0.1) × 102
>1 × 104
(2.3 0.3) × 103
(5.4 0.6) × 103
>1 × 104
S. pneumoniae (3.7 0.6) × 103 (3.3 0.4) × 103
>1 × 104
NEU2
18 1a
43
2
(1.5 0.3) × 102
13
4
IC50 (μM) with Neu5Acα2–6GalβpNP as sialidase substrate
C. perfringens 13
V. cholerae
NEU2
1
40
8.7 1.0
31
4
(1.4 0.4) × 103
13
(1.9 0.7) × 102
(1.2 0.2) × 103
(1.1 0.2) × 102
(3.1 0.6) × 102
(1.2 0.3) × 102
(4.1 0.6) × 102
>1 × 103
10 1a
32 6a
2
>1 × 103
6
13
3
(2.4 0.8) × 103
a Data are from ref. 11.
NEU2 and V. cholerae sialidase. Although with a mildly
decreased IC50 value compared to Neu5Ac2en (1) in inhibiting
V. cholerae sialidase, Neu5Gc9N32en (4) was shown to be a
selective inhibitor against this enzyme.
and human NEU2, respectively, the structure of Neu5Ac9N32en
(3), Neu5Gc9N32en (4), or Neu5AcN39N32en (5) was docked
into the active site of human NEU2 or V. cholerae sialidase
using docking program FRED. Most docking programs are
usually effective but highly inconsistent, while the pose of the
ligand structure is predictable and reproducible and the virtual
screening is accurate using FRED.45 Three structure-based
scoring functions PLP (Piecewise Linear Potential),46 chem-
gauss3,47 and Oechemscore,48 implemented in the program
FRED were used to optimize the multiple docked poses. The
best scoring pose was selected as the final position of the ligand
in the crystal of protein.
As shown in Fig. 4A3 and 4A5, the C9-azido group in
Neu5Ac9N32en (3) and Neu5AcN39N32en (5) fits well in the
deep pocket of human NEU2 and points out towards the neigh-
bouring cavity close to C9 of the inhibitors. However, replacing
the C5-N-acetyl group in Neu5Ac9N32en (3) by the N-glycolyl
group in Neu5Gc9N32en (4) causes a 180° flipping of the inhibi-
tor (Fig. 4A4 and S1‡). This may be due to the less favorable
interaction between the inner neutral cavity (mainly composed of
Met85, Ile103, Ile105, and Ala158) and the N-glycolyl. The
bigger outside positive cavity (mainly contributed by Arg83)
may allow more favorable interaction with the N-glycolyl
group11 (Fig. S1‡). Switching the positions of C5 and C9 groups
may lead to lower inhibition efficiency of Neu5Gc9N32en (4)
against human NEU2. Compared to Neu5Ac9N32en (3) in
Also as expected, Neu5AcN39N32en (5) was a selective
inhibitor against human NEU2 with a slightly improved IC50
compared to Neu5Ac2en (1) when either Neu5Acα2–3GalβpNP
or Neu5Acα2–6GalβpNP was used as the substrate. The inhibi-
tory efficiency was comparable to other reported human sialidase
inhibitors.27–29 These results indicate that Neu5AcN39N3-con-
taining sialosides are most likely suitable substrates for human
NEU2. A previous report using a library of octyl sialyllactosides
with modifications at the Neu5Ac residue as sialidase substrates
also showed that Neu5Ac with an azide at C5 or C9 could be
cleaved by human membrane-associated sialidases NEU3.29
Neu5Ac9OMe2en (6) and Neu5AcN39NPro2en (7) with a
methoxy and a propylamine substitution, respectively, at C9 of
Neu5Ac2en (1) were shown to be weak inhibitors against all five
sialidases tested. The inhibitory activities of similar compounds
as 7 with amide-linked C9-modified Neu5Ac2en analogues were
studied against NEU1–NEU4.27 All the compounds were found
to be poor inhibitors of the NEU2–NEU4. However, selective
inhibitors with slightly improved inhibition compared to
Neu5Ac2en were observed for those containing a methyl,
propyl, butyl, or phenyl group.
Inhibition studies of compounds 1, 3, 4, 5, 6, and 7 against
β-galactosidase were also carried out. Using 1 mM of GalβpNP
as the substrate for β-galactosidase, no inhibitory effect was
observed for any of the compounds with a final concentration
of 100 μM or 1 mM (data not shown). This confirms that
the selective inhibitory activities of Neu5Gc9N32en (4) and
Neu5AcN39N32en (5) against V. cholerae sialidase and human
NEU2, respectively, are not due to inhibition of the compounds
against β-galactosidase in the assays.
Fig. 4A3 (IC50 = 43
2 μM when Neu5Acα2–3GalβpNP
was used as sialidase substrate and IC50 = 31 6 μM when
Neu5Acα2–6GalβpNP was used as sialidase substrate),
Neu5AcN39N32en (5) with an N3-substitution at C5-N-acetyl
group in Fig. 4A5 fits the binding pocket of NEU2 better, which
explains an improved IC50 value for human NEU2 by
Neu5AcN39N32en (5) (IC50 = 13 4 μM when Neu5Acα2–
3GalβpNP was used as sialidase substrate and IC50 = 13 3 μM
when Neu5Acα2–6GalβpNP was used as sialidase substrate).
