Synthesis and biological evaluation of non-hydrolyzable 1,2,3-triazole-linked sialic acid derivatives as neuraminidase inhibitors

Article abstract of DOI:10.1002/ejoc.200900117

αa-Sialic acid, azide 1 has been used as a substrate for the efficient preparation of 1,2,3-triazole derivatives of sialic acid using the copper-catalyzed azide-alkyne Huisgen cycloaddition ("click chemistry"). Our approach is to generate nonnatural N-glycosides of sialic acid, that are resistant to neuraminidase-catalyzed hydrolysis as opposed, to the natural Oglycosides. These N-glycosides would act as neuraminidase inhibitors to prevent the release of new virions. As a preliminary study, a small library of 1,2,3-triazole-linked sialic acid, derivatives has been synthesized in 71-89% yield. A. disaccharide mimic of sialic acid, has also been prepared using the α-sialic acid azide 1 and a C-8 propargyl sialic acid acceptor in 68 % yield. A model sialic acid coated dendrimer was also synthesized from a perpropargylated pentaerythritol acceptor. These novel sialic acid derivatives were then evaluated, as potential neuraminidase inhibitors using a 96-well plate fluorescence assay; micromolar IC50 values were observed, comparable to the known sialidase inhibitor Neu5Ac2en.

Full text of DOI:10.1002/ejoc.200900117

FULL PAPER
DOI: 10.1002/ejoc.200900117
Synthesis and Biological Evaluation of Non-Hydrolyzable 1,2,3-Triazole-
Linked Sialic Acid Derivatives as Neuraminidase Inhibitors
Michel Weïwer,[a] Chi-Chang Chen,[a] Melissa M. Kemp,[b] and Robert J. Linhardt*[a,b,c]
Keywords: Sialic acids / Cycloaddition / N-Glycosides / Inhibitors / Viruses
α-Sialic acid azide 1 has been used as a substrate for the
efficient preparation of 1,2,3-triazole derivatives of sialic acid
using the copper-catalyzed azide–alkyne Huisgen cycload-
dition (“click chemistry”). Our approach is to generate non-
natural N-glycosides of sialic acid that are resistant to neur-
aminidase-catalyzed hydrolysis as opposed to the natural O-
glycosides. These N-glycosides would act as neuraminidase
inhibitors to prevent the release of new virions. As a prelimi-
nary study, a small library of 1,2,3-triazole-linked sialic acid
derivatives has been synthesized in 71–89% yield. A disac-
charide mimic of sialic acid has also been prepared using the
α-sialic acid azide 1 and a C-8 propargyl sialic acid acceptor
in 68% yield. A model sialic acid coated dendrimer was also
synthesized from a perpropargylated pentaerythritol ac-
ceptor. These novel sialic acid derivatives were then evalu-
ated as potential neuraminidase inhibitors using a 96-well
plate fluorescence assay; micromolar IC50 values were
observed, comparable to the known sialidase inhibitor
Neu5Ac2en.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
Influenza viruses use their hemagglutinin to bind to sialic
acid residues located on the surface of the host cell to gain
entry into the cell. Once the cell is infected, the new virions
use their neuraminidases to escape the infected cell.[6] Thus,
neuraminidases (or sialidases) have been targeted to stop
the viral infection by blocking the virus inside the infected
cell. Neuraminidase inhibitors have been developed and
rapidly introduced to the market. Although M2 protein in-
hibitors (adamantane structures, amantadine and rimanta-
dine) have also been developed and proven to be efficient
against influenza A, they have to be given early in infection
and they are ineffective against influenza B. Thus, neur-
aminidase inhibitors (oseltamivir and zanamivir) are still
the preferred drugs for influenza infection. They act as
transition-state mimics, inhibiting influenza neuraminidases
and preventing new viruses from being released from an
infected cell. With only two effective neuraminidase inhibi-
tors on the market, the different forms of influenza viruses
have developed resistance through genetic mutations.[7] The
wide variety of influenza viruses is a result of their capacity
to develop resistance. The small number of effective drugs
for the treatment of influenza makes the discovery of new
neuraminidase inhibitors a very important goal for increas-
ing the control of known serotypes and to prevent the
spread of a new pandemic.
Introduction
Every year, during the flu season, influenza viruses circu-
late worldwide and are the cause of a great number of fatal-
ities, particularly in young children and the elderly. Influ-
enza viruses are composed of three major classes: influenza
virus A, influenza virus B and influenza virus C. Influenza
virus A can be subdivided into different serotypes according
to the antibody response triggered by these viruses and as
many as ten of them have been identified in humans.[1]
Among these, H1N1 caused the Spanish flu in 1918,[2]
H3N2 caused the Hong Kong flu in 1968,[3] and H5N1 (av-
ian influenza A, bird flu) was responsible for the pandemic
threat in 2007.[4] From December 2003, through July 2007,
319 human cases of avian influenza A (H5N1) infection
were reported, 60% were fatal. All cases reported were from
Asia and Africa. These viruses are highly susceptible to mu-
tations, which can also be a threat by decreasing the efficacy
of the existing drugs on the market.[5] In this context, there
is an urgent need to develop new drugs for the treatment of
influenza.
[a] Department of Chemistry and Chemical Biology, Rensselaer
Polytechnic Institute,
110, 8th Street, Troy, NY 12180, USA
[b] Department of Biology, Rensselaer Polytechnic Institute,
110, 8th Street, Troy, NY 12180, USA
Different analogs of zanamivir have been synthesized to
improve efficacy. Modifications of the side chain of sialic
acid include the synthesis of dihydropyrancarboxamide de-
rivatives,[8] 7-substituted derivatives,[9] and dimeric conju-
gates.[10] Recently, the synthesis of 4-triazole-modified zana-
mivir analogs have been reported by Li et al. and led to the
[c] Department of Chemical and Biological Engineering, Rensse-
laer Polytechnic Institute,
110, 8th Street, Troy, NY 12180, USA
Fax: +1-518-276-3405
E-mail: linhar@rpi.edu
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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FULL PAPER
discovery of some analogs possessing EC50 values compar-
able to zanamivir.[11] The biologically compatible 1,2,3-tri-
azole group is thus able to replace the guanidine group in
the C-4 position of zanamivir without loss of activity.
In our laboratory, we have been focused on the samar-
ium-catalyzed synthesis of non-hydrolyzable C-glycosides of
sialic acid, like the sTn-KLH, which showed remarkable im-
munogenicity.[12] These derivatives might be useful in pre-
paring immunogens for active immunization against neur-
aminic acid containing glycoconjugates in the design and
preparation of anti-cancer vaccines with increased bio-
logical half-life. In view of the synthetic complexity associ-
ated with the preparation of C-disaccharides of sialic acid,
mainly due to the multi-step synthesis of the sialic acid al-
dehyde acceptor moiety, we decided to examine the possibil-
ity of synthesizing a new class of non-hydrolyzable deriva-
tives of sialic acid, N-glycosides, using “click chemistry”.
Our approach is to develop a simple and efficient strategy
that would allow the straightforward synthesis of non-hy-
drolyzable N-glycosides of sialic acid as potential neur-
aminidase inhibitors. The simplicity of the click reaction
would ultimately allow rapid access to a large library of
non-hydrolyzable N-glycosides of sialic acid. The copper(I)-
catalyzed azide–alkyne Huisgen cycloaddition leading to
the formation of 1,2,3-triazole derivatives has been widely
used during the last decade to develop new pathways to
biologically active molecules.[13] It has also been applied
successfully in the field of carbohydrate chemistry.[14] We
developed a new, rapid, high-yield, highly regioselective
strategy to access N-glycosides of sialic acid by reacting the
azido sialic acid donor 1 with a variety of different alkynes
using the copper(I)-catalyzed azide–alkyne Huisgen cyclo-
addition. The sialic acid 1,2,3-triazoles constitute a new
class of neuraminidase inhibitors. The availability of a wide
variety of alkynes should allow the access to a broad library
of this new class of compounds. In this work, a small li-
brary of 1,2,3-triazole-linked sialic acids has been synthe-
sized and the strategy has then been applied to the synthesis
of a 1,2,3-triazole-linked disaccharide mimic of sialic acid,
as well as a dendrimeric structure containing four sialic acid
residues. The efficiency of the click reaction affords good
yields ranging from 68% to 89%. Three of the synthesized
Results and Discussion
Development of a Clickable Sialic Acid
In 1991, Tropper and co-workers described the synthesis
of the α-sialic acid azide 3 (Scheme 1) starting from the cor-
responding β-chloride, using a phase-transfer catalysis pro-
cess and NaN3.[15] We first considered the use of 3 as a
potential starting material to synthesize 1,2,3-triazole-
linked sialic acids using the copper(I)-catalyzed azide–
alkyne Huisgen cycloaddition (Scheme 1).
