Identification of selective inhibitors for human neuraminidase isoenzymes using C4,C7-modified 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) analogues

Article abstract of DOI:10.1021/jm301892f

In the past two decades, human neuraminidases (human sialidases, hNEUs) have been found to be involved in numerous pathways in biology. The development of selective and potent inhibitors of these enzymes will provide critical tools for glycobiology, help to avoid undesired side effects of antivirals, and may reveal new small-molecule therapeutic targets for human cancers. However, because of the high active site homology of the hNEU isoenzymes, little progress in the design and synthesis of selective inhibitors has been realized. Guided by our previous studies of human NEU3 inhibitors, we designed a series of C4,C7-modified analogues of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) and tested them against the full panel of hNEU isoenzymes (NEU1, NEU2, NEU3, NEU4). We identified inhibitors with up to 38-fold selectivity for NEU3 and 12-fold selectivity for NEU2 over all other isoenzymes. We also identified compounds that targeted NEU2 and NEU3 with similar potency.

Full text of DOI:10.1021/jm301892f

Article
pubs.acs.org/jmc
Identification of Selective Inhibitors for Human Neuraminidase
Isoenzymes Using C4,C7-Modified 2‑Deoxy-2,3-
didehydro‑N‑acetylneuraminic Acid (DANA) Analogues
Yi Zhang,† Amgad Albohy,† Yao Zou,† Victoria Smutova,‡,§ Alexey V. Pshezhetsky,‡,§
and Christopher W. Cairo*,†
†Alberta Glycomics Center, Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
‡Division of Medical Genetics, Centre Hospitalier
̀
e Universitaire Sainte-Justine, University of Montreal, Montreal, Quebec, Canada
§Department of Anatomy, Cell Biology, Faculty of Medicine, McGill University, Montreal, Quebec, Canada
S
* Supporting Information
ABSTRACT: In the past two decades, human neuraminidases
(human sialidases, hNEUs) have been found to be involved in
numerous pathways in biology. The development of selective and
potent inhibitors of these enzymes will provide critical tools for
glycobiology, help to avoid undesired side effects of antivirals, and
may reveal new small-molecule therapeutic targets for human
cancers. However, because of the high active site homology of the
hNEU isoenzymes, little progress in the design and synthesis of
selective inhibitors has been realized. Guided by our previous studies of human NEU3 inhibitors, we designed a series of C4,C7-
modified analogues of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) and tested them against the full panel of hNEU
isoenzymes (NEU1, NEU2, NEU3, NEU4). We identified inhibitors with up to 38-fold selectivity for NEU3 and 12-fold
selectivity for NEU2 over all other isoenzymes. We also identified compounds that targeted NEU2 and NEU3 with similar
potency.
enzymes, the neuraminidase enzymes (NEU1, NEU2, NEU3,
and NEU4) have been shown to participate in a variety of
signaling pathways and pathologies including inflammation,14,15
adhesion,7,16 tumorigenesis,17 and cancer metastasis.18,19
Notably, sialic acid content in cells and serum is altered in
autoimmune diseases; for example, multiple sclerosis patients
have decreased levels of sialic acid on their cell membranes20
and increased free sialic acid in serum.21 These and other
examples support the hypothesis that hNEUs are important
regulators in health and disease, and are therefore potential
therapeutic targets. Yet there are few examples of specific
inhibitors for this family of enzymes.
The design of specific chemical inhibitors for hNEU must
take into account the similarities between isoenzymes. The four
hNEU enzymes are classified as family 33 GH22 and exo-
sialidases (EC 3.2.1.18).23 They vary in their subcellular
location and substrate specificity. NEU1 is localized at the
lysosomal and plasma membranes.24,25 NEU2 is a soluble
protein found in the cytosol.26−28 NEU3 is a membrane-
associated protein localized in the caveolae microdomains of
plasma membranes,29 as well as endosomal and lysosomal
membranes.30 The NEU4 gene can be differentially spliced,
resulting in the appearance of two NEU4 isoforms that differ in
the first 12 N-terminal amino acids.31,32 The short isoform of
INTRODUCTION
■
The glycosylation of proteins can alter cell signaling, protein−
ligand and protein−protein interactions, and protein stabil-
ity.1−3 Most of glycan biosynthesis occurs in the endoplasmic
reticulum and Golgi compartments of the cell.1 However,
enzymatic modification of glycan structures also occurs after
glycoprotein biosynthesis, a process known as glycan
remodeling. The effects of this process can be seen in measures
of the relative half-life of glycan structures versus the protein
backbone.4,5 Glycan remodeling is gaining recognition as an
important regulatory function within mammalian cells.6 While
glycan remodeling is implicated in cell signaling,7 there remains
a lack of chemical tools to probe the specific enzymes that
participate in these processes. Two likely instigators of this
process are glycosyl hydrolase (GH) and glycosyl transferase
(GT) enzyme classes, which degrade and synthesize the
glycosidic linkage, respectively. Thus, strategies for chemical
inhibition of mammalian GH and GT are of interest for
exploring cell signaling pathways.8 The challenge, however, is to
design potent inhibitors with selectivity for specific isoenzymes
within each class in order to avoid potentially toxic effects.
Membrane-associated GH regulate signaling pathways
through cleavage of glycolipids and glycoproteins. A number
of membrane-associated glycosidases are known in mammalian
cells including β-galactosidase,9 β-glucosidase,10 α- and β-
mannosidase,11 α-fucosidase,12 and four isoenzymes of
neuraminidase (also known as sialidase).13 Among these
Received: December 22, 2012
Published: March 26, 2013
© 2013 American Chemical Society
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NEU4 is found predominantly on the ER membranes,31,32
whereas the long form is targeted to both mitochondria31,32 and
lysosomes.33 Genetic defects in NEU1 result in autosomal
pediatric sialidosis disease associated with lysosomal storage of
sialylated glycopeptides and oligosaccharides.34 NEU3 is
selective for glycolipids over glycoproteins33 and has been
reported to be modulated by signaling events such as protein
kinase C activation in immune cells.35
Importantly, we found that modification of the DANA scaffold
at the C7 side chain can impart selectivity for inhibitors among
members of the hNEU family. We identified a NEU3 inhibitor
with nearly 40-fold selectivity over other isoenzymes (5c) and a
NEU2 inhibitor with 12-fold selectivity (8b). We propose that
these results can be used to accelerate the identification of more
potent and isoenzyme-selective hNEU inhibitors.
Only a few investigations have explored the substrate
specificity of hNEU beyond native glycolipids,36,37 providing
limited data for the design of synthetic substrates or inhibitors.
Inhibitor selectivity for human NEU2 over bacterial enzymes
has been investigated and seems to suggest that these two
enzymes have significantly different preferences.37,38 Magesh et
al. have reported compounds that selectively target NEU1 with
micromolar activity.39 However, inhibitors that target NEU2,
NEU3, and NEU4 enzymes remain to be identified.