For V. cholerae sialidase, both Neu5Ac9N32en (3) and
Neu5Gc9N32en (4) fit the substrate binding pocket of the
enzyme similarly well (Fig. 4B3 and 4B4), which explains
similar IC50 values of these two inhibitors. In comparison, the extra
N3-substitution at C5-N-acetyl group in Neu5AcN39N32en (5)
Docking studies of compounds 3, 4, and 5 in the crystal
structures of human NEU2 and V. cholerae sialidase
To help the understanding of the selectivity of Neu5Gc9N32en
(4) and Neu5AcN39N32en (5) in inhibiting V. cholerae sialidase
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 6112–6120 | 6115

View Article Online
sialidases is a valuable tool for identifying unique structural
features of selective inhibitors. However, drastic improvement of
inhibitory activity of bacterial and human sialidases will require
more significant structural changes which could be facilitated by
screening of focused compound libraries.
Experimental
Materials
All chemicals were obtained from commercial suppliers and
used without further purification unless otherwise noted. Anhy-
drous solvents were used to carry out organic reactions under
inert argon or nitrogen environment. Neu5Acα2–3GalβpNP
and Neu5Acα2–6GalβpNP were synthesized as reported pre-
viously.49 1H NMR (400 and 600 MHz) and 13C NMR
(151 MHz) spectra were recorded on a Varian Inova 400 and
600 MHz spectrometer. High resolution electrospray ionization
(ESI) mass spectra were obtained at the Mass Spectrometry
Facility in the University of California, Davis. Optical rotation
values were recorded on an Autopol IV Automatic polarimeter
at 589 nm wavelength. Infrared spectra were recorded on a
PerkinElmer Spectrum 100 ATR-FTIR. Silica gel 60
Å
(200–425 mesh, Fisher Chemical) was used for flash column
chromatography. Thin-layer chromatography (TLC) was per-
formed on silica gel plates 60 GF254 (Sorbent technologies)
using anisaldehyde sugar stain for detection. Clostridium per-
fringens sialidase (type VI), Vibrio cholerae sialidase (type III),
and β-galactosidase from Aspergillus oryzae were purchased
from Sigma. Salmonella typhimurium sialidase and Streptococcus
pneumoniae sialidase NanB were bought from Prozyme. All of
these enzymes were used without further purification. NEU2
was expressed in E. coli and purified as described previously.17
384-Well plates for sialidase assays were from Fisher Biotech.
Fig. 4 Docking studies of Neu5Ac9N32en (3), Neu5Gc9N32en (4),
and Neu5AcN39N32en (5) in the crystal structures of human NEU2 (A)
and V. cholerae sialidase (B). The surface is coloured according to the
electrostatic potential. Positive potentials are shown in blue, negative
potentials are in red, and neutral are in white.
Chemical synthesis of sialidase inhibitors
causes flipping and ill fitting of the inhibitor (Fig. 4B5 and S1‡),
which increases the IC50 values for about 10-fold when either
Neu5Acα2–3GalβpNP or Neu5Acα2–6GalβpNP was used as
sialidase substrate.
5-Acetamido-2,6-anhydro-9-azido-3,5,9-trideoxy-D-glycero-D-
galacto-non-2-enonic acid (Neu5Ac9N32en, 3). To a suspension
of 850 (1.12 g, 2.36 mmol) in dry MeOH (15 mL) was added a
catalytic amount of NaOMe and the mixture was stirred at room
temperature for 3 h. The reaction was neutralized with Dowex
(H+) resin, filtrated, concentrated, and purified by silica gel flash
column chromatography (EtOAc–MeOH–H2O
= 8 : 1 : 0.5)
Conclusions
1
to afford 9 (0.56 g, 78%). H NMR (600 MHz, D2O) δ 6.04 (s,
1H), 4.50 (d, J = 9.0 Hz, 1H), 4.27 (d, J = 11.4 Hz, 1H), 4.08 (t,
J = 9.6 Hz, 1H), 3.91–3.85 (m, 2H), 3.81 (s, 3H), 3.66–3.63 (m,
2H), 2.06 (s, 3H); 13C NMR (151 MHz, D2O) δ 177.56, 167.08,
146.16, 115.30, 78.83, 72.67, 70.74, 69.79, 65.69, 55.64, 52.30,
24.82.
A stirred solution of 9 (0.56 g, 1.83 mmol) in anhydrous pyri-
dine (10 mL) was treated with a solution of p-toluenesulfonyl
chloride (0.52 g, 2.75 mmol) at 0 °C and the mixture was stirred
overnight at room temperature. The reaction was stopped by
adding MeOH (2 mL) and concentrated. The compound was
purified by column chromatography (EtOAc–MeOH = 9 : 1) to
give 9-OTs Neu5Ac2en (0.54 g, 65%) which was then dissolved
in water (10 mL) and acetone (10 mL) containing NaN3 (0.76 g,
11.75 mmol) and stirred at 70 °C overnight. The mixture was
In conclusion, guided by sialidase substrate specificity studies
and sialidase crystal structural analysis, several C9- or both
C9- and C5-modified Neu5Ac2en analogues were synthesized
and their inhibitory activities were tested. Neu5Gc9N32en (4)
and Neu5AcN39N32en (5) were identified as selective inhibitors
against V. cholerae sialidase and human NEU2, respectively,
using Neu5Acα2–3GalβpNP or Neu5Acα2–6GalβpNP as the
substrate for human cytosolic sialidase NEU2 and four commer-
cially available bacterial sialidases. Nevertheless, their IC50
values were not improved compared to Neu5Ac2en. Docking of
Neu5Ac9N32en (3), Neu5Gc9N32en (4), or Neu5AcN39N32en
(5) to the crystal structures of human NEU2 and V. cholerae
sialidase helped to illustrate the selectivity of sialidase inhibitors
4 and 5. Therefore, substrate specificity study of various
6116 | Org. Biomol. Chem., 2012, 10, 6112–6120
This journal is © The Royal Society of Chemistry 2012

View Article Online
concentrated and purified by silica gel flash column chromato-
graphy (EtOAc–MeOH = 9.5 : 0.5) to give product 10 (0.27 g,
70%). 1H NMR (600 MHz, D2O) δ 6.04 (s, 1H), 4.51 (d,
J = 9.1 Hz, 1H), 4.27 (d, J = 8.4 Hz, 1H), 4.08–4.07 (m, 2H),
3.81 (s, 3H), 3.65–3.63 (m, 2H), 3.51 (s, 1H), 2.06 (s, 3H);
13C NMR (151 MHz, D2O) δ 177.56, 167.08, 146.16, 115.30,
78.83, 72.67, 70.74, 69.79, 65.69, 55.64, 52.30, 24.82.