3-Butyn-1-ol was used as a model alkyne and different
conditions were examined to obtain the 1,2,3-triazole deriv-
ative 4. None of the conditions tested (CuSO4/sodium as-
corbate or CuI/DiPEA) provided the desired product and
in all cases, the starting material could be completely reco-
vered by extraction. We speculated that the linkage of the
azido group to a quaternary carbon might make it too hin-
dered to react with the alkyne. Additionally, stereoelec-
tronic effects from electron-withdrawing acetyl-protecting
groups or an inappropriate conformation of azido sialic
acid might not be optimal for reaction with the alkyne.
Thus, we decided to change the protecting groups and be-
cause the click reaction is compatible with the presence of
hydroxy groups, the unprotected azido sialic acid 1 was ini-
tially tested as a potential donor (Scheme 1). Zemplen con-
ditions were used to deprotect 3 affording unprotected
azido sialic acid 1, which was engaged in the click reaction
with 3-butyn-1-ol in the presence of copper(II) sulfate and
sodium ascorbate as the reducing agent (Scheme 1). The de-
sired 1,2,3-triazole 2, was obtained in 81% yield and with
complete regiospecificity. When the protecting group of the
methyl ester was removed from 1, prior to the click reac-
tion, to directly afford the fully unprotected target 1,2,3-
triazole, the reaction was much slower. Thus, the azido
sialic acid donor 1 was selected for subsequent click reac-
tions involving sialic acid.
Synthesis of a Small Library of 1,2,3-Triazole-Linked
Sialic Acid
A number of alkynes were next used to examine the
compounds were found to have a better IC50 than the scope of the click reaction with 1, and to produce a small
known neuraminidase inhibitor, Neu5Ac2en, in an in-vitro library of 1,2,3-triazole derivatives of sialic acid (Table 1).
neuraminidase inhibition assay.
As a preliminary study, we considered diverse alkynes,
Scheme 1. Use of a peracetylated (3) or unprotected (1) α-sialic acid azide donor.
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Triazole-Linked Sialic Acids as Neuraminidase Inhibitors
short, long, rigid and flexible side chains as well as different acetal (5:2), was reductively aminated with an amino-pro-
functional groups, in order to gain some information on the tected lysine to convert this mixture to a single compound,
nature of the R group that would favor binding to neur- 2h, in 71% yield (Scheme 2). This suggests that aldehyde-
aminidases. The desired 1,2,3-triazole-containing sialic acid containing sialic acid derivatives might find applications for
compounds were obtained in excellent 71–89% yields and further conjugation by reductive amination to surfaces or
again with complete regiospecificity.
to KLH-carrier proteins for the production of antibodies
for example.
Table 1. Synthesis of a small library of 1,2,3-triazole-linked sialic
acid.[a]
Synthesis of a 1,2,3-Triazole-Linked Sialic Acid
Disaccharide Mimic
We next undertook the synthesis of multivalent 1,2,3-tri-
azole-linked sialic acid derivatives in order to increase the
affinity of these inhibitors for neuraminidase. A sialic acid
disaccharide mimic was first synthesized using the click re-
action. This synthesis relied on the azido donor 1 and a C-
8 propargyl sialic acid acceptor 10 (Scheme 3). The known
sialic acid sulfide 5[12c] was first protected at the C-8 and
C-9 positions as a p-methoxybenzylidene, followed by
acetylation at C-4 and C-7, to give compound 6 in 86%
yield (exo/endo ≈ 1:1). Regioselective reductive ring opening
of the p-methoxybenzylidene freed the C-8 position, and
compound 7 was obtained in 78% yield. The C-8 hydroxy
group was then propargylated by treatment with propargyl
bromide in the presence of barium oxide and barium hy-
droxide. The C-8 propargyl compound 8 was obtained as
the major product in 36% yield as a propargyl ester that
lost its C-4 and C-7 acetyl protecting groups due to the
presence of barium hydroxide. A mixture of partially de-
acetylated methyl ester, propargyl ester and carboxylic acid
[a] The unprotected azido sialic acid donor 1 (0.2 mmol, 70 mg),
the alkyne (0.22 mmol), CuSO4 (0.02 mmol, 3.2 mg), sodium as- by-products were obtained during this reaction. These by-
corbate (0.1 mmol, 20 mg) in a mixture of H2O/tBuOH/DCM,
products were easily converted into the desired compound
1:2:1 (4 mL) was covered with aluminum foil and heated to 60 °C
9 by acetylation and/or methylation. The acetylation of 8
overnight. After purification on silica gel, the corresponding 1,2,3-
also allowed us to confirm the position of the propargyl
triazole derivatives were obtained and further treated with 0.2 
KOH for 12 h at room temperature. [b] 1H and 13C NMR shows
the presence of 2 diastereoisomers in a 5:3 ratio, due to the presence
of a chiral nitrogen atom. [c] Sialoside 2g was isolated as a mixture
of aldehyde and the corresponding hydrated acetal in a 5:2 ratio.
group on the C-8 hydroxy, along with 2D NMR experi-
ments, because it shifted 4-H and 7-H downfield (see Sup-
porting Information). Removal of the PMB protecting
group using dichlorodicyanoquinone (DDQ) followed by
treatment with sodium methoxide in methanol gave the de-
Interestingly, an alkyne substrate containing a conju- sired C-8 propargyl acceptor 10 in 67% yield.
gated keto group (Entry 4) and another one containing a Using the click conditions previously described, 1 and 10
masked aldehyde (Entry 7) were clicked and deprotected were coupled and the disaccharide mimic 11 was obtained
in excellent yield. The aldehyde-containing compound 2g, after de-methyl esterification using 0.2  KOH in 68% yield
isolated as a mixture of the aldehyde and the corresponding over 2 steps (Scheme 3).
Scheme 2. Reductive amination of 2g using a lysine residue.
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M. Weïwer, C.-C. Chen, M. M. Kemp, R. J. Linhardt
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Scheme 3. Synthesis of a 1,2,3-triazole-linked disaccharide mimic of sialic acid 11.
Synthesis of a Model Dendrimer of Sialic Acid
compound 13. Product 13 was obtained in 32% isolated
yield, corresponding to an average 75% yield for the click
reaction occurring at each site. Treatment of 13 with a cata-
lytic amount of sodium methoxide in methanol followed by
saponification of the methyl ester groups using 0.2  KOH
afforded the fully deprotected dendrimer 14 in 94% yield.
Because of their multivalency leading to higher local con-
centration, carbohydrate-coated dendrimers can show in-
creased activity compared to their corresponding monosac-
charides.[16] Roy and co-workers, for example, have shown
the potential of sialic acid coated dendrimers to cross-link
and precipitate Limax flavus lectin (LFA).[17] We expect that
sialic acid coated dendrimers might exhibit enhanced neur-
aminidase inhibitory activity. We first functionalized penta-
erythritol with propargyl groups to obtain a model den-
Neuraminidase Inhibitor Assay
A 96-well plate neuraminidase inhibitor assay, adapted
drimer scaffold 12 (Scheme 4). This alkyne-containing scaf- from Gubareva et al.,[18] was used to evaluate the biological
fold was then clicked with the azido sialic acid donor 1. activity of these novel sialic acid structures. This fluores-
To facilitate product purification, the reaction mixture was cence assay measures the release of 4-methylumbelliferone
peracetylated with acetic anhydride and pyridine to afford (λex = 360 nm, λem = 440 nm) produced by the hydrolysis
Scheme 4. Synthesis of a model dendrimer of sialic acid 14.