We set out to generate a small panel of 2-deoxy-2,3-
didehydro-N-acetylneuraminic acid (DANA) analogues that
contained modified C7 side chains. Previous reports have
suggested that a basic functional group at the C4 position may
increase inhibitory activity.40 Therefore, C4-modified com-
pounds were included in our scheme to probe the combination
of these two strategies (Scheme 1). Upon testing of these
RESULTS
■
Inhibitor Synthesis. We set out to synthesize DANA (1)
analogues containing C4 and C7 modifications individually or
in combination (Scheme 1). To introduce an azide at C4, we
employed established methods via nucleophilic opening of an
oxazoline intermediate, producing compound 2 after depro-
tection.41,42 Subsequent reduction of the azide to an amine
provided compound 3. Modification at C7 was accomplished
via oxidative cleavage at C7−C8 using sodium periodate.43 The
resulting aldehyde could then be used for a variety of coupling
chemistries including reductive amination and oxime or
hydrazide formation. DANA analogues containing a C4-azide
were generated by coupling to 4 (Scheme 2),44−46 while
analogues with the C4-hydroxyl were generated by coupling to
7 (Scheme 3).43 Reductive coupling of aldehyde 4 was
performed with several nucleophiles, including aromatic amines
(5a, 5b), phenylhydrazine (5e), and benzylic hydroxylamines
(5c, 5d). Reduction of the C4-azide of compound 5a with
Lindlar’s catalyst afforded the corresponding amine 6. Although
we expected that the oxime linkage initially formed in the
coupling of 5c would be stable, we chose to reduce the oxime
to avoid the formation of diastereomers.47 Coupling of
compound 7 followed an identical protocol to compound 4
using aniline (8a), benzylamine (8b), and biphenylmethylhy-
drazine (8c) nucleophiles.
Scheme 1. Inhibitor Targets
Inhibition Assays. To determine the potency of DANA
analogues against separate isoenzymes of hNEU, enzyme
samples were produced either by recombinant expression or
by purification from human cells. NEU1 was purified as a
protein complex with β-galactosidase and cathepsin A,
following previous protocols.48 Human NEU2,49 NEU3,50
DANA derivatives against the four human neuraminidase
isoenzymes, as well as a bacterial isoenzyme (Vibrio cholera
neuraminidase, vcNEU), we confirmed that NEU2 and NEU3
tolerate large hydrophobic groups at the C7 position.
Scheme 2. Synthesis of C4- and C4,C7-Subsituted DANA Derivatives
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Scheme 3. Synthesis of C7-Substituted DANA Derivatives
Table 1. Inhibition of Neuraminidase Isoenzymes
a
Coloring of potency values is by relative ranking within each isoenzyme. Darker shades of red indicate higher potency compounds with activity
below 500 μM or that are ranked in 1−7 among compounds tested. Darker shades of blue indicate weaker potency, with activities typically above
b
500 μM or that are ranked 8−12 among compounds tested. Relative activity was determined by dividing the potency of the compound for its next
weakest target by that of its primary target. For cases where multiple isoenzymes are listed as the target, the average of these was used.
and NEU451 were expressed as fusion proteins following
previous reports. With all four human isoenzymes in hand, we
first compared the pH profiles of each enzyme using a
fluorogenic substrate, 2′-(4-methylumbelliferyl)-α-D-N-acetyl-
neuraminic acid (4MU-NANA). All four enzymes had optimal
enzymatic activity in acidic environments. NEU1, NEU3, and
NEU4 showed narrow activity ranges between pH 4 and pH 5.
In contrast, NEU2 gave a wide range of activity between pH 5
and pH 6. We then tested the potency of DANA against each
enzyme using the 4MU-NANA substrate. We found that
DANA was indeed a general and nonselective inhibitor with
low micromolar (6−90 μM) activity against hNEU (Table 1).
For comparison, we tested the activity of DANA against a
bacterial enzyme, vcNEU. DANA was at least 4-fold more
potent against all members of the hNEU family, relative to
vcNEU. The inhibition constants (Ki) of DANA against three
of the human isoenzymes were determined for comparison
(NEU2, 5 2 μM; NEU3, 2.2 0.5 μM; NEU4, 1.0 0.6
μM).
Testing of the remaining compounds revealed that in
comparison to DANA (1), modification of the C4 substituent
from a hydroxyl to a bulkier azide (2) reduced potency against
NEU1 and NEU4. This effect was most pronounced in the case
of NEU1 for the C4-amino derivative 3. Interestingly, NEU2
was the most tolerant of C4 modifications; compound 3 was 2-
fold more potent than 1 against NEU2 and showed 4-fold
selectivity over other isoenzymes. These results suggested that
modifications to the C4 position could be used to gain inhibitor
selectivity for NEU2 and NEU3 over other isoenzymes.
The activity of compounds with C4,C7-modifications
confirmed that these changes could impart NEU2 and NEU3
selectivity to DANA analogues. Comparison of the activity of
compounds where the glycerol side chain of DANA was
replaced with a phenylamine (5a, 6, and 8a) illustrated that the
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Figure 1. IC50 curves of 5c and 8b for the four human neuraminidase isoenzymes. The experiments were performed as described in the Experimental
Section. 5c was 38 times more selective for NEU3, and 8b was 12 times more selective for NEU2 over the other hNEU.
C4-azido, -amino, and -hydroxy groups cannot always
compensate for significant modifications at the C7 position.
All three compounds had significantly reduced potency in
comparison to DANA. Taken together with the results above
for compounds 1−3, we concluded that the C7-aniline was not
able to gain significant contacts in the binding pocket, thus
masking any apparent gains from modification at C4. Extension
of the C7 group to a phenylhydrazide (5e) resulted in a
remarkable gain in potency for NEU2 and NEU3. Presumably,
the addition of a H-bond acceptor/donor site was partly
responsible for the observed 33-fold selectivity for NEU2 and
NEU3 over other isoenzymes. Modification of the C7-site to a
hydroxyamino group ablated one H-bond donor site from the
side chain (5c). We observed that 5c was remarkably selective
for the NEU3 isoenzyme (24 2 μM, 38-fold selective) over
all other enzymes tested. To the best of our knowledge, this
compound is the first confirmed NEU3-specific inhibitor
reported.
hydrazide group. The C4-azido-C7-biphenylamino analogue
5b targeted both NEU2 and NEU3 with mid-micromolar
activity. The mild selectivity of 5b was similar to that of the C4-
hydroxy-C7-biphenylmethylamino analogue 8c. Both 5b and 8c
had approximately 3-fold selectivity for NEU2 and NEU3, with
only moderate activity for NEU4. Finally, we determined the Ki
values for the two most potent compounds identified (with
IC50 < 25 μM) against their target enzyme, NEU3. We found
that 5c had a Ki of 8 1 μM against NEU3 and that 5e had
similar activity at 11 3 μM (Table 2).