Compound 10 (0.27 g, 0.82 mmol) was dissolved in an
aqueous solution of NaOH (10 mL, 0.1 M) and stirred at room
temperature for 1 h. The reaction was neutralized with Dowex
(H+) resin, filtrated, concentrated, and purified using silica gel
column chromatography (EtOAc–MeOH–H2O = 8 : 3 : 0.5) to
give final product 3 (0.22 g, 85%). [α]D24 = +39.00 (c = 1.0
H2O). νmax (film)/cm−1 3205 (m, broad, OH, NH, C–H alkene,
COOH), 2101 (s, N3), 1646 (s, CO carboxylic acid), 1557 (m,
CO amide, CC alkene), 1145 (s, C–N). 1H NMR (600 MHz,
D2O) δ 5.67 (s, 1H, H-3), 4.41 (d, J = 8.4 Hz, 1H, H-6), 4.15 (d,
J = 10.8 Hz, 1H, H-4), 4.03–3.97 (m, 2H, H-5 and H-7),
3.58–3.54 (m, 2H, H-8 and H-9), 3.44 (dd, J = 12.6, 5.4 Hz, 1H,
H-9′), 2.00 (s, 3H, NHCOMe); 13C NMR (151 MHz, D2O)
δ 174.83 (COONa), 169.16 (NHCOMe), 147.39 (C-2), 108.51
(C-3), 75.32 (C-6), 68.69 (2C, C-7 and C-8), 67.63 (C-4), 53.81
(C-5), 49.92 (C-9), 22.26 (NHCOMe). HRMS (ESI) calculated
for C11H16N4NaO7 (M + H) 339.0917, found 339.0910.
dissolved in water (15 mL) and acetone (15 mL) containing
NaN3 (2.22 g, 34.20 mmol) and stirred at 70 °C overnight. The
mixture was concentrated and purified by silica gel flash column
chromatography (EtOAc–hexane = 7 : 3) to give compound
1
13 (1.06 g, 80%). H NMR (600 MHz, CD3OD) δ 5.83 (d, J =
3.0 Hz, 1H), 4.28 (dd, J = 9.0, 3.0 Hz, 1H), 4.01–3.95 (m, 2H),
3.68 (s, 3H), 3.59 (dd, J = 10.8, 8.4 Hz, 1H), 3.55 (d, J =
9.0 Hz, 1H), 3.46 (dd, J = 12.6, 2.4 Hz, 1H), 3.35 (dd, J = 13.2,
6.0 Hz, 1H), 1.38 (s, 9H); 13C NMR (151 MHz, CD3OD)
δ 164.31, 159.35, 144.99, 113.82, 81.10, 78.33, 70.53, 70.08,
67.79, 55.67, 52.81, 52.49, 28.65. Compound 13 (1.06 g,
2.73 mmol) was dissolved in a mixture of CH2Cl2 (30 mL) and
an aqueous solution of trifluoroacetic acid (90%, 10 mL) and
stirred at room temperature for 30 min. The mixture was concen-
trated, co-evaporated with toluene, and dried. To a solution of
the resulted mixture in CH2Cl2 (20 mL) was added hydroxy-
benzotriazole (HOBT) (0.44 g, 1.2 mmol), 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDCI) (0.51 g, 1.2 mmol),
and N,N-diisopropylethylamine (DIPEA) (0.57 mL, 1.2 mmol)
at 0 °C. Acetoxyacetyl chloride (0.23 mL, 2.12 mmol) was
added drop-wisely and the reaction mixture was stirred at room
temperature for 2.5 h. The reaction solution was concentrated
and diluted with CH2Cl2, washed with water, saturated NaHCO3,
and water then dried and concentrated. Silica gel column chrom-
atography (EtOAc–MeOH–H2O = 9 : 2 : 0.5) afforded compound
14 (0.91 g, 85%). To a suspension of 14 (0.91 g, 2.32 mmol) in
dry MeOH (10 mL) was added NaOMe (40 mg) and the mixture
was stirred for 1 h at room temperature. Water was then added to
the solution and pH was adjusted to about 9 by adding NaOMe.
The reaction mixture was stirred at room temperature for 1 h.
The solution was neutralized with Dowex (H+) resin, filtrated,
concentrated, and purified by silica gel flash column chromato-
graphy (EtOAc–MeOH–H2O = 7.5 : 3 : 0.5) to afford product 4
(0.77 g, 95%). [α]2D4 = +16.50 (c = 1.0 H2O). νmax (film)/cm−1
3184 and 3058 (s, broad, OH, NH, C–H alkene, COOH),
2101 (s, N3), 1665 (s, CO carboxylic acid), 1570 (s, CO amide,
2,6-Anhydro-9-azido-3,5,9-trideoxy-5-(2-hydroxyacetamido)-
D-glycero-D-galacto-non-2-enonic acid (Neu5Gc9N32en, 4). To a
solution of compound 8 (2.47 g, 5.21 mmol) in dry THF
(70 mL) was added (Boc)2O (4.55 g, 20.86 mmol) and
4-dimethylaminopyridine (DMAP) (0.19 g, 1.56 mmol) and the
mixture was refluxed for 4 h. The reaction mixture was cooled
to room temperature, diluted with CH2Cl2, washed with HCl
(0.5 M), water, saturated NaHCO3, and water. The solution was
dried, concentrated, and purified by flash column chromato-
graphy (EtOAc–hexane = 3 : 7) to afford 11 (2.69 g, 90%).