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Triazole-Linked Sialic Acids as Neuraminidase Inhibitors
of the substrate [2Ј-(4-methylumbelliferyl)-α--N-acetyl-
In our assay, Neu5Ac2en (Entry 1) was found to have an
neuraminic acid, MUNANA] by the enzyme [neuramini- IC50 of 67 µ, which is in the range of literature values.[20]
dase from Clostridium perfringens (C. welchii)] (Scheme 5). The azido sialic acid (N3, Entry 2), obtained by hydrolysis
N-Acetyl-2,3-dehydro-2-deoxyneuraminic acid (Neu5- of 1 using 0.2  KOH, was found to be a weak neuramini-
Ac2en), a commercially available and known neuraminidase dase inhibitor, with an IC50 of 406 µ. When the azido
inhibitor, was used as a positive control.[19] Serial, half-log group was replaced by a 1,2,3-triazole group (inhibitors 2a–
dilutions (1 m to 3.16 n) of each inhibitor were pre- 2h), the IC50 values were variable, depending on the nature
pared and tested in triplicate in a 96-well plate format and of the side chain on the C-4 position of the triazole ring.
IC50 values were calculated. The results are presented in Although general structure–activity relationship of those
Table 2.
compounds would require a much larger library, some in-
Scheme 5. Neuraminidase inhibitor assay.
Table 2. Evaluation of the IC50 values of inhibitors.
Entry
Inhibitor
IC50/µ
1
2
3
4
5
6
7
8
Neu5Ac2en
67
N3
2a
2b
2c
2d
2e
2f
2g
2h
11
14
406
290
Ͼ 1000
28
549
Ͼ 1000
343
–
9
10
11
12
133
17
20
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Methyl [5-Acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-2-azido-
D-
formation can be derived from these results. The presence
of a short chain (2a, b, d, e, f, Entries 3, 4, 6, 7, 8) afforded
weak neuraminidase inhibitors having IC50 values ranging
from 290 µ to Ͼ 1 m. The presence of a longer and more
flexible side chain (2c, h, Entries 5, 10) afforded inhibitors
with significantly lower IC50. These results are in agreement
with previous studies done in our lab on the synthesis and
biological evaluation of C-glycoside-type neuraminidase in-
hibitors.[21] As expected, the disaccharide mimic 11 (Entry
glycero-α- -galacto-non-2-ulopyranoside]onate (3): Methyl [5-acet-
D
amido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-2-chloro--glycero-β--
galacto-non-2-ulopyranoside]onate (10.0 mmol, 5.0 g), tetrabu-
tylammonium hydrogen sulfate (10.0 mmol, 3.4 g) and sodium az-
ide (50.0 mmol, 3.26 g) were dissolved in a mixture of CH2Cl2/aq.
NaHCO3, 1:1 (100 mL) and stirred vigorously for 2 h at room tem-
perature. Dichloromethane was added and the two phases were sep-
arated. The organic phase was then washed with saturated aq.
NaHCO3, dried with sodium sulfate and filtered. Evaporation of
11), probably because it can better mimic the non-reducing the solvent under diminished pressure afforded compound 3 as a
white solid in 86 % yield (4.8 g). 1H NMR (500 MHz, CDCl3,
end of the glycoproteins present on the surface of epithelial
25 °C): δ = 5.4–5.3 (m, 3 H, 4-H, 6-H, 7-H), 5.0 (ddd, J = 11.6, J
cells, and dendrimer 14 (Entry 12), due to its multivalency,
= 10.3, J = 4.8 Hz, 1 H, 8-H), 4.4 (dd, J = 11.6, J = 3.0 Hz, 1 H,
afforded the lowest IC50 values. In order to improve our
understanding of the structure-activity relationship of these
molecules, a larger library of these novel neuraminidase in-
hibitors will be prepared as well as higher generations of
sialic acid coated dendrimers.
1 9-H), 4.2–4.1 (m, 2 H, 5-H, 9-H), 3.9 (s, 3 H, COOMe), 2.6
(dd, J = 13.0, J = 4.8 Hz, 1 H, 3eq-H), 2.15, 2.12, 2.04, 2.03 (4s,
4 CH3COO), 1.9 (s, 3 H, CH3CON), 1.8–1.7 (m, 1 H, 3ax-H) ppm.
13C NMR (125 MHz, CDCl3, 25 °C): δ = 170.8, 170.6, 170.2, 170.0,
169.9 (4 CH3COO, C-1), 167.1 (CH3CONH), 89.0 (C-2), 73.9 (C-
6), 69.5 (C-8), 68.8 (C-4), 67.4 (C-7), 62.1 (C-9), 53.5 (C-5), 49.2
(COOCH3), 36.5 (C-3), 23.1 (CH3CONH), 21.0, 20.8, 20.73, 20.71
(4 CH3COO) ppm. ESIMS m/z, calcd. for C20H28N4NaO12 [M +
Na]+ 539.2, found 539.2.
Conclusions
This preliminary study demonstrates that the click reac-
tion can be applied to the synthesis of a library of 1,2,3-
triazole derivatives of sialic acid using alkyne-containing
Methyl [5-Acetamido-3,5-dideoxy-2-azido-D-glycero-α-D-galacto-
non-2-ulopyranoside]onate (1): Compound 3 (7.66 mmol, 3.95 g)
was dissolved in methanol (16 mL) under argon and cooled to 0 °C.
molecules. It allows access to multivalent structures of sialic A solution of NaOMe/MeOH 0.5  (7.6 mL) was added dropwise
and the reaction was stirred at room temperature for 3 h. After
completion of the reaction, Amberlite IR-120 (H+ form) was added
to neutralize the reaction mixture. Evaporation of the solvent under
diminished pressure afforded compound 1 as a white solid in quan-
acid like disaccharide analogs and sialic acid coated dendri-
mers. Screening of a larger library of 1,2,3-triazole deriva-
tives of sialic acid is underway to improve our understand-
ing of the structure–activity relationship of these molecules
and to develop a novel class of neuraminidase inhibitors for
evaluation as agents for the treatment of influenza. Ad-
ditional dendrimer scaffolds are also being prepared, with
increased valency, as well as sialic acid coated surfaces and
materials. The synthesis of an azido sialic acid donor of
1
titative yield (2.7 g). H NMR (500 MHz, MeOD, 25 °C): δ = 3.9
(s, 3 H, COOMe), 3.9–3.8 (m, 4 H, 4,5,6-H and 9-H), 3.7 (dd, J =
11.8, J = 5.5 Hz, 1 H, 9-H), 3.5 (m, 2 H, 7,8-H), 2.6 (dd, J = 12.8,
J = 4.3 Hz, 1 H, 3eq-H), 2.0 (s, 3 H, AcNH), 1.6 (dd, J = 12.8, J
= 11.2 Hz, 1 H, 3ax-H) ppm. 13C NMR (125 MHz, MeOD, 25 °C):
δ = 175.9 (CH3CONH), 170.5 (C-1), 91.0 (C-2), 76.6 (C-6), 72.4
type 1 that contains a propargyl group at C-8 or C-9, as (C-8), 70.8 (C-7), 68.9 (C-4), 65.5 (C-9), 54.7 (C-5), 54.2 (CO-
OCH3), 41.1 (C-3), 23.6 (CH3CONH) ppm. HRMS m/z, calcd. for
a potential building block for the click-based synthesis of
polysialic acid analogs is also underway and will be re-
ported in due course. Due to its high efficiency, the click
reaction is expected to be transferred to a microarray for-
mat for the on-ship synthesis and screening of large libraries
C12H20N4NaO8 [M + Na]+ 371.1179, found 371.1181.