Table 2. Ki Determinations
compd
enzyme
Ki (μM)
DANA (1)
DANA (1)
DANA (1)
5c
NEU2
NEU3
NEU4
NEU3
NEU3
5
2
2.2 0.5
1.0 0.6
8
1
5e
11
3
Examination of the potency data for 8b and 5c reveal a
dramatic switch in selectivity from NEU2 to NEU3 (Figure 1).
Compounds 8b and 5c differ in the C4 substituent and the
composition of the C7 moiety. Although the addition of a C4-
amino group improved potency for compound 3 against NEU2,
compound 5c had negligible activity against the enzyme. In
contrast, the C4-hydroxy-C7-benzylamino analogue (8b) was
12-fold selective for NEU2. The apparent switch of selectivity
between NEU2 and NEU3 is, therefore, not strictly tied to
either the C4 or C7 substituents. Chavas et al. have previously
observed that, at least in NEU2, the recognition of substrate is
dynamic and requires significant rearrangement of the active
site.52 Even so, we were surprised to find that additional steric
bulk in the form of an N-benzyl group on 5d did not disrupt
NEU3 activity. Instead, this modification reduced NEU3
selectivity (7-fold selective) while improving activity against
NEU2.
Molecular Modeling. We used molecular modeling to gain
insight into the selectivity switch of 5c and 8b between the
NEU3 and NEU2 active sites. We used the structural data
reported by Chavas et al. to model ligand interactions with
NEU2 (PDB code 1VCU).52 As no X-ray structure is currently
available for NEU3, we employed a homology model of the
enzyme that was previously developed in our group.50 To
provide a model of protein−ligand interactions, we performed
molecular docking using Autodock 4.2,54,55 followed by an
unrestrained 10 ns molecular dynamics simulation. We
evaluated the binding of 5c with NEU3 and of 8b with
NEU2 using this strategy.
The binding model of 5c supported our hypothesis that the
NEU3 active site can accommodate large hydrophobic groups
(Figure 2). The model maintained key contacts between the
C1-carboxylate and the arginine triad, as well as those of the
N5-Ac pocket. The C4-azido group was well accommodated by
the O4 pocket (I26, D50, M87, and N88). The C7 side chain of
5c contains a four-atom linker to the phenyl group and contains
two H-bond acceptors and one H-bond donor. The distance
Next we examined the effect of a large aromatic group at the
C7 position. In our previous studies of NEU3, we observed that
large hydrophobic groups could be tolerated at the C9 position
of DANA.53 We wanted to determine if the loss of the glycerol
side chain could be compensated by incorporation of a
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Figure 2. Molecular models of the NEU2 and NEU3 active sites. (a) A model of the protein−ligand complex was generated using docking and
molecular dynamics (see Experimental Section). The large hydrophobic pocket of NEU3 accommodates the C7 side chain of compound 5c. (b) The
active site topology of NEU2 (PDB code 1VCU)52 is shown for comparison and reveals a smaller pocket for the glycerol side chain of DANA (C7−
C9). Electrostatic potential surfaces were generated with DelPhi63 and visualized using PyMOL.62
Figure 3. (a) A molecular model of compound 8b bound to NEU2 postulates a binding mode with the phenyl group interacting with the
hydrophobic portion of Q270 and interacting with L217 and P192. (b) The electrostatic potential surface of NEU2 shows that the region interacting
with the phenyl ring is relatively nonpolar. (c) A molecular model of 8b in the active site of NEU3 shows that while the C7 side chain can be
accommodated sterically, the linker positions the phenyl ring in a region of the binding pocket where it cannot gain any significant contacts. (d) An
electrostatic potential surface of NEU3 reveals that the phenyl ring of 8b is positioned in a relatively polar region of the binding pocket.
between the pyranoside ring and the phenyl group would not
allow for interactions with H277 (the groups are ∼5−6 Å
apart). However, the phenyl group could gain hydrophobic
interactions with several residues of the active site (V224, V222,
P247, P198). An inspection of the NEU2 active site revealed
that the side chain of 5c, were it in the same conformation as
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predicted for NEU3, would result in a steric clash with Q270.
We propose that this difference between the active site
topology results in the selectivity observed for inhibitors with
larger C7 groups. Additionally, the model predicts that a
shorter C7 group would be better accommodated in the NEU2
active site.
utility in vivo where they will compete against the full battery of
hNEUs.38,53,58
Known inhibitors of hNEU have primarily been based upon
the 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (1) core.
The sequence homology of hNEU enzymes suggests that many
recognition elements of the active site are preserved across the
family.59 Although X-ray structural data are only available for
NEU2, DANA analogues with fewer H-bond donor groups in
place of the glycerol side chain are known to disrupt the H-
bond network of the active site and reduce potency against
NEU2.52,58 The recognition of the glycerol side chain of sialic
acid by hNEUs involves contacts from multiple tyrosine
residues.50,52,59 This arrangement is in contrast to the viral
enzyme and could potentially be exploited to improve inhibitor
selectivity.51 Modifications at the C9 position have yielded
NEU1 selective inhibitors.39 We53 and others37,38 have
explored C9- and N5-Ac-modified analogues of DANA and
their inhibitory potency against NEU3 and NEU2 isoenzymes,
respectively. Modification of DANA by inclusion of a C4-
guanidino group improved potency against NEU258 and
NEU3.57
Although the best compounds identified here have relatively
low potency (Ki ≈ 8−11 μM) for in vivo studies, they represent
the first panel of compounds to selectively target NEU2 and
NEU3. The most interesting trends observed are the apparent
selectivity switch observed between NEU2 and NEU3. The
activity of several compounds suggests similarities between
substrate recognition by these two isoenzymes. Compounds 2,
5b, 5e, and 8c show little distinction between NEU2 and
NEU3. In contrast, 5c and 8b selectively target NEU3 and
NEU2, respectively, with only minor structural differences
between the compounds. Molecular modeling suggested that
the differences between the recognition of these two active sites
can be attributed to the topology of the glycerol side chain
binding pocket. The NEU3 binding pocket is more
accommodating to larger groups and contains several polar
contacts that are distal to the position of the C9-hydroxyl of the
native substrate. In the case of NEU2, the binding pocket relies
on hydrophobic packing with smaller side chains. The NEU2
Q270 residue may act as a gatekeeper that prevents the binding
of larger groups in this pocket.