1H NMR (600 MHz, CDCl3) δ 5.99 (s, 1H), 5.87 (d, J = 2.4 Hz,
1H), 5.25–4.60 (m, 5H), 4.12–4.04 (m, 2H), 3.75 (s, 3H), 2.32 (s,
3H), 2.01–1.95 (m, 12H), 1.54 (s, 9H); 13C NMR (151 MHz,
CDCl3) δ 170.75, 170.31, 169.93, 161.93, 146.46, 109.25, 85.38,
71.10, 67.63, 66.97, 62.03, 52.73, 50.55, 28.08, 20.95.
Compound 11 (2.69 g, 4.69 mmol) was dissolved in dry
MeOH (30 mL) containing a catalytic amount of NaOMe. The
mixture was stirred at room temperature for 2 h and neutralized
using Dowex (H+) resin. The resulting suspension was filtrated
and concentrated. Silica gel column chromatography (EtOAc–
MeOH = 9.5 : 0.5) of the crude product afforded product 12
(1.62 g, 95%). 1H NMR (600 MHz, CD3OD) δ 5.91 (d, J =
1.8 Hz, 1H), 4.35 (dd, J = 8.4, 1.8 Hz, 1H), 4.11 (d, J =
10.8 Hz, 1H), 3.89–3.82 (m, 2H), 3.76 (s, 3H), 3.70–3.62 (m,
3H), 1.45 (s, 9H); 13C NMR (151 MHz, CD3OD) δ 164.41,
159.32, 145.13, 113.68, 81.01, 78.60, 71.11, 70.19, 67.94,
64.98, 52.75, 52.52, 28.69.
1
CC alkene), 1153 (s, C–N). H NMR (400 MHz, D2O) δ 5.69
(d, J = 1.6 Hz, 1H, H-3), 4.55 (dd, J = 6.4, 1.6 Hz, 1H, H-6),
4.31 (d, J = 7.2 Hz, 1H, H-4), 4.15–4.09 (m, 4H, NHCO-
CH2OH, H-5, and H-7), 3.66–3.59 (m, 2H, H-9 and H-8), 3.50
(dd, J = 8.8, 4.0 Hz, 1H, H-9′); 13C NMR (151 MHz, D2O)
d 175.75 (COONa), 165.58 (NHCOCH2OH), 143.72 (C-2),
112.86 (C-3), 75.78 (C-6), 68.87 (C-7), 68.47 (C-8), 67.19 (C-4),
61.18 NHCOCH2OH), 53.81 (C-5), 49.37 (C-9). HRMS (ESI) cal-
culated for C11H16N4NaO8 (M + H) 355.0866, found 355.0858.
2,6-Anhydro-9-azido-5-(2-azidoacetamido)-3,5,9-trideoxy-D-
glycero-D-galacto-non-2-enonic acid (Neu5AcN39N32en, 5). To a
solution of compound 13 (0.21 g, 0.56 mmol) in pyridine
(10 mL) was added acetic anhydride (0.7 mL) at 0 °C and the
mixture was stirred at room temperature overnight. The solvent
was removed under reduced pressure and the crude product was
purified using silica gel column chromatography (EtOAc–hexane
= 3 : 7) to give compound 15 (0.22 g, 80%). 1H NMR
(600 MHz, CDCl3) δ 5.93 (d, J = 4.2 Hz, 1H), 5.53–5.48
(m, 1H), 5.20–5.17 (m, 1H), 4.79 (d, J = 15.6 Hz, 1H), 4.29 (dd,
J = 15.0, 4.2 Hz, 1H), 4.10–4.01 (m, 1H), 3.89 (dd, J = 20.4,
3.6 Hz, 1H), 3.78 (s, 3H), 3.45 (dd, J = 20.4, 12.0 Hz, 1H), 2.11
(s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.37 (s, 9H); 13C NMR
A stirred solution of 12 (1.62 g, 4.46 mmol) in anhydrous
pyridine (50 mL) was treated with a solution of p-toluenesulfo-
nyl chloride (2.55 g, 13.37 mmol) at 0 °C and the mixture was
stirred overnight at room temperature. The reaction was stopped
by adding MeOH (3 mL) and concentrated. Purification of the
residue by flash column chromatography (EtOAc–hexane = 7 : 3)
afforded 9-tosylated product (1.77 g, 77%) which was then
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 6112–6120 | 6117

View Article Online
(151 MHz, CDCl3) δ 170.97, 170.57, 169.96, 161.72, 155.13,
145.10, 108.64, 80.51, 77.36, 72.93, 69.21, 68.34, 52.71, 50.29,
47.93, 28.20, 20.99, 20.91, 20.79.
9.0 Hz, 1H), 3.99–3.97 (m, 1H), 3.79 (s, 3H), 3.69 (dd, J = 10.8,
2.4 Hz, 1H), 3.61 (d, J = 9.0 Hz, 1H), 3.55 (dd, J = 10.8,
6.0 Hz, 1H), 3.69 (s, 3H), 2.03 (s, 3H); 13C NMR (151 MHz,
D2O) δ 174.79, 164.40, 143.51, 112.69, 76.12, 73.75, 68.39,
68.09, 67.26, 58.65, 53.04, 49.67, 22.23.