Typical Procedure for the Click Reaction: Unprotected azido sialic
acid donor 1 (0.2 mmol, 70 mg), alkyne (0.22 mmol), CuSO4
(0.02 mmol, 3.2 mg) and sodium ascorbate (0.1 mmol, 20 mg) were
of triazole-linked sialic acid derivatives, allowing for a bet- successively added to a reaction flask. A mixture of H2O/tBuOH/
DCM, 1:2:1 (4 mL) was then added and the reaction flask was
surmounted with a reflux condenser, covered with aluminum foil
and warmed to 60 °C. After stirring at this temperature overnight,
the mixture was cooled to room temperature and the solvents were
evaporated. The mixture was then loaded on a silica gel column
and eluted with a gradient of CH2Cl2/MeOH, 15:1 to 5:1. The cor-
responding 1,2,3-triazole derivative was obtained and further
treated with 0.2  KOH (1 mL for 0.1 mmol) for 12 h at room tem-
perature. Neutralization with Amberlite IR-120 (H+ form) followed
by filtration and evaporation of the solvents afforded the desired
sialic acid derivatives.
ter understanding of the structure–activity relationships
governing their neuraminidase inhibitory effect.
Experimental Section
General Methods: Solvents were dried and distilled by classical pro-
cedures and stored on molecular sieves (4 Å). Nuclear magnetic
resonance (1H NMR and 13C NMR) spectra were recorded at room
temperature, in CDCl3, MeOD or D2O (500 MHz). Chemical shifts
(δ) are indicated in ppm and coupling constants (J) in Hz. ESIMS
were recorded with a LC/MSD trap. HRMS were performed with
an orbitrap mass spectrometer using electrospray ionization. Thin-
layer chromatography (TLC) was carried out using plates of silica
gel 60 with fluorescent indicator and revealed with UV light
(254 nm) when possible and Von’s reagent [Ce(SO4)2/(NH4)6Mo7-
O24·4H2O/H2SO4]. Flash chromatography was performed using
silica gel 230–400 mesh.
5-Acetamido-3,5-dideoxy-2-[4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-
1
yl]-
D
-glycero-α-
D
-galacto-non-2-ulopyranosidic Acid (2a): H NMR
(500 MHz, D2O, 25 °C): δ = 7.9 (s, 1 H, 1Ј-H), 3.9–3.7 (m, 7 H,
4,5,6,7,8,9-H), 3.5–3.4 (m, 2 H, 2ϫ4Ј-H), 3.1 (dd, J = 13.0, J =
4.5 Hz, 1 H, 3eq-H), 2.8 (t, J = 6.5 Hz, 2 H, 2ϫ3Ј-H), 2.1 (t, J =
13.0 Hz, 1 H, 3ax-H), 1.9 (s, 3 H, AcNH) ppm. 13C NMR
(125 MHz, D2O, 25 °C): δ = 175.0 (C-1), 170.4 (CH3CO), 144.9 (C-
2616
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 2611–2620

Triazole-Linked Sialic Acids as Neuraminidase Inhibitors
2Ј), 121.8 (C-1Ј), 90.8 (C-2), 74.2 (C-6), 71.2 (C-8), 68.1 (C-4), 68.0
33.3 (2 C-3), 21.9 and 20.6 (2 C-6Ј), 20.5 and 20.2 (CH3CONH)
(C-7), 62.8 (C-9), 60.5 (C-4Ј), 51.6 (C-5), 39.6 (C-3), 27.8 (C-3Ј), ppm. HRMS m/z, calcd. for C17H27N5NaO9 [M + Na]+ 468.1701,
22.2 (CH3CO) ppm. HRMS m/z, calcd. for C15H24N4NaO9 [M +
found 468.1706.
Na]+ 427.1435, found 427.1440.
5-Acetamido-3,5-dideoxy-2-(4-formyl-1H-1,2,3-triazol-1-yl)-D-gly-
cero-α-D
-galacto-non-2-ulopyranosidic Acid (2g): 1H NMR (500
5-Acetamido-3,5-dideoxy-2-[4-(2-hydroxymethyl)-1H-1,2,3-triazol-
1-yl]-D-glycero-α-D
-galacto-non-2-ulopyranosidic Acid (2b): 1H
MHz, D2O, 25 °C): δ = 9.9 (s, 1 H, CHO), 8.8 (s, 1 H, H-1Јald), 8.1
(s, 1 H, H-1Јacetal), 6.1 (s, 1 H, H-3Јacetal), 4.0–3.7 (m, 10 H,
2ϫ4,5,6,7-H, 2ϫ9-H), 3.5–3.4 (m, 4 H, 2ϫ8-H, 2ϫ9-H), 3.2 (dd,
J = 12.8, J = 3.8 Hz, 1 H, 3eqald-H), 3.1 (dd, J = 13.0, J = 4.0 Hz,
1 H, 3eqacetal-H), 2.2–2.1 (m, 2 H, 3axald/acetal-H), 1.9 (s, 6 H,
AcNHald/acetal) ppm. 13C NMR (125 MHz, D2O, 25 °C): δ = 186.1
(CHO), 174.9 (C-1), 170.2 (CH3COacetal), 169.9 (CH3COald), 145.9
(C-2Ј), 127.2 (C-1Јald), 120.6 (C-1Јacetal), 91.0 (C-2ald), 90.6
(C-2acetal), 84.7 (C-3Јacetal), 74.2 (C-6ald), 74.1 (C-6acetal), 71.0 (C-
NMR (500 MHz, D2O, 25 °C): δ = 8.1 (s, 1 H, 1Ј-H), 4.6 (s, 2 H,
2H-3Ј), 3.8 (s, 3 H, COOMe), 3.8–3.6 (m, 4 H, 4,5,6-H, 9-H), 3.5–
3.4 (m, 3 H, 7,8-H, 9-H), 3.1 (m, 1 H, 3eq-H), 2.1 (m, 1 H, 3ax-
H), 1.9 (s, 3 H, AcNH) ppm. 13C NMR (125 MHz, D2O, 25 °C): δ
= 174.8 (C-1), 169.3 (CH3CON), 146.4 (C-2Ј), 121.8 (C-1Ј), 90.0
(C-2), 74.1 (C-6), 70.6 (C-8), 67.9 (C-4), 67.3 (C-7), 62.7 (C-9), 54.3
(C-3Ј), 51.3 (C-5), 39.1 (C-3), 21.9 (CH3CO) ppm. HRMS m/z,
calcd. for C14H22N4NaO9 [M + Na]+ 413.1279, found 413.1283.
8
acetal), 70.96 (C-8ald), 67.9 (C-4ald), 67.8 (C-4,7acetal), 67.7 (C-7ald),
5-Acetamido-3,5-dideoxy-2-(4-octyl-1H-1,2,3-triazol-1-yl)-
D-gly-
cero-α-
D
-galacto-non-2-ulopyranosidic Acid (2c): 1H NMR (500
62.9 (C-9acetal), 62.7 (C-9ald), 51.39 (C-5ald), 51.35 (C-5acetal), 39.6
(C-3ald), 39.4 (C-3acetal), 22.0 (CH3CO) ppm. HRMS m/z, calcd. for
C14H19N4O9 [M – H]– 387.1158, found 387.1164.
MHz, D2O, 25 °C): δ = 7.9 (s, 1 H, H-1Ј), 3.9–3.4 (m, 7 H, H-
4,5,6,7,8,9), 3.0 (m, 1 H, H-3eq), 2.4 (m, 2 H, 2ϫ-3Ј-H), 2.1 (m, 1
H, 3ax-H), 1.9 (s, 3 H, AcNH), 1.4 (m, 2 H, 2ϫ4Ј-H), 1.0 (broad
s, 10 H, 10ϫ5Ј–9Ј-H), 0.6 (t, J = 7.0 Hz, 3 H, CH3) ppm. 13C
NMR (125 MHz, D2O, 25 °C): δ = 174.9 (C-1), 168.6 (CH3CO),
147.0 (C-2Ј), 120.7 (C-1Ј), 90.0 (C-2), 74.3 (C-6), 70.4 (C-8), 67.7
(C-4), 67.2 (C-7), 62.4 (C-9), 51.4 (C-5), 39.4 (C-3), 31.7, 29.1, 28.6,
24.6, 22.0 [(CH2)7CH3], 22.4 (CH3CO), 13.7 [(CH2)7CH3] ppm.