We next examined the selective binding of 8b to the NEU2
active site (Figure 3). The model of 8b maintained the same
contacts to the active site for the C1-carboxylate, O4, and N5Ac
groups as observed with DANA and NEU2. The C7 side chain
was the only point of departure from DANA. In this case the
C7 side chain contained a phenyl group; however, the linker
between the pyranoside ring and the arene has been truncated
to three atoms and contains only one H-bond donor and
acceptor. In this binding mode, the linker could gain a H-bond
contact with Y181 (3.4 Å) and hydrophobic interaction with
L217 and Q270(Cβ). Thus, the relatively small C7 side chain,
with its shorter linker, packs into the restricted space available
in the NEU2 pocket and gains contacts that improve its activity
for the enzyme. In contrast, the NEU3 pocket is too large for
effective packing of the 8b side chain, resulting in a loss of one
H-bond contact and leaving the phenyl ring more solvent
exposed. The C7 side chain of 8b positions the phenyl group
near relatively polar residues (H277, Y181, and R245), where it
is unable to contact the hydrophobic groups that benefit 5c
binding in the NEU3 pocket.
DISCUSSION
■
In this study we have found that modification of the DANA
inhibitor scaffold at the C4 and C7 positions resulted in
compounds with improved specificity against the hNEU
isoenzymes NEU2 and NEU3. Importantly, we identified lead
compounds with 38-fold (5c) and 12-fold (8b) selectivity for
these two isoenzymes, respectively. These are the first reported
compounds that have been confirmed to be selective for NEU2
and NEU3 over other hNEUs. While our data confirmed that
DANA (1) was a nonselective inhibitor of hNEU, we also
identified compounds that could target a subset of hNEU
isoenzymes. For example, compounds 2, 5b, 5e, and 8c inhibit
NEU2 and NEU3 with comparable potency. These compounds
provide an important starting point for the future design of
more potent and selective compounds targeting individual
human neuraminidases.
CONCLUSION
■
There are very few reports of isoenzyme-selective hNEU
inhibitors, and yet selective small molecule inhibitors are
needed to expand our understanding of this important family of
enzymes. Despite the fact that nanomolar inhibitors of viral
sialidases are known,56 the most potent inhibitors reported for
hNEU have inhibitory constants in the low micromolar
range.39,53 Interestingly, nanomolar inhibitors of influenza
viral sialidase are significantly less active against hNEU.51,57
Zanamivir (a C4-guanidino-modified DANA analogue) is
reported to be the most potent of the viral inhibitors against
hNEU, with moderate selectivity for NEU2.57 We observed that
a C4-azido analogue of DANA (2) had similar selectivity for
both NEU2 and NEU3 isoenzymes. Few other reports have
tested inhibitors designed to target individual hNEUs among
the four isoenzymes. Several compounds selective for NEU1
have been identified.39 Our group has previously identified
oseltamivir analogues that were weakly selective for NEU3 over
NEU4.51 Although other studies have examined the activity of
inhibitors against hNEU individually, without data on their
activity against other isoenzymes it is difficult to judge their
On the basis of our results, we conclude that derivatives of the
DANA scaffold can act as isoenzyme-selective hNEU inhibitors.
Furthermore, the binding pocket for the glycerol side chain
(C7−C9 of the sialic acid substrate) is a site of diverse
enzyme−substrate interactions that can be exploited to improve
the potency and selectivity of inhibitors. The human
neuraminidase enzymes are of growing biological interest, and
new tools are required to understand their roles in
glycobiology. Previously, genetic approaches, such as gene
targeting in mice or siRNA, have been used to study the
biological roles of human neuraminidase enzymes. However,
small molecule inhibitors have inherent advantages because of
their rapid onset and reversibility of their effects. Herein, we
have described a panel of DANA analogues that achieve
selective inhibition of one or multiple hNEU isoenzymes at low
micromolar potency. These findings should guide future design
of inhibitors with improved potency and selectivity for this
family of enzymes. Future work will focus on the development
of analogues with improved potency and selectivity, as well as
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2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonic
acid methyl ester (119 mg, 0.36 mmol)44−46 in methanol/water (3:1,
v/v, 4 mL) was added sodium periodate (156 mg, 0.729 mmol), and
the reaction mixture was stirred at room temperature for 30 min. The
precipitate was removed by filtration, and the filtrate was evaporated in
vacuo to give a white solid. The aldehyde was typically isolated as a
mixture of the aldehyde and the methanolic hemiacetal. 1H NMR (500
MHz, CD3OD) δ 5.98−5.97 (m, 1H, H-3), 4.71−4.65 (m, 1H, H-7),
4.31−4.15 (m, 2H, H-4, H-5), 4.08−4.03 (m, 1H, H-6), 3.83−3.81
(m, 3H, COOCH3), 1.99−1.98 (d, J = 4.5 Hz, 3H, CH3). 13C NMR
(125 MHz, CD3OD) δ 172.2−171.9 (COCH3), 162.5−162.3
(COOH), 144.39−144.38 (C2), 107.2−106.4 (C3), 95.0−94.1 (C7),
78.6−78.4 (C6), 57.1−56.2 (C4), 51.7−51.5 (COOMe), 48.4−48.2
(C5), 21.27−21.20 (COCH3). HRMS (ESI) calcd for the methyl
hemiacetal sodium adduct, C11H16N4NaO6 [M + Na]+, 323.0962;
found, 323.0959.
5-Acetamido-4-azido-2,6-anhydro-3,4,5-trideoxy-6-
((phenylamino)methyl)-L-gluco-hex-2-enonic Acid (5a). Com-
pound 5a was prepared by coupling of compound 4 and aniline
following the general procedure given above. The product was
obtained in three steps in 36% yield. 1H NMR (600 MHz, CD3OD) δ
7.37−7.34 (m, 2H, ArH), 7.15−7.09 (m, 3H, ArH), 6.26−6.25 (d, J =
5.4 Hz, 1H, H-3), 4.35−4.30 (m, 2H, H-4, H-6), 4.12−4.10 (m, 1H,
H-5), 3.63−3.61 (m, 1H, H-7), 3.51−3.49 (m, 1H, H-7), 2.02 (s, 3H,
CH3). 13C NMR (125 MHz, DMSO) δ 174.9 (COCH3), 163.1
(COOH), 162.8 (ArC1), 130.4 (C2), 129.5 (ArC3), 122.2 (ArC2),
117.5 (ArC4), 106.4 (C3), 72.4 (C6), 57.5 (C4), 49.0 (C7), 43.9 (C5),
21.9 (COCH3). HRMS (ESI) calcd for C15H16N5O4 [M − H]−,
330.1208; found, 330.1218.
5-Acetamido-4-azido-2,6-anhydro-3,4,5-trideoxy-6-(([1,1′-
biphenyl]-4-ylamino)methyl)-L-gluco-hex-2-enonic Acid (5b).