Compound 17 (0.12 g, 0.37 mmol) was dissolved in an
aqueous solution of NaOH (10 mL, 0.05 M) and stirred at room
temperature for 30 min. The solution was neutralized with
Dowex (H+) resin, filtrated, concentrated, and purified by silica
To a solution of compound 15 (0.22 g, 0.43 mmol) in CH2Cl2
(6 mL) was added 90% aqueous trifluoroacetic acid (2 mL) at
0 °C. The reaction mixture was stirred at room temperature for
30 min. It was then concentrated, co-evaporated with toluene,
and dried under vacuum. To the resulting ammonium salt in
CH2Cl2 (3 mL) were added N3CH2COOH (52 μL, 0.51 mmol),
HOBT (0.08 g, 0.51 mmol), EDCI (0.10 g, 0.51 mmol), and
DIPEA (0.22 mL, 1.29 mmol) at 0 °C and the reaction mixture
was stirred at room temperature for 2 h. The mixture was diluted
with CH2Cl2, washed with water, saturated NaHCO3, and water
then dried and concentrated. Silica gel column chromatography
(EtOAc–hexane = 4 : 6) afforded compound 16 (0.17 g, 80%).
1H NMR (600 MHz, CDCl3) δ 6.79 (d, J = 9.6 Hz, 1H), 5.93
(d, J = 1.8 Hz, 1H), 5.63 (dd, J = 8.4, 3.0 Hz, 1H), 5.47 (t, J =
3.0 Hz, 1H), 5.17–5.15 (m, 1H), 4.46–4.39 (m, 2H), 3.93 (dd,
J = 13.2, 1.8 Hz, 1H), 3.83 (d, J = 5.4 Hz, 2 H), 3.79 (s, 3H),
3.46 (dd, J = 13.8, 8.4 Hz, 1H), 2.13 (s, 3H), 2.10 (s, 3H), 2.07
(s, 3H); 13C NMR (151 MHz, CDCl3) δ 171.28, 170.97, 170.50,
167.54, 161.54, 145.32, 108.68, 77.17, 73.41, 68.75, 68.03,
52.78, 52.61, 50.20, 46.41, 20.88.
gel flash column chromatography (EtOAc–MeOH–H2O
=
8 : 3 : 0.7) to afford product 6 (107 mg, 95%). [α]D24 = +25.30
(c = 1.0 H2O). νmax (film)/cm−1 3278 (s, broad, OH, NH, C–H
alkene, COOH), 1590 (s, broad, CO carboxylic acid, CO amide,
1
CC alkene), 1092 (s, C–N). H NMR (600 MHz, D2O) δ 5.69
(d, J = 2.4 Hz, 1H, H-3), 4.45 (dd, J = 9.0, 2.4 Hz, 1H, H-6),
4.20 (d, J = 11.4 Hz, 1H, H-4), 4.07–4.03 (m, 2H, H-5 and
H-7), 3.73 (dd, J = 12.0, 2.4 Hz, 1H, H-9), 3.61–3.57 (m, 2H,
H-8 and H-9′), 3.40 (s, 3H, OMe), 2.06 (s, 3H, NHCOMe);
13C NMR (151 MHz, D2O) δ 174.85 (COONa), 169.72
(NHCOMe), 148.01 (C-2), 107.91 (C-3), 75.40 (C-6), 73.91
(C-9), 68.41 (C-7), 68.28 (C-8), 67.79 (C-4), 58.66 (OMe),
50.01 (C-5), 22.30 (NHCOMe). HRMS (ESI) calculated for
C12H19NNaO8 (M + H) 328.1008, found 328.1005.
To a suspension of 16 (0.17 g, 0.35 mmol) in dry MeOH
(10 mL) was added NaOMe (40 mg) and the mixture was stirred
for 2 h at room temperature. After adding water to the reaction
mixture, the pH was adjusted to 9 and the solution was stirred
for 1 h at room temperature. The reaction was neutralized with
Dowex (H+) resin, filtrated, concentrated, and purified by silica
5-Acetamido-9-aminopropyl-2,6-anhydro-3,5,9-trideoxy-D-glycero-
D-galacto-non-2-enonic acid (Neu5Ac9NPro2en, 7). Compound
10 (0.33 g, 1.0 mmol) was dissolved in MeOH (20 mL) and
Pd–C (100 mg) was added. The resulting mixture was hydrogen-
ated for 1.5 h with stirring under H2 at atmospheric pressure and
then the catalyst was removed by filtration over Celite and
washed with MeOH and water. The concentrated product
was purified by flash column chromatography (EtOAc–MeOH–
H2O = 5 : 4 : 3) to afford 18 (0.26 g, 80%). To compound 18
(42 mg, 0.14 mmol) in dry THF (15 mL) was added NaBH3CN
(52 mg, 0.83 mmol), acetic acid to adjust pH to about 5, and
propanal (50 μL, 0.7 mmol). The mixture was stirred at room
temperature overnight. The mixture was concentrated and
purified by silica gel flash column chromatography (EtOAc–
MeOH–H2O = 5 : 3 : 1) to afford the target compound intermedi-
ate (33.5 mg, 70%) which was then dissolved in an aqueous
solution of NaOH (10 mL, 0.05 M) and stirred at room tempera-
ture for 30 min. The solution was neutralized with Dowex (H+)
resin, filtrated, concentrated, and purified by silica gel flash
column chromatography (EtOAc–MeOH–H2O = 5 : 3 : 1) to
afford product 7 (30.8 mg, 90%). [α]D24 = +33.20 (c = 1.0 H2O).