HRMS m/z, calcd. for C21H36N4NaO8 [M + Na]+ 495.2425, found
495.2428.
5-Acetamido-3,5-dideoxy-2-(4-{[5-(tert-butoxycarbonylamino)-5-
carboxypentylamino]methyl}-1H-1,2,3-triazol-1-yl)-D-glycero-α-D-
galacto-non-2-ulopyranosidic Acid (2h): Compound 2g (21 µmol,
8 mg) and Nα-(tert-butoxycarbonyl)--lysine (23 µmol, 6 mg) were
dissolved in MeOH/H2O, 1:1 (200 µL) and stirred at room tempera-
ture for 30 min to form the Schiff base. NaCNBH3 (0.3  in
MeOH, 36 µL) was added and the mixture was stirred overnight at
room temperature. Solvents were evaporated and the mixture was
redissolved in 1 mL of water, loaded on a SAX column and eluted
with a gradient of NaCl (0 to 1 ). Fractions containing the prod-
uct were combined and desalted on a P2 biogel to afford 2h in 71%
yield (9 mg). 1H NMR (500 MHz, D2O, 25 °C): δ = 8.2 (s, 1 H, 1Ј-
H), 4.3 (s, 2 H, 3Ј-H), 4.0–3.7 (m, 6 H, 4,5,6,7-H, 9-H, 8Ј-H), 3.6–
3.5 (m, 2 H, 8,9-H), 3.2 (dd, J = 12.8, J = 4.2 Hz, 1 H, 3eq-H), 3.0
(t, J = 7.6 Hz, 2 H, 2ϫ4Ј-H), 2.1 (dd, J = 12.8, J = 11.2 Hz, 1 H,
3ax-H), 1.9 (s, 3 H, AcNH), 1.8–1.6 (m, 4 H, 2ϫ5Ј-H, 2ϫ7Ј-H),
1.4–1.3 (m, 2 H, 2 ϫ 6Ј-H), 1.3 (s, 9 H, Boc) ppm. 13C NMR
(125 MHz, D2O, 25 °C): δ = 176.9 (C-1), 175.1 (C-5ЈЈ), 170.5
(CH3CONH), 157.8 (OCONH), 138.0 (C-2Ј), 123.9 (C-1Ј), 91.0 (C-
2), 81.5 (OCMe3), 74.2 (C-6), 71.3 (C-8), 68.0, 67.9 (C-4, C-7), 62.8
(C-9), 53.7 (C-5ЈЈ), 51.5 (C-5), 46.8 (C-3Ј), 41.3 (C-1ЈЈ), 39.5 (C-3),
30.1, 29.6 (C-2ЈЈ, C-4ЈЈ), 27.6 (CMe3), 24.9 (C-3ЈЈ), 22.1
(CH3CONH) ppm. HRMS m/z, calcd. for C25H42N6NaO12 [M +
Na]+ 641.2753, found 641.2746.
5-Acetamido-3,5-dideoxy-2-(4-acetyl-1H-1,2,3-triazol-1-yl)-
D-gly-
cero-α-
D
-galacto-non-2-ulopyranosidic Acid (2d): 1H NMR (500
MHz, D2O, 25 °C): δ = 8.7 (s, 1 H, 1Ј-H), 3.9–3.8 (m, 3 H, 4,6-H,
9-H), 3.8–3.7 (m, 2 H, 5,7-H), 3.6–3.5 (m, 2 H, 8-H, 9-H), 3.2 (dd,
J = 12.7, J = 3.9 Hz, 1 H, H-3eq), 2.5 (s, 3 H, 3ϫ4Ј-H), 2.1 (dd,
J = 12.7, J = 11.0 Hz, 1 H, 3ax-H), 1.9 (s, 3 H, AcNH) ppm. 13C
NMR (125 MHz, D2O, 25 °C): δ = 194.8 (C-3Ј), 174.9 (C-1), 169.4
(CH3CON), 146.0 (C-2Ј), 126.3 (C-1Ј), 90.6 (C-2), 74.2 (C-6), 70.7
(C-8), 67.9 (C-4), 67.5 (C-7), 62.7 (C-9), 51.3 (C-5), 39.5 (C-3), 26.9
(C-4Ј), 22.0 (CH3CO) ppm. HRMS m/z, calcd. for C15H21N4O9
[M – H]– 401.1314, found 401.1317.
5-Acetamido-3,5-dideoxy-2-[4-(1-hydroxycyclopentyl)-1H-1,2,3-tri-
azol-1-yl]-D-glycero-α-D
-galacto-non-2-ulopyranosidic Acid (2e): 1H
NMR (500 MHz, D2O, 25 °C): δ = 8.1 (s, 1 H, 1Ј-H), 3.9–3.8 (m,
3 H, 4,6-H, 9-H), 3.8–3.7 (m, 2 H, 5,7-H), 3.5–3.4 (m, 2 H, 8-H,
9-H), 3.1 (dd, J = 12.7, J = 3.8 Hz, 1 H, 3eq-H), 2.1 (m, 1 H, 3ax-
H), 2.0–1.8 (m, 4 H, 4ϫ4Ј-H), 1.9 (s, 3 H, AcNH), 1.8–1.6 (m, 4
H, 4ϫ5Ј-H) ppm. 13C NMR (125 MHz, D2O, 25 °C): δ = 174.8
(C-1), 169.6 (CH3CO), 153.1 (C-2Ј), 119.9 (C-1Ј), 90.3 (C-2), 78.3
(C-3Ј), 74.1 (C-6), 70.7 (C-8), 67.9 (C-4), 67.5 (C-7), 62.7 (C-9),
51.3 (C-5), 39.8 (2 C-4Ј), 39.3 (C-3), 22.8 (2 C-5Ј), 21.9 (CH3CO)
ppm. HRMS m/z, calcd. for C18H28N4O9Na [M + Na]+ 467.1748,
found 467.1752.