Compound 5b was prepared by coupling of compound 4 and [1,1′-
biphenyl]-4-amine following the general procedure given above. The
product was obtained in three steps in 54% yield. 1H NMR (500 MHz,
CD3OD) δ 7.79−7.78 (d, J = 7.0 Hz, 2H, ArH), 7.70−7.69 (d, J = 6.5
Hz, 2H, ArH), 7.54−7.51 (t, J = 6.0 Hz, 2H, ArH), 7.46−7.44 (d, J =
7.5 Hz, 3H, ArH), 6.05−6.05 (d, J = 4.0 Hz, 1H, H-3), 4.22−4.18 (m,
2H, H-4, H-6), 4.06−4.05 (d, J = 4.0 Hz, 1H, H-5), 3.71−3.69 (d, J =
6.0 Hz, 2H, H-7), 1.98 (s, 3H, CH3). 13C NMR (125 MHz, CD3OD)
δ 172.3 (COCH3), 167.7 (COOH), 152.2 (ArC1′), 147.8 (ArC1),
141.2 (C2), 130.1 (ArC3′), 128.2 (ArC3), 127.2 (ArC2′), 127.1 (ArC4′),
125.6 (ArC2), 113.2 (ArC4), 98.4 (C3), 72.0 (C6), 55.6 (C4), 48.4
(C7), 43.9 (C5), 20.9 (COCH3). HRMS (ESI) calcd for C21H20N5O4
[M − H]−, 406.1521; found, 406.1526.
compounds that can selectively target NEU4, the only
remaining isoenzyme with no reported selective inhibitors.
EXPERIMENTAL SECTION
■
General Synthetic Methods. All reagents, including hydrazines
and amines, were purchased from Sigma-Aldrich (Oakville, Ontario,
Canada) or Acros Organics unless otherwise noted. Compounds were
used without further purification unless otherwise noted. All reactions
were carried out under argon at room temperature unless otherwise
indicated. Reactions were monitored by analytical TLC on silica gel
60-F254 (0.25 mm, Silicycle, Quebec, Canada), and spots were
visualized under UV light (254 nm) or stained by charring with
ceric ammonium molybdate (CAM). Compounds were purified by
flash column chromatography with silica gel (230−400 mesh, Silicycle,
Quebec, Canada) or by HPLC. 1H NMR spectra were obtained with a
Varian 500, 600, or 700 MHz instrument as noted. 13C NMR spectra
were recorded at 125, 150, or 175 MHz. Electrospray ionization mass
spectra were recorded on an Agilent Technologies 6220 TOF
spectrometer. Target compounds 2 and 3 and intermediate aldehydes
4 and 7 were synthesized according to known protocols.43−46
Compounds used for inhibitor assays were confirmed to be of
≥95% purity by HPLC (see Supporting Information for details).
General Procedure for Synthesis of 5a−e and 8a−c. To a
solution of aldehyde 4 (134 mg, 0.5 mmol) in MeOH (5 mL) was
added the appropriate amine or hydrazine (2.5 mmol, 5 equiv) and
HOAc (50 μL). The mixture was stirred until TLC (hexane/ethyl
acetate, 1:2) showed no starting material remained. Sodium
cyanoborohydride (400 mg, 3 mmol) was added and the mixture
stirred overnight. The solvent was then concentrated in vacuo. The
residue was diluted with ethyl acetate and washed with saturated
aqueous NaHCO3 and brine. The organic layer was dried on Na2SO4
and concentrated in vacuo. The ester was isolated by flash column
chromatography (hexane/ethyl acetate, 1:2). The ester was then
hydrolyzed by treatment with 0.1 M LiOH in MeOH (5 mL) for 1 h at
room temperature. The product was neutralized with Amberlite IR-
120 (H+ form) followed by filtration and evaporation of the solvent.
Yields of 5a−e ranged from 36% to 58% as noted below.
The synthesis of 8a−c followed a procedure identical to that for
5a−e, with the replacement of aldehyde 4 with aldehyde 7 (122 mg,
0.5 mmol). Yields of 8a−c ranged from 42% to 74% as noted below.
5-Acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-
non-2-enonic Acid (1). 1H NMR (500 MHz, CD3OD) δ 5.64−5.63
(d, J = 2.5 Hz, 1H, H-3), 4.34−4.32 (dd, J = 8.5, 2.0 Hz, 1H, H-4),
4.10−4.00 (d, J = 1.0 Hz, 1H, H-6), 4.08−3.98 (m, 1H, H-5), 3.89−
3.82 (m, 1H, H-8), 3.81−3.78 (dd, J = 11.5, 3.0 Hz, 1H, H-9), 3.63−
3.60 (m, 1H, H-7), 3.50−3.48 (dd, J = 9.5, 1.0 Hz, 1H, H-9), 2.01 (s,
3H, CH3). 13C NMR (125 MHz, CD3OD) δ 178.5 (COCH3), 173.2
(COOH), 148.8 (C2), 106.6 (C3), 75.7 (C6), 69.8 (C8), 68.7 (C9),
67.3 (C4), 63.5 (C7), 50.5 (C5), 22.5 (COCH3). HRMS (ESI) calcd
for C11H16NO8 [M − H]−, 290.0881; found, 290.0874.
5-Acetamido-4-azido-2,6-anhydro-3,4,5-trideoxy-6-
(((benzyloxy)amino)methyl)-L-gluco-hex-2-enonic Acid (5c).
Compound 5c was prepared by coupling of compound 4 and O-
benzylhydroxylamine following the general procedure given above.
The product was found to have two rotamers present in an 11:1 ratio
as confirmed by analytical HPLC (see Supporting Information). The
1
product was isolated in three steps with 47% yield. H NMR (500
5-Acetamido-4-azido-2,6-anhydro-3,4,5-trideoxy-D-glycero-
1
MHz, CD3OD) δ 7.33−7.22 (m, 6H, ArH, NH), 5.70−5.69 (d, J = 2.5
Hz, 1H, H-3), 4.65 (s, 2H, CH2), 4.17−4.08 (m, 2H, H-6, H-4), 3.86−
3.83 (dd, J = 10.0, 8.5 Hz, 1H, H-5), 3.14−3.11 (dd, J = 14.0, 2.0 Hz,
1H, H-7), 2.94−2.90 (dd, J = 14.0, 9.5 Hz, 1H, H-7), 1.94 (s, 3H,
CH3). 13C NMR (125 MHz, CD3OD) δ 172.0 (COCH3), 167.5
(COOH), 150.1 (ArC1), 138.0 (C2), 128.0 (ArC3), 127.8 (ArC2),
127.3 (ArC4), 101.9 (C3), 75.1 (C6), 73.6 (OCH2Ph), 58.6 (C4), 52.1
(C7), 49.7 (C5), 21.3 (COCH3). HRMS (ESI) calcd for
C16H19N5NaO5 [M + Na]+, 384.1278; found, 384.1281.