νmax (film)/cm−1 3255 (s, broad, OH, NH, C–H alkene, COOH),
1583 (s, broad, CO carboxylic acid, CO amide, CC alkene),
1148 (s, C–N). 1H NMR (600 MHz, D2O) δ 5.70 (d, J = 1.8 Hz,
1H, H-3), 4.48 (dd, J = 9.0, 2.4 Hz, 1H, H-6), 4.33 (t, J =
9.0 Hz, 1H, H-4), 4.21 (d, J = 10.8 Hz, 1H, H-5), 4.05–4.02 (m,
1H, H-7), 3.57–3.54 (m, 2H, H-8 and H-9), 3.21–3.17 (m, 3H,
CH2-1 propyl, H-9′), 2.07 (s, 3H, NHCOMe), 1.78–1.72 (m, 2H,
CH2-2 propyl), 0.97 (t, J = 7.8 Hz, 3H, CH3 propyl); 13C NMR
(151 MHz, D2O) δ 175.07 (COOH), 169.77 (NHCOMe), 148.06
(C-2), 108.08 (C-3), 75.20 (C-6), 70.57 (C-7), 67.61 (C-8),
64.52 (C-4), 56.17 (C-5), 55.39 (C-9), 50.00 (CH2-1 propyl),
22.26 (NHCOMe), 16.92 (CH2-2 propyl), 10.31 (CH3 propyl).
HRMS (ESI) calculated for C14H24N2O7 (M + H) 333.1662,
found 333.1670.
gel flash column chromatography (EtOAc–MeOH–H2O
=
8 : 3 : 0.5) to afford 5 (0.12 g, 95%). [α]D24 = +35.50 (c = 1.04
H2O). νmax (film)/cm−1 3293 (s, broad, OH, NH, C–H alkene,
COOH), 2109 (s, N3), 1596 (s, CO carboxylic acid), 1542 (s,
CO amide, CC alkene), 1072 (s, C–N). 1H NMR (600 MHz,
D2O) δ 5.68 (d, J = 1.8 Hz, 1H, H-3), 4.50 (dd, J = 8.4, 3.0 Hz,
1H, H-6), 4.28 (d, J = 11.4 Hz, 1H, H-4), 4.14–4.07 (m, 4H,
NHCOCH2N3, H-5, and H-7), 3.63 (dd, J = 13.2, 2.4 Hz, 1H,
H-9), 3.59 (d, J = 9.6 Hz, 1H, H-8), 3.49 (dd, J = 13.2, 6.0 Hz,
1H, H-9′); 13C NMR (151 MHz, D2O) d 171.08 (COONa),
169.65 NHCOCH2N3), 148.04 (C-2), 107.91 (C-3), 75.03 (C-6),
68.76 (2C, C-7 and C-8), 67.65 (C-4), 53.86 (NHCOCH2N3),
52.15 (C-5), 50.13 (C-9). HRMS (ESI) calculated for
C11H15N7NaO7 (M + H) 380.0931, found 380.0924.
5-Acetamido-2,6-anhydro-3,5-dideoxy-9-O-methyl-D-glycero-D-
galacto-non-2-enonic acid (Neu5Ac9Me2en, 6). A solution of
NaOMe (0.17 g, 3.14 mmol) in MeOH (5 mL) was prepared.
A 2 mL portion of the prepared solution was added to compound
9 (0.22 g, 0.48 mmol) and stirred at room temperature for
20 min. The second portion of NaOMe in MeOH (2 mL) was
added to the reaction mixture and stirred at room temperature for
30 min. Dowex (H+) resin was added to the solution to adjust the
pH to about 2 and the mixture was stirred at room temperature
for 2 h. The reaction was neutralized using Et3N, filtrated, and
washed with MeOH and H2O. The concentrated crude product
was purified by silica gel flash column chromatography (EtOAc–
1
MeOH = 9 : 1) to afford compound 17 (0.12 g, 80%). H NMR
(600 MHz, D2O) δ 6.01 (d, J = 2.4 Hz, 1H), 4.47 (dd, J = 8.4,
2.4 Hz, 1H), 4.25 (d, J = 10.8 Hz, 1H), 4.06 (dd, J = 10.8,
6118 | Org. Biomol. Chem., 2012, 10, 6112–6120
This journal is © The Royal Society of Chemistry 2012

View Article Online
4 B. L. Cantarel, P. M. Coutinho, C. Rancurel, T. Bernard, V. Lombard and
B. Henrissat, Nucleic Acids Res., 2009, 37, D233.
Inhibition assays of bacteria sialidases and human NEU2
5 M. Kuro-o, Biochim. Biophys. Acta, 2009, 1790, 1049.
6 T. C. Camilli, M. Xu, M. P. O’Connell, B. Chien, B. P. Frank, S. Subaran,
F. E. Indig, P. J. Morin, S. M. Hewitt and A. T. Weeraratna, Pigm. Cell
Melanoma Res., 2011, 24, 175.
7 J. Cheng, H. Yu, K. Lau, S. Huang, H. A. Chokhawala, Y. Li,
V. K. Tiwari and X. Chen, Glycobiology, 2008, 18, 686.
8 J. Cheng, S. Huang, H. Yu, Y. Li, K. Lau and X. Chen, Glycobiology,
2010, 20, 260.