Methyl [Phenyl 5-Acetamido-3,5-dideoxy-4,7-di-O-acetyl-8,9-(p-
methoxybenzylidenyl)-2-thio-D-glycero-α-D-galacto-non-2-ulopyr-
anoside]onate (6): Neu5Ac phenyl sulfide 5 (2.41 mmol, 1.0 g) was
dissolved in MeCN (100 mL), and benzylidene dimethyl acetal
(19.2 mmol, 2.9 mL) and camphorsulfonic acid (0.2 mmol, 56 mg)
were added. The reaction mixture was stirred for 15 h at room tem-
perature and neutralized with trimethylamine. The solvent was
evaporated under reduced pressure. The residue was dissolved in
5-Acetamido-3,5-dideoxy-2-{4-[(N-methylacetamido)methyl]-1H-
1,2,3-triazol-1-yl}-D-glycero-α-D-galacto-non-2-ulopyranosidic Acid dichloromethane (100 mL) and washed with saturated aqueous
1
(2f): H NMR (500 MHz, D2O, 25 °C): δ = 8.1 and 8.0 (2s, 2 H,
2ϫ1Ј-H), 4.52 and 4.45 (2s, 4 H, 4ϫ3Ј-H), 3.8 (broad s, 6 H,
4ϫ4,6-H, 2ϫ9-H), 3.7–3.6 (m, 4 H, 4ϫ5,7-H), 3.5–3.4 (m, 4 H,
2ϫ8-H, 2ϫ9-H), 3.1 (m, 2 H, 2ϫ3eq-H), 2.9 and 2.7 (2s, 6 H,
6 ϫ 4Ј-H), 2.1 (m, 2 H, 2 ϫ H-3ax), 2.0 and 1.9 (2s, 6 H, 2
NaHCO3 (60 mL) and water (3ϫ25 mL). The organic phase was
dried with anhydrous MgSO4 and filtered. The filtrate was concen-
trated under vacuum. The obtained residue was dissolved in pyr-
idine (5 mL) and acetic anhydride (3 mL) and the mixture was
stirred at room temperature overnight. Pyridine and acetic anhy-
CH3CONMe), 1.88 and 1.86 (2s, 6 H, 2 CH3CONH) ppm. 13C dride were evaporated under vacuum and the obtained residue was
NMR (125 MHz, D2O, 25 °C): δ = 174.8 (C-1), 174.1 and 174.0 (2
C-5Ј), 169.2 and 169.1 (2 CH3CONH), 143.1 and 142.9 (2 C-2Ј),
121.9 and 121.7 (2 C-1Ј), 89.99 and 89.95 (2 C-2), 74.1 (2 C-6),
70.5 (2 C-8), 67.9 (2 C-4), 67.3 (2 C-7), 62.8 (2 C-9), 51.3 and 48.7
(2 C-5), 45.5 and 42.1 (2 C-3Ј), 39.22 and 39.17 (2 C-4Ј), 36.1 and
purified by flash chromatography (petroleum ether/ethyl acetate,
1:1 to 0:1) to afford 6 as light yellow foam in 86% yield (1.25 g,
endo/exo ≈ 1:1). 1H NMR (500 MHz, CDCl3, 25 °C): δ = 6.80–7.54
(m, 18 H, 10HArSPh, 8HArPMP), 5.5–4.9 (m, 8 H, 2 CHPh,
2ϫ4,6,7-H), 4.6–4.0 (m, 8 H, 2ϫ5,8-H, 4ϫ9-H), 3.83, 3.81 (2s, 6
Eur. J. Org. Chem. 2009, 2611–2620
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2617

M. Weïwer, C.-C. Chen, M. M. Kemp, R. J. Linhardt
FULL PAPER
H, 2 COOMe), 3.80, 3.6 (2s, 6 H, 2 PhOMe), 2.8 (m, 1 H, 3eq-H),
(s, 3 H, AcNH), 2.0–1.9 (m, 1 H, 3ax-H) ppm. 13C NMR
2.7 (dd, J = 12.8, J = 4.7 Hz, 1 H, 3eq-H), 2.2–1.8 (m, 20 H, 4 ( 1 25 M H z , M e O D, 2 5 ° C ): δ = 17 2. 8 ( CO OP r) , 168. 2
MeCOO, 2 MeCONH, 2 ϫ 3ax-H) ppm. 13C NMR (125 MHz,
CDCl3, 25 °C): δ = 171.4, 170.3, 170.2, 170.0, 169.3, 168.54, 168.47,
168.2 (2 CH3CONH, 4 CH3COO, 2 COOCH3), 160.3, 159.8 (2
CHOMe), 136.84, 136.81, 136.2, 130.2, 130.1, 130.0, 129.6, 129.4,
128.79, 128.76, 128.72, 128.68, 127.83, 127.81, 127.77, 127.6 (12
CArSPh, 6 CArPMP), 113.7, 113.6, 113.4 (4 CHPMP), 103.8, 103.4,
101.2 (2 CHPh), 87.4, 86.8 (2 C-2), 76.0, 75.8, 75.7, 74.8, 73.3, 69.9,
69.5, 69.0, 68.0, 67.8, 66.8, 62.8 (2ϫC-4,6,7,8,9), 55.3 (2 PhOMe),
52.8, 52.5 (2 C-5), 49.5, 49.1 (2 COOMe), 37.8, 37.4 (2 C-3), 23.4,
23.2 (2 CH3CONH), 21.0, 20.95, 20.85, 20.7 (4 OCOMe) ppm.
HRMS m/z, calcd. for C30H35NNaO11S [M + Na]+ 640.1823,
found 640.1821.
(CH3CONH), 159.0 (C-5Ј), 136.8, 130.5, 130.3, 129.2, 128.8,
128.1 (6 CSPh, 3 CPMB), 113.7 (2 C-3Ј), 85.9 (C-2), 79.9 (C-8),
76.9 (C-6), 76.5, 76.0 (2 C-4arylpropargyl), 75.3, 74.1 (2 CHpropargyl),
72.8 (OCH2Ph), 71.1 (C-4), 70.1 (C-7), 68.7 (C-9), 56.3
(OCH2 propargyl ester), 55.2 (MeOPh), 53.3 (OCH2,propargyl ether), 50.7
(C-5), 36.9 (C-3), 23.1 (MeCONH) ppm. HRMS m/z, calcd. for
C31H35NNaO9S [M + Na]+ 620.1930, found 620.1938.
Methyl [Phenyl 5-acetamido-9-O-(p-methoxybenzyl)-3,5-dideoxy-
4,7-di-O-acetyl-8-O-propargyl-2-thio-D-glycero-α-D-galacto-non-2-
ulopyranoside]onate (9): Compound 8 (0.129 mmol, 77 mg) was dis-
solved in methanol (2 mL) and cooled to 0 °C. A solution of so-
dium methoxide in methanol (0.5 , 0.3 mL) was then added and
the mixture was stirred for 1 h. The reaction was quenched with
Amberlite (H+ form), washed with methanol and the solvents evap-
orated. The residue obtained was treated with pyridine (1 mL) and
acetic anhydride (0.5 mL) at room temperature for 5 h. Evapora-
tion of the volatiles afforded 9 in 95% yield (80 mg). 1H NMR
(500 MHz, MeOD, 25 °C): δ = 7.6–7.2 (m, 7 H, 5ϫHSPh, 2ϫ3ЈЈ-
H), 6.9 (d, J = 8.5 Hz, 2ϫ4Ј-HЈ), 5.5 (m, 1 H, 6-H), 5.4 (m, 1 H,
4-H), 5.3 (m, 1 H, 7-H), 4.5 (d, J = 11.5 Hz, 2ϫ1ЈЈ-H), 4.3 (d, J
= 11.0 Hz, 5-H), 4.3–4.1 (m, 2 H, 2ϫ1Ј-H), 4.0–3.8 (m, 2 H, 8,9-
H), 3.8 (s, 4 H, 3Ј-H, MeOPh), 3.5 (s, 3 H, COOMe), 3.4–3.3 (m,
1 H, 9-H), 3.0 (dd, J = 12.5 Hz, J = 4.4 Hz, 1 H, 3eq-H), 2.0 (2s,
6 H, 2 OAc), 1.96 (s, 3 H, AcNH), 1.7 (dd, J = 12.5 Hz, J =
11.6 Hz, 1 H, 3ax-H) ppm. 13C NMR (125 MHz, MeOD, 25 °C):
Methyl [Phenyl 5-acetamido-9-O-(p-methoxybenzyl)-3,5-dideoxy-
4,7-di-O-acetyl-2-thio-D-glycero-α-D-galacto-non-2-ulopyranoside]-
onate (7): BH3·NH3 (5.0 mmol, 365 mg) and AlCl3 (4.86 mmol,
648 mg) were added to a solution of 6 (0.81 mmol, 500 mg) in anhy-
drous THF (10 mL) with activated molecular sieves (4 Å, 2.50 g)
at 0 °C. After stirring at 0 °C for 1 h, the reaction mixture was
filtered through a pad of Celite and the solids were washed with
MeCN. The combined filtrate was concentrated and the residue
was dissolved in ethyl acetate and washed with saturated aqueous
NaHCO3 and water. The organic phase was dried with MgSO4,
filtered, and concentrated. The residue was purified by flash col-
umn chromatography (petroleum ether/ethyl acetate, 1:1 to 0:1),
and afforded 7 as a snow white foam in 78% yield (391 mg). 1H
δ = 170.6, 170.5, 170.3 (COOMe, 2 OCOMe), 168.0 (CH3CONH),
NMR (500 MHz, CDCl3, 25 °C): δ = 7.5–7.2 (m, 7 H, 5ϫHSPh
,
aryl
159.2 (C-5ЈЈ), 136.2 (2 CHSPh), 136.0 (C-2ЈЈ), 129.9 (4
C ),
SPh
2ϫHPMB), 6.9 (d, J = 8.6 Hz, 2 H, 2ϫHPMB), 5.9 (d, J = 8.3 Hz,
1 H, 6-H), 5.1 (dt, Jd = 9.2 Hz, Jt = 2.6 Hz, 1 H, 7-H), 4.9 (ddd, J
= 11.6 Hz, J = 11.5 Hz, J = 4.7 Hz, 1 H, 4-H), 4.5–4.4 (m, 2 H,
OCH2Ph), 4.1–4.0 (m, 1 H, 5-H), 3.9–3.8 (m, 2 H, 8,9-H), 3.8 (s, 3
H, MeOPh), 3.5 (dd, J = 10.5 Hz, J = 1.4 Hz, 1 H, 9-H), 3.4 (s, 3
H, COOMe), 2.8 (dd, J = 12.8 Hz, J = 4.7 Hz, 1 H, 3eq-H), 2.1,
2.0 (2s, 6 H, 2 OAc), 2.1–2.0 (m, 1 H, 3ax-H), 1.9 (s, 3 H, AcNH)
ppm. 13C NMR (125 MHz, CDCl3, 25 °C): δ = 172.4, 171.9, 169.7,
167.6 (COOMe, 2 OCOMe, CH3CONH), 159.0 (C-5Ј), 135.9,
130.3, 129.5, 129.2, 128.8, 128.7 (6 CSPh, 3 CPMB), 113.6 (2 C-3Ј),
87.2 (C-2), 75.7 (C-6), 72.8 (OCH2Ph), 71.3 (C-7), 69.1 (C-4), 67.7
(C-8), 66.3 (C-9), 55.1 (MeOPh), 52.4 (C-5), 51.4 (COOMe), 37.9
(C-3), 23.0 (MeCONH), 21.1 and 20.9 (2 MeCOO) ppm. HRMS
m/z, calcd. for C30H37NNaO11S [M + Na]+ 642.1980, found
642.1979.