5-Acetamido-4-azido-2,6-anhydro-3,4,5-trideoxy-6-((benzyl-
(benzyloxy)amino)methyl)-L-gluco-hex-2-enonic Acid (5d).
Compound 5d was prepared by coupling of compound 4 and N,O-
dibenzylhydroxylamine60 following the general procedure given above.
The product was obtained in three steps in 52% yield. 1H NMR (600
MHz, D2O) δ 7.45−7.43 (m, 5H, ArH), 7.35−7.33 (m, 3H, ArH),
7.22−7.20 (m, 2H, ArH), 5.81−5.80 (d, J = 3.0 Hz, 1H, H-3), 4.43−
4.35 (m, 2H, OCH2), 4.17−4.14 (dd, J = 8.4, 2.4 Hz, 1H, H-4), 4.14−
4.11 (d, J = 13.2 Hz, 1H, H-6), 4.00−3.97 (t, J = 9.0 Hz, 1H, H-5),
3.92−3.87 (m, 2H, NCH2), 3.12−3.08 (dd, J = 13.8, 8.4 Hz, 1H, H-7),
D-galacto-non-2-enonic Acid (2). H NMR (500 MHz, D2O) δ
6.01−6.00 (d, J = 1.5 Hz, 1H, H-3), 4.38−4.33 (m, 2H, H-4, H-6),
4.24−4.21 (t, J = 8.5 Hz, 1H, H-5), 3.93−3.90 (m, 1H, H-8), 3.87−
3.85 (dd, J = 10.0, 2.0 Hz, 1H, H-9), 3.67−3.27 (m, 2H, H-7, H-9),
2.06 (s, 3H, CH3). 13C NMR (125 MHz, D2O) δ 175.5 (COCH3),
166.4 (COOH), 146.4 (C2), 108.7 (C3), 76.7 (C6), 70.8 (C8), 68.7
(C9), 63.9 (C4), 61.1 (C7), 59.7 (C5), 23.0 (COCH3). HRMS (ESI)
calcd for C11H17N2O7 [M − Na]−, 289.1041; found, 289.1041.
5-Acetamido-4-amino-2,6-anhydro-3,4,5-trideoxy-D-glycero-
1
D-galacto-non-2-enonic Acid (3). H NMR (500 MHz, D2O) δ
5.66−5.65 (d, J = 2.0 Hz, 1H, H-3), 4.34−4.33 (m, 2H, H-5, H-6),
4.22−4.18 (m, 1H, H-4), 3.98−3.85 (m, 1H, H-8), 3.86−3.84 (m, 1H,
H-7), 3.66−3.63 (m, 2H, H-9), 2.06 (s, 3H, CH3). 13C NMR (125
MHz, D2O) δ 175.7 (COCH3), 169.4 (COOH), 151.6 (C2), 100.9
(C3), 76.0 (C6), 70.6 (C8), 68.7 (C9), 64.0 (C4), 51.1 (C7), 46.8 (C5),
22.8 (COCH3). HRMS (ESI) calcd for C11H16N4NaO7 [M + Na]+,
339.0911; found, 339.0910.
Methyl 5-Acetamido-4-azido-2,6-anhydro-3,4,5-trideoxy-6-
formyl-L-gluco-hex-2-enonate (4). To a solution of 5-acetamido-
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2.91−2.89 (m, 1H, H-7), 1.96 (s, 3H, CH3). 13C NMR (125 MHz,
CD3OD) δ 171.8 (COCH3), 167.8 (COOH), 150.2 (C2), 139.2
(ArC), 137.1 (ArC), 136.9 (ArC), 129.9 (ArC), 128.7 (ArC), 128.1
(ArC), 128.0 (ArC), 127.8 (ArC), 127.7 (ArC), 127.4 (ArC), 127.0
(ArC), 126.7 (ArC), 100.6 (C3), 75.4 (OCH2Ph), 74.7 (C6), 63.2
(NCH2Ph), 58.2 (C7), 57.5 (C4), 52.1 (C5), 21.3 (COCH3). HRMS
(ESI) calcd for C23H24N5O5 [M − H]−, 450.1783; found, 450.1779.
5-Acetamido-4-azido-2,6-anhydro-3,4,5-trideoxy-6-((2-
phenylhydrazinyl)methyl)-L-gluco-hex-2-enonic Acid (5e).
Compound 5e was prepared by coupling of compound 4 and
phenylhydrazine following the general procedure given above. The
6.25−6.23 (d, J = 5.5 Hz, 1H, H-3), 4.38−4.35 (dd, J = 8.5, 4.0 Hz,
1H, H-6), 4.30 (s, 2H, CH2), 4.10 (s, 1H, H-5), 4.07−4.06 (d, J = 5.5
Hz, H-4), 3.36−3.34 (m, 2H, H-7), 1.94 (s, 3H, CH3). 13C NMR (125
MHz, D2O) δ 174.5 (COCH3), 165.2 (COOH), 144.5 (ArC1), 130.3
(C2), 130.0 (ArC3), 129.8 (ArC2), 129.3 (ArC4), 109.8 (C3), 69.8
(C6), 62.2 (C4), 51.4 (CH2Ph), 49.0 (C5), 47.5 (C7), 21.6 (COCH3).
HRMS (ESI) calcd for C16H21N2O5 [M + H]+, 321.1445; found,
321.1439.
5-Acetamido-2,6-anhydro-3,5-dideoxy-6-((2-([1,1′-biphen-
yl]-4-ylmethyl)hydrazinyl)methyl)-L-gluco-hex-2-enonic Acid
(8c). Compound 8c was prepared by coupling of compound 7 and
([1,1′-biphenyl]-4-ylmethyl)hydrazine following the general procedure
given above. The product was obtained in three steps in 42% yield. 1H
NMR (600 MHz, D2O) δ 7.75−7.71 (m, 4H, ArH), 7.58−7.52 (m,
4H, ArH), 7.46−7.43 (m, 1H, ArH), 5.99−5.98 (d, J = 2.0 Hz, 1H, H-
3), 4.41−4.31 (m, 3H, H-4, CH2), 4.20−4.20 (m, 1H, H-6), 3.95−3.92
(t, J = 7.5 Hz, 1H, H-5), 3.33−3.32 (m, 2H, H-7), 2.02 (s, 3H, CH3).