9 H. Yu, H. Chokhawala, R. Karpel, H. Yu, B. Wu, J. Zhang, Y. Zhang,
Q. Jia and X. Chen, J. Am. Chem. Soc., 2005, 127, 17618.
10 T. Yamamoto, Y. Hamada, M. Ichikawa, H. Kajiwara, T. Mine,
H. Tsukamoto and Y. Takakura, Glycobiology, 2007, 17, 1167.
11 Y. Li, H. Cao, H. Yu, Y. Chen, K. Lau, J. Qu, V. Thon, G. Sugiarto and
X. Chen, Mol. BioSyst., 2011, 7, 1060.
12 X. Chen and A. Varki, ACS Chem. Biol., 2010, 5, 163.
13 L. M. Chavas, C. Tringali, P. Fusi, B. Venerando, G. Tettamanti, R. Kato,
E. Monti and S. Wakatsuki, J. Biol. Chem., 2005, 280, 469.
14 M. Vonitzstein, W. Y. Wu, G. B. Kok, M. S. Pegg, J. C. Dyason, B. Jin,
T. V. Phan, M. L. Smythe, H. F. White, S. W. Oliver, P. M. Colman,
J. N. Varghese, D. M. Ryan, J. M. Woods, R. C. Bethell, V. J. Hotham,
J. M. Cameron and C. R. Penn, Nature, 1993, 363, 418.
15 M. von Itzstein, Nat. Rev. Drug Discovery, 2007, 6, 967.
16 M. C. Mann, R. J. Thomson, J. C. Dyason, S. McAtamney and M. von
Itzstein, Bioorg. Med. Chem., 2006, 14, 1518.
17 W. F. Diven, W. J. Doyle and B. Vietmeier, Ann. Oto. Rhinol. Laryngol.,
1988, 97, 6.
18 M. Kawano, H. Bando, T. Yuasa, K. Kondo, M. Tsurudome, H. Komada,
M. Nishio and Y. Ito, Virology, 1990, 174, 308.
19 T. Corfield, Glycobiology, 1992, 2, 509–521.
20 A. Albohy, M. D. Li, R. B. Zheng, C. Zou and C. W. Cairo, Glyco-
biology, 2010, 20, 1127.
21 E. Monti, M. T. Bassi, R. Bresciani, S. Civini, G. L. Croci, N. Papini,
M. Riboni, G. Zanchetti, A. Ballabio, A. Preti, G. Tettamanti,
B. Venerando and G. Borsani, Genomics, 2004, 83, 445.
22 A. Caciotti, M. Di Rocco, M. Filocamo, S. Grossi, F. Traverso,
A. d’Azzo, C. Cavicchi, A. Messeri, R. Guerrini, E. Zammarchi,
M. A. Donati and A. Morrone, J. Neurol., 2009, 256, 1911.
23 P. M. Colman, J. N. Varghese and W. G. Laver, Nature, 1983, 303, 41.
24 P. M. Mitrasinovic, Curr. Drug Targets, 2010, 11, 315.
25 J. N. Varghese, J. L. McKimm-Breschkin, J. B. Caldwell, A. A. Kortt
and P. M. Colman, Proteins: Struct., Funct., Genet., 1992, 14, 327.
26 C. U. Kim, W. Lew, M. A. Williams, H. T. Liu, L. J. Zhang,
S. Swaminathan, N. Bischofberger, M. S. Chen, D. B. Mendel, C. Y. Tai,
W. G. Laver and R. C. Stevens, J. Am. Chem. Soc., 1997, 119, 681.
27 S. Magesh, S. Moriya, T. Suzuki, T. Miyagi, H. Ishida and M. Kiso,
Bioorg. Med. Chem. Lett., 2008, 18, 532.
All sialidase assays were carried out at 37 °C in duplicates in
384-well plates in a final volume of 20 μL containing
Neu5Acα2–3GalβpNP or Neu5Acα2–6GalβpNP (0.3 mM), and
the β-galactosidase (12 μg, 126 mU) with or without inhibitors.
The amount of the β-galactosidase required to completely hydro-
lyze the GalβpNP within the time frame of the assay was pre-
determined and confirmed by assays with GalβpNP (0.3 mM).
The assay conditions for various sialidases were as follows:
C. perfringens sialidase (1.6 mU), MES buffer (100 mM,
pH 5.0); V. cholerae sialidase (0.5 mU), NaCl (150 mM),
CaCl2 (10 mM), and sodium acetate buffer (100 mM, pH 5.5);
S. typhimurium sialidase (0.6 mU), NaCl (100 mM) and sodium
acetate buffer (100 mM, pH 5.5); S. pneumoniae sialidase NanB
(0.2 mU), sodium acetate buffer (100 mM, pH 6.0); NEU2 (6 μg
for Neu5Acα2–3GalβpNP and 9.6 μg for Neu5Acα2–
6GalβpNP), MES buffer (100 mM, pH 5.0). The reactions were
carried out for 30 min. The assay was stopped by adding CAPS
buffer (N-cyclohexyl-3-aminopropane sulfonic acid, 40 μL,
0.5 M, pH 10.5). The amount of the para-nitrophenolate formed
was determined by measuring the A405 nm of the reaction mix-
tures using a microplate reader. Reactions without inhibitors
were used as controls.
IC50 values were obtained by varying the concentrations of
inhibitors from 0 to 50 mM (twelve concentrations of each
inhibitor were used to determine an IC50) with a fixed concen-
tration (0.3 mM) of Neu5Acα2–3GalβpNP or Neu5Acα2–
6GalβpNP. Typical concentration–response plots were obtained
from the average values of duplicate assay results. The values of
IC50 were obtained by fitting the averages to the Michaelis–
Menten equation using Grafit 5.0.