129.6 (2 C-3ЈЈ), 129.0 (1 CHSPh), 128.8 (2 CHSPh), 113.7 (2 C-4ЈЈ),
87.5 (C-2), 79.6 (C-2Ј), 74.6 (C-8), 73.4 (C-6), 72.9 (C-3Ј), 70.5
(OCH2Ph), 68.7 (C-4, C-7), 67.7 (C-9), 56.7 (MeOPh), 55.3 (C-5,
C-1Ј), 52.5 (COOMe), 38.2 (C-3), 23.7 (MeCONH), 21.1 and 20.9
(2 MeCOO) ppm. HRMS m/z, calcd. for C33H39NNaO11S [M +
Na]+ 680.2136, found 680.2138.
Methyl [Phenyl 5-acetamido-3,5-dideoxy-8-O-propargyl-2-thio-
D-
glycero-α- -galacto-non-2-ulopyranoside]onate (10): DDQ (20 µmol,
D
5 mg) was added to a solution of compound 9 (19 µmol, 13 mg) in
DCM/H2O, 20:1 (0.5 mL) at 0 °C. The reaction mixture was stirred
vigorously for 1 h and poured into a saturated aqueous NaHCO3
solution. The mixture was extracted with DCM followed by drying
over Na2SO4, filtration and evaporation under reduced pressure.
The obtained residue was dissolved in anhydrous methanol
(0.5 mL), cooled to 0 °C and sodium methoxide in methanol solu-
tion (0.5 , 0.1 mL) was added. After stirring for 2 h at room tem-
perature, the reaction was quenched with Amberlite (H+ form),
washed with methanol and the solvents evaporated. The residue
was purified by flash column chromatography (DCM/methanol,
Propargyl [Phenyl 5-acetamido-9-O-(p-methoxybenzyl)-3,5-dideoxy-
8-O-propargyl-2-thio-D-glycero-α-D-galacto-non-2-ulopyranoside]-
onate (8): Compound 7 (0.3 mmol, 200 mg), barium oxide
(0.91 mmol, 140 mg), and barium hydroxide (0.6 mmol, 188 mg)
were dissolved in DMF (10 mL). A solution of propargyl bromide
in toluene (80%, 0.32 mL) was then added and the mixture was
stirred at room temperature for 10 h. The reaction was quenched
with pTosOH and filtered through a pad of Celite. The solids were
washed with MeCN, and the filtrates were combined and the sol-
vents evaporated. The residue was purified by flash column
chromatography (ethyl acetate/methanol, 1:0 to 4:1), to afford 8 as
a white solid in 36% yield (77 mg). 1H NMR (500 MHz, MeOD,
25 °C): δ = 7.6–7.2 (m, 7 H, 5ϫHSPh, 2ϫHPMB), 6.9 (d, J = 8.6 Hz,
2ϫHPMB), 5.8 (d, J = 7.4 Hz, 1 H, 6-H), 4.6 (d, J = 2.4 Hz, 2 H,
COOCH2propargyl), 4.6–4.5 (m, 2 H, OCH2Ph), 4.3 (dd, J = 16.3 Hz,
J = 2.4 Hz, 1 H, 1 H-OCH2propargyl), 4.2 (dd, J = 16.3 Hz, J =
2.3 Hz, 1 H, 1 H-OCH2propargyl), 4.0–3.8 (m, 2 H, 4,7-H), 3.8 (s, 3
H, MeOPh), 3.8–3.7 (m, 1 H, 5-H), 3.7–3.6 (m, 1 H, 9-H), 3.6–3.5
(m, 1 H, 8-H), 3.4 (dd, J = 10.4 Hz, J = 1.4 Hz, 1 H, 9-H), 3.1–3.0
(m, 2 H, 2ϫ3eq-H, 1 CCH), 2.5 (d, J = 2.4 Hz, 1 H, 1 CCH), 2.0
1
15:1 to 7:1), to afford 10 as a white solid in 67% yield (5 mg). H
NMR (500 MHz, MeOD, 25 °C): δ = 7.6–7.3 (m, 5 H, 5ϫHSPh),
4.3 (s, 2 H, 2ϫ1Ј-H), 3.9 (t, J = 10.3 Hz, 1 H, 6-H), 3.9–3.8 (m, 3
H, 4,7,9-H), 3.7 (s, 3 H, COOMe), 3.7–3.6 (m, 2 H, 5,9-H), 3.5–
3.4 (m, 2 H, 8,3Ј-H), 3.1 (dd, J = 12.8 Hz, J = 4.6 Hz, 1 H, 3eq-
H), 2.0 (s, 3 H, AcNH), 1.8 (dd, J = 12.8 Hz, J = 11.3 Hz, 1 H,
3ax-H) ppm. 13C NMR (125 MHz, MeOD, 25 °C): δ = 175.5 (C-
1), 171.7 (CH3CONH), 138.7 (2 CHSPh), 132.1 (CHSPh), 131.0
(4arylCSPh), 130.8 (2 CHSPh), 88.7 (C-2), 81.0 (C-2Ј), 78.1 (C-8), 77.1
(C-6), 73.8 (C-3Ј), 70.9 (C-4), 65.3 (C-7), 58.6 (C-9), 54.2 (C-5),
52.6 (C-1Ј), 50.7 (COOCH3), 39.8 (C-3), 23.6 (CH3CONH) ppm.
HRMS m/z, calcd. for C21H27NNaO8S [M + Na]+ 476.1361, found
476.1352.