13C NMR (125 MHz, CD3OD) δ 172.0 (COCH3), 168.5 (COOH),
1
product was obtained in three steps and 58% yield. H NMR (500
MHz, CD3OD) δ 7.19−7.16 (m, 2H, ArH3), 6.93−6.91 (d, J = 8.5 Hz,
2H, ArH2), 6.80−6.78 (m, 1H, ArH4), 5.90−5.89 (d, J = 2.5 Hz, 1H,
H-3), 4.24−3.99 (m, 2H, H-4, H-6), 4.00−3.97 (t, J = 9.5 Hz, 1H, H-
5), 3.56−3.33 (m, 2H, H-7), 3.28−3.13 (m, 2H, H-7), 1.90 (s, 3H,
CH3). 13C NMR (125 MHz, CD3OD) δ 173.5 (COCH3), 153.4
(COOH), 148.8 (ArC1), 146.5 (C2), 129.8 (ArC3), 121.2 (ArC2),
115.4 (ArC4), 106.8 (C3), 76.9 (C6), 59.2 (C4), 50.9 (C7), 50.7 (C5),
22.7 (COCH3). HRMS (ESI) calcd for C15H17N6O4 [M − H]−,
345.1307; found, 345.1317. IR (film) ν 3271, 3061, 2929, 2098, 1712,
1656, 1601, 1555, 1497, 1406, 1375, 1313, 1256, 1148, 1093, 1032,
895, 886, 825, 786, 774, 752, 715, 696, 678, 663 cm−1. [α]D20 112.96° (c
0.55, CH3OH).
148.5 (C2), 140.9 (ArC), 139.8 (ArC), 137.8 (ArC), 129.5 (ArC),
128.3 (ArC), 126.7 (ArC), 126.5 (ArC), 126.3 (ArC), 106.7 (C3), 75.5
(C6), 66.8 (C4), 60.0 (CH2Ph), 52.3 (C5), 50.3 (C7), 21.4 (COCH3).
HRMS (ESI) calcd for C22H26N3O5 [M + H]+, 412.1867; found,
412.1867.
General Protocol for Inhibition Assays. NEU3 and NEU2 were
expressed in E. coli as N-terminal MBP fusion proteins and purified as
described previously.50 NEU4 was expressed as a GST fusion protein
and purified as described.51 NEU1 was purified as previously
described.48 Assays were conducted in 0.1 M sodium acetate buffer
at the enzyme optimum pH (pH 4.5 for NEU1, NEU3, and NEU4;
pH 5.5 for NEU2), using a similar amount of enzymatic activity for all
four proteins, as determined by assay with 4MU-NANA. Inhibitors
were subjected to 3-fold serial dilutions starting from a final
concentration of 1 mM. Dilutions were performed in reaction buffer
(20 μL). The mixture was then incubated for 15 min at 37 °C.
Fluorogenic substrate (4MU-NANA, 50 μM final concentration) was
added to the reaction buffer (20 μL) and incubated at 37 °C for 30
min. The reaction was quenched with 200 μL of 0.2 M sodium
glycinate buffer, pH 10.7, and enzyme activity was determined by
measuring fluorescence (λex = 365 nm excitation; λem = 445 nm
emission) in a 384-well plate using a plate reader (Molecular Devices,
Sunnyvale CA). Assays were performed with four replicates for each
point. Error bars indicate the standard deviation. Reported IC50 values
were determined by nonlinear regression using SigmaPlot 12. For
curves that showed less than a 50% decrease in signal, fits were
conducted using the maximum inhibition values found for DANA.
Ki Determinations. Solutions of 4MU-NANA in sodium acetate
buffer (0.1 M) at the optimum pH of the target enzyme were prepared
with concentrations of 20, 40, 60, 80, and 100 μM. Each substrate
solution (25 μL) was mixed with an equal volume of a solution
containing serial concentrations of the inhibitor and the target enzyme.
The inhibitor concentrations were selected as a range that included the
determined IC50 value. The reaction mixture (50 μL) was transferred
to a 384-well plate. The rate of the product formation at 37 °C was
followed by measuring the fluorescence (λex = 365 nm excitation; λem
= 445 nm emission) every 30 s for 60 min using a plate reader
(Molecular Devices, Sunnyvale CA). Pseudo-first-order rates were
determined using the initial linear part of the resulting curves. The
double reciprocal plot (1/rate versus 1/[S]) for each inhibitor
concentration was used to determine a slope, and a plot of the slopes
versus inhibitor concentration was fit to determine Ki.
5-Acetamido-4-amino-2,6-anhydro-3,4,5-trideoxy-6-
((phenylamino)methyl)-L-gluco-hex-2-enonic Acid (6). Acid 5a
(5 mg) was dissolved in MeOH (2 mL) followed by addition of
Lindlar’s catalyst (5 mg). The system was purged three times with
argon, and the resulting mixture was stirred under positive hydrogen
pressure at room temperature overnight. The system was purged again
with argon, and the reaction mixture was filtered through a plug of
Celite. The filtrate was then evaporated under reduced pressure to give
1
the title compound 6 as a white solid (4 mg, quant). H NMR (700
MHz, D2O) δ 7.54−7.43 (m, 5H, ArH), 6.01−6.00 (d, J = 3.5 Hz, 1H,
H-3), 4.67−4.63 (m, 1H, H-6), 4.70−4.52 (t, J = 6.3 Hz, 1H, H-5),
4.22−4.21 (m, 1H, H-4), 3.77−3.72 (m, 2H, H-7), 1.99 (s, 3H, CH3).
13C NMR (175 MHz, D2O) δ 174.9 (COCH3), 165.1 (COOH), 146.0
(ArC1), 134.4 (C2), 130.5 (ArC), 129.6, 122.2, 117.1, 115.5, 102.9
(C3), 70.5 (C6), 50.5 (C7), 44.9 (C5), 44.0 (C4), 21.8 (COCH3).
HRMS (ESI) calcd for C17H19F3N3O6 [M + CF3COO]−, 418.1231;
found, 418.1235.
Methyl 5-Acetamido-2,6-anhydro-3,5-dideoxy-6-formyl-L-
gluco-hex-2-enonate (7). Aldehyde 7 was prepared according to
known protocols.43 The compound was typically isolated as a mixture
1
of the aldehyde and the methanolic hemiacetal. H NMR (500 MHz,
CD3OD) δ 8.19−8.13 (m, 1H, COOH), 6.02−5.97 (m, 1H, H-3),
4.72−4.69 (m, 1H, H-7), 4.39−4.00 (m, 3H, H-4, H-6, H-5), 4.01−
3.78 (m, 3H, COOCH3), 1.96−1.92 (m, 3H, CH3). 13C NMR (125
MHz, CD3OD) δ 172.4−171.7 (COCH3), 163.1−160.4 (COOH),
145.2−142.8 (C2), 111.7−105.6 (C3), 95.6−94.3 (C7), 78.7−78.5
(C5), 66.5−64.5 (C4), 51.7−51.4 (COOCH3), 49.7−49.4 (C6), 21.3-
21.1 (COCH3). HRMS (ESI) calcd for the methanolic hemiacetal
sodium adduct, C11H17NNaO7 [M + Na]+, 298.0897; found, 298.0896.