Docking studies
The monomeric structure of NEU2 was modelled based on the
dimeric structure of NEU2 (pdb: 1VCU).13 Neu5Ac9N32en (3),
Neu5Gc9N32en (4), or Neu5AcN39N32en (5) was docked into
the active sites of human NEU2 and Vibrio cholerae sialidase
(pdb: 1W0O), respectively, using molecular building and opti-
mizing softwares Gaussview 5.0 and Gaussview 09W and
docking software OpenEye. The designation of the protein active
site was performed using Fred Receptor 2.2.5. Docking the mol-
ecule into the designated active site was achieved using Fred as
well as 3D displaying and analyzing software Vida 4.0.0. The
electrostatic potential maps were built by PyMOL.
28 S. Magesh, V. Savita, S. Moriya, T. Suzuki, T. Miyagi, H. Ishida and
M. Kiso, Bioorg. Med. Chem., 2009, 17, 4595.
29 Y. Zou, A. Albohy, M. Sandbhor and C. W. Cairo, Bioorg. Med. Chem.
Lett., 2010, 20, 7529.
30 A. Albohy, S. Mohan, R. B. Zheng, B. M. Pinto and C. W. Cairo, Bioorg.
Med. Chem., 2011, 19, 2817.
31 M. C. Mann, R. J. Thomson, J. C. Dyason, S. McAtamney and M. von
Itzstein, Bioorg. Med. Chem., 2006, 14, 1518.
32 P. H. Nguyen, T. N. Nguyen, K. W. Kang, D. T. Ndinteh, J. T. Mbafor,
Y. R. Kim and W. K. Oh, Bioorg. Med. Chem., 2010, 18, 3335.
33 H. Hinou and S. Nishimura, Curr. Top. Med. Chem., 2009, 9, 106.
34 R. J. Russell, L. F. Haire, D. J. Stevens, P. J. Collins, Y. P. Lin,
G. M. Blackburn, A. J. Hay, S. J. Gamblin and J. J. Skehel, Nature, 2006,
443, 45.
35 H. Hinou, R. Miyoshi, Y. Takasu, H. Kai, M. Kurogochi, S. Arioka,
X. D. Gao, N. Miura, N. Fujitani, S. Omoto, T. Yoshinaga, T. Fujiwara,
T. Noshi, H. Togame, H. Takemoto and S. Nishimura, Chem.–Asian J.,
2011, 6, 1048.
Acknowledgements
This work was supported by NIH grant 1R43AI092874. X.C. is
a Camille Dreyfus Teacher-Scholar and a UC-Davis Chancellor’s
Fellow.
36 H. Cao, Y. Li, K. Lau, S. Muthana, H. Yu, J. Cheng, H. A. Chokhawala,
G. Sugiarto, L. Zhang and X. Chen, Org. Biomol. Chem., 2009, 7,
5137.
37 S. Crennell, E. Garman, G. Laver, E. Vimr and G. Taylor, Structure,
1994, 2, 535.
Notes and references
1 G. Taylor, Curr. Opin. Struct. Biol., 1996, 6, 830.
38 G. Xu, J. A. Potter, R. J. Russell, M. R. Oggioni, P. W. Andrew and
G. L. Taylor, J. Mol. Biol., 2008, 384, 436.
39 G. Xu, X. Li, P. W. Andrew and G. L. Taylor, Acta Crystallogr., Sect. F:
Struct. Biol. Cryst. Commun., 2008, 64, 772.
2 A. Buschiazzo and P. M. Alzari, Curr. Opin. Chem. Biol., 2008, 12, 565.
3 K. Stummeyer, A. Dickmanns, M. Muhlenhoff, R. Gerardy-Schahn and
R. Ficner, Nat. Struct. Mol. Biol., 2005, 12, 90.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 6112–6120 | 6119

View Article Online
40 S. L. Newstead, J. A. Potter, J. C. Wilson, G. Xu, C. H. Chien,
A. G. Watts, S. G. Withers and G. L. Taylor, J. Biol. Chem., 2008, 283,
9080.
41 S. J. Crennell, E. F. Garman, C. Philippon, A. Vasella, W. G. Laver,
E. R. Vimr and G. L. Taylor, J. Mol. Biol., 1996, 259, 264.
42 X. Xu, X. Zhu, R. A. Dwek, J. Stevens and I. A. Wilson, J. Virol., 2008,
82, 10493.
43 Z. Khedri, M. M. Muthana, Y. Li, S. M. Muthana, H. Yu, H. Cao and
X. Chen, Chem. Commun., 2012, 48, 3357.
44 T. Q. Gregar and J. Gervay-Hague, J. Org. Chem., 2004, 69, 1001.
45 M. McGann, J. Chem. Inf. Model., 2011, 51, 578.
46 G. M. Verkhivker, D. Bouzida, D. K. Gehlhaar, P. A. Rejto, S. Arthurs,
A. B. Colson, S. T. Freer, V. Larson, B. A. Luty, T. Marrone and
P. W. Rose, J. Comput.-Aided Mol. Des., 2000, 14, 731.
47 M. R. McGann, H. R. Almond, A. Nicholls, J. A. Grant and F. K. Brown,
Biopolymers, 2003, 68, 76.
48 M. D. Eldridge, C. W. Murray, T. R. Auton, G. V. Paolini and R. P. Mee,
J. Comput.-Aided Mol. Des., 1997, 11, 425.
49 H. A. Chokhawala, H. Yu and X. Chen, ChemBioChem, 2007, 8, 194.
50 T. Ercegovic and G. Magnusson, J. Org. Chem., 1995, 60, 3378–3384.
6120 | Org. Biomol. Chem., 2012, 10, 6112–6120
This journal is © The Royal Society of Chemistry 2012