5-Acetamido-2,6-anhydro-3,5-dideoxy-6-((phenylamino)-
methyl)-L-gluco-hex-2-enonic Acid (8a). Compound 8a was
prepared by coupling of compound 7 and aniline following the
general procedure given above. The product was obtained in three
steps and 74% yield. 1H NMR (500 MHz, D2O) δ 7.59−7.56 (m, 3H,
ArH), 7.55−7.51 (m, 2H, ArH), 6.24−6.24 (d, J = 4.5 Hz, 1H, H-3),
4.30−4.28 (m, 1H, H-6), 4.10 (s, 1H, H-5), 4.09−4.07 (dd, J = 4.5, 2.0
Hz, 1H, H-4), 3.75−3.74 (m, 2H, H-7), 1.98 (s, 3H, CH3). 13C NMR
(125 MHz, D2O) δ 175.3 (COCH3), 166.5 (COOH), 163.9 (ArC1),
145.9 (C2), 131.4 (ArC3), 131.0 (ArC2), 123.4 (ArC4), 110.3 (C3),
69.8 (C6), 63.2 (C4), 52.5 (C7), 49.8 (C5), 22.5 (COCH3). HRMS
(ESI) calcd for C15H17N2O5 [M − H]−, 305.1143; found, 305.1144.
5-Acetamido-2,6-anhydro-3,5-dideoxy-6-((benzylamino)-
methyl)-L-gluco-hex-2-enonic Acid (8b). Compound 8b was
prepared by coupling of compound 7 and benzylamine following the
general procedure given above. The product was obtained in three
steps and 70% yield. 1H NMR (500 MHz, D2O) δ 7.47 (s, 5H, ArH),
Molecular Modeling. The homology model of NEU3 was the
same as reported previously.50 Molecular docking to the protein
models was performed using Autodock 4.2 using a 603 Å grid centered
on the active site with 0.375 Å resolution.54,55 The 200 lowest energy
ligand poses were evaluated by cluster analysis, and the lowest energy
conformer that maintained key contacts to the active site was selected
as the starting point for further analysis by molecular dynamics
calculations. Molecular dynamics were performed using MacroModel
9.9 from an initial protein−ligand complex obtained from docking.
The complex was first equilibrated for 100 ps at 300 K, followed by 10
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Article
ns dynamics at 300 K with 1.5 fs time steps. The force field used was
AMBER* with a GB/SA continuum solvation model.61 All simulations
were observed to converge (see Supporting Information). The
converged structure was then subjected to unconstrained minimiza-
tion, providing the final model of the protein−ligand complex. Models
were visualized in PyMOL,62 and protein surfaces were calculated
using DelPhi.63
(5) Tauber, R.; Park, C. S.; Reutter, W. Intramolecular heterogeneity
of degradation in plasma membrane glycoproteinsevidence for a
general characteristic. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 4026−
4029.
(6) Parker, R. B.; Kohler, J. J. Regulation of intracellular signaling by
extracellular glycan remodeling. ACS Chem. Biol. 2010, 5, 35−46.
(7) Pshezhetsky, A. V.; Hinek, A. Where catabolism meets signalling:
neuraminidase 1 as a modulator of cell receptors. Glycoconjugate J.
2011, 28, 441−452.
(8) Gloster, T. M.; Vocadlo, D. J. Developing inhibitors of glycan
processing enzymes as tools for enabling glycobiology. Nat. Chem. Biol.
2012, 8, 683−694.
(9) Goi, G.; Bairati, C.; Massaccesi, L.; Lovagnini, A.; Lombardo, A.;
Tettamanti, G. Membrane anchoring and surface distribution of
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473, 89−94.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR data, HRMS spectra, and HPLC traces for all
tested compounds, SDS−PAGE of purified human neuramini-
dases, IC50 curves, pH profiles of the human neuraminidases,
and Ki determinations. This material is available free of charge
via the Internet at http://pubs.acs.org.
(10) Aureli, M.; Masilamani, A. P.; Illuzzi, G.; Loberto, N.;
Scandroglio, F.; Prinetti, A.; Chigorno, V.; Sonnino, S. Activity of
plasma membrane beta-galactosidase and beta-glucosidase. FEBS Lett.
2009, 583, 2469−2473.
(11) Porwoll, S.; Loch, N.; Kannicht, C.; Nuck, R.; Grunow, D.;
Reutter, W.; Tauber, R. Cell surface glycoproteins undergo
postbiosynthetic modification of their N-glycans by stepwise
demannosylation. J. Biol. Chem. 1998, 273, 1075−1085.
(12) Cordero, O. J.; Merino, A.; de la Cadena, M. P.; Bugía, B.;
Accession Codes
PDB code used: 1VCU, reported by Chavas et al.52
AUTHOR INFORMATION
Corresponding Author
*Phone: 780 492 0377. Fax: 780 492 8231. E-mail: ccairo@
ualberta.ca.
■
Author Contributions
Nogueira, M.; Vinuela, J. E.; Martínez-Zorzano, V. S.; de Carlos, A.;
̃
Y. Zhang synthesized and characterized compounds. A.A.
produced NEU2, NEU3, and NEU4, performed molecular
modeling, and conducted enzyme assays. Y. Zou developed
synthetic methodology. V.S. prepared NEU1. A.V.P. designed
experiments and wrote the manuscript. C.W.C. designed
experiments and wrote the manuscript. All authors have given
approval to the final version of the manuscript.
Rodríguez-Berrocal, F. J. Cell surface human alpha-L-fucosidase. Eur. J.
Biochem. 2001, 268, 3321−3331.
(13) Monti, E.; Bonten, E.; D’Azzo, A.; Bresciani, R.; Venerando, B.;
Borsani, G.; Schauer, R.; Tettamanti, G. Sialidases in Vertebrates: A
Family of Enzymes Tailored for Several Cell Functions. In Advances in
Carbohydrate Chemistry and Biochemistry, Academic Press: New York,
2010; Vol. 64, pp 403−479.
(14) Feng, C.; Zhang, L.; Almulki, L.; Faez, S.; Whitford, M.; Hafezi-
Moghadam, A.; Cross, A. S. Endogenous PMN sialidase activity
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge the support by a grant from the
Natural Sciences and Engineering Research Council (NSERC)
and the Alberta Glycomics Centre. Infrastructure support was
provided by the Canadian Foundation for Innovation.
ABBREVIATIONS USED
■
NEU, neuraminidase enzyme; hNEU, human neuraminidase
enzyme; vcNEU, Vibrio cholera sialidase; DANA, 2-deoxy-2,3-
didehydro-N-acetylneuraminic acid; GH, glycosyl hydrolase;
GT, glycosyl transferase; SAR, structure−activity relationship;
4MU-NANA, 2′-(4-methylumbelliferyl)-α-D-N-acetylneura-
minic acid
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