Disaccharide Mimic 11: In a reaction flask was successively added
the unprotected azido sialic acid donor 1 (13 µmol, 4.5 mg), the C-
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Triazole-Linked Sialic Acids as Neuraminidase Inhibitors
8 propargyl acceptor 10 (11 µmol, 5 mg), CuSO4 (1.2 µmol, 0.2 mg)
and sodium ascorbate (6 µmol, 1.2 mg). A mixture of H2O/tBuOH/
DCM, 1:2:1 (0.4 mL) was then added and the reaction flask was
covered with aluminum foil and warmed to 60 °C. After stirring at
this temperature overnight, the mixture was cooled to room tem-
perature and the solvents were evaporated. The mixture was then
loaded on a silica gel column and eluted with a gradient of CH2Cl2/
MeOH, 10:1 to 5:1. The disaccharide intermediate was obtained as
a white solid in 68% (6 mg). It was then treated with 0.2  KOH
(0.1 mL) for 12 h at room temperature. Neutralization with Am-
berlite IR-120 (H+ form) followed by filtration and evaporation of
the solvents afforded the desired sialic acid disaccharide mimic 11
in quantitative yield (5 mg). 1H NMR (500 MHz, D2O, 25 °C): δ =
8.1 (s, 1 H, 1ЈЈ-H), 7.5 (d, 2 H, J = 7.5 Hz, 2HSPh), 7.4 (d, J =
7.5 Hz, 1 H, 1 HSPh), 7.3 (t, 2 H, 2HSPh), 4.7 (d, J = 8.5 Hz, 1 H,
1 3ЈЈ-H), 4.6 (d, J = 8.5 Hz, 1 H, 3ЈЈ-H), 3.9–3.4 (m, 14 H,
2ϫ4,5,6,7,8-H, 4ϫ9-H), 3.2 (dd, J = 12.5, J = 4.0 Hz, 1 H, 3eq-
H), 2.9 (dd, J = 12.9, J = 4.5 Hz, 1 H, 3eq-H), 2.1 (dd, J = 12.5,
J = 11.5 Hz, 1 H, 3ax-H), 1.9, 1.8 (2s, 6 H, 2 AcNH), 1.9–1.8 (m,
1 H, 3ax-H) ppm. 13C NMR (125 MHz, D2O, 25 °C): δ = 175.0,
173.7 (2 C-1), 171.8, 170.3 (2 CH3CONH), 143.6 (C-2ЈЈ), 136.4,
130.3, 129.0, 127.7, 127.3 (6 CSPh), 122.9 (C-1ЈЈ), 90.8, 87.2 (2 C-
2), 75.9, 75.0, 74.2, 71.9, 71.2, 70.4, 68.1, 66.8 (2 C-4, 2 C-6, 2 C-
7, 2 C-8), 63.2, 62.6 (2 C-9), 61.3 (C-3ЈЈ), 51.5, 49.8 (2 C-5), 38.9,
37.5 (2 C-3), 22.1 (2 CH3CONH) ppm. HRMS m/z, calcd. for
C31H42N5O16S [M – H]– 772.2353, found 772.2346.
90.9 (4 C-2), 75.7 (4 C-6), 71.2 (4 C-2ЈЈ), 71.14 (4 C-3Ј), 71.11 (4
C-8), 70.0 (4 C-4), 69.0 (4 C-7), 66.3 (4 C-9), 64.3 (4 C-5), 55.6 (4
COOMe), 50.6 (4 C-1ЈЈ), 38.0 (4 C-3), 23.6, 22.29, 22.28, 21.8, 21.6
(2 OCH3CO) ppm. HRMS m/z, calcd. for C97H133N16O52 [M +
H]+ 2353.8185, found 2353.8189.
Deprotected Dendrimer 14: Compound 13 (8.5 µmol, 20 mg) was
dissolved in anhydrous methanol (0.1 mL) and cooled to 0 °C. So-
dium methoxide in methanol (0.5 , 0.04 mL) was added dropwise
and the reaction mixture was stirred at room temperature for 5 h.
After neutralization with Amberlite (H+ form), filtration and evap-
oration of the solvents, the partially deprotected dendrimer was
treated with 0.2  KOH (0.5 mL) for 12 h at room temperature.
Neutralization with Amberlite (H+ form) followed by filtration and
1
evaporation of the solvents afforded 14 in 94% yield (13 mg). H
NMR (500 MHz, CDCl3, 25 °C): δ = 8.1 (s, 4 H, 4ϫ1Ј-H), 4.4–
4.3 (m, 4 H, 4ϫ6-H), 4.3 (s, 8 H, 8ϫ3Ј-H), 3.9 (s, 8 H, 8ϫ2ЈЈ-H),
3.8–3.6 (m, 8 H, 4ϫ4,9-H), 3.5–3.4 (m, 8 H, 4ϫ5,7-H), 3.3–3.2
(m, 8 H, 4ϫ8,9-H), 3.1 (m, 4 H, 4ϫ3eq-H), 2.1 (dd, J = 12.8 Hz,
J = 10.9 Hz, 4 H, 4ϫ3ax-H), 1.9 (s, 12 H, 4 CH3CONH) ppm.
13C NMR (125 MHz, CDCl3, 25 °C): δ = 174.8 (4 C-1), 169.2 (4
CH3CONH), 143.7 (4 C-2Ј), 122.8 (4 C-1Ј), 90.0 (4 C-2), 74.1 (4
C-6), 70.6 (4 C-2ЈЈ), 67.9 (4 C-8), 67.7 (4 C-4), 67.4 (4 C-7), 63.0
(4 C-3Ј), 62.7 (4 C-9), 51.3 (4 C-5), 44.3 (4 C-1ЈЈ), 39.2 (4 C-3),
22.0 (4 CH3CONH) ppm. HRMS m/z, calcd. for C61H93N16O36 [M
+ H]+ 1625.5933, found 1625.5935.
Neuraminidase Inhibition Assay: 4-Methylumbelliferyl-N-acetyl-
neuraminic acid (MUNANA), N-Acetyl-2,3-dehydro-2-deoxyneur-
aminic acid (Neu5Ac2en), neuraminidase from Clostridium per-
fringens (C. welchii) and sodium acetate were purchased from
Sigma and used as received. 96-Well plates, white polystyrene, flat
bottom, non-treated, non-sterile, Costar, were from Corning Inc.
3-{3-(Prop-2-ynyloxy)-2,2-bis[(prop-2-ynyloxy)methyl]propoxy}-
prop-1-yne (12): Pentaerythritol (5 mmol, 681 mg), KOH (76 mmol,
4.25 g) and DMF (15 mL) were stirred at 0 °C for 5 min. An 80%
solution of propargyl bromide in toluene (60 mmol, 10 mL) was
added dropwise and the mixture was kept at 50 °C overnight. After
cooling to room temperature, the mixture was extracted with di-
ethyl ether, dried with Na2SO4, filtered and the solvents evaporated.
After flash chromatography on silica gel (petroleum ether/ethyl ace-
tate, v/v, 1:1 to 0:1), compound 12 was obtained as light yellow
The assays were performed at a total volume of 100 µL in a 96-well
plate. Serial half-log dilutions of each inhibitor and a commercially
available inhibitor, Neu5Ac2en, were prepared from 3.16 n to
1 m in 0.1 m sodium acetate buffer, pH 5.0 in triplicate, to deter-
mine IC50 of each inhibitor. Neuraminidase (0.5 mU) and different
concentrations of the inhibitors were added to each well and al-
lowed to incubate for 30 min at room temperature. The substrate
(0.5 m) was added and the reaction mixture was incubated for 1 h
at 37 °C. Immediately following incubation, the fluorescence was
read on a Molecular Devices SpectraMax M5, with an excitation
wavelength at 360 nm and an emission wavelength at 440 nm. The
percent of neuraminidase activity was plotted against inhibitor con-
centration to determine the IC50 using software from GraphPad
Prism version 5.00 for Windows, GraphPad Software, San Diego
California USA.
1
syrup in 42% yield (608 mg). H NMR (500 MHz, CDCl3, 25 °C):
δ = 4.1 (s, 8 H, 8ϫ2-H), 3.5 (s, 8 H, 8ϫ1-H), 2.4 (s, 4 H, 4ϫ3-
H) ppm. 13C NMR (125 MHz, CDCl3, 25 °C): δ = 80.3 (C-4), 74.4
(C-5), 69.2 (C-2), 58.9 (C-3), 45.0 (C-1) ppm. ESIMS m/z, calcd.
for C17H20NaO4 [M + Na]+ 826.6, found 826.6.
Protected Dendrimer 13: In a reaction flask was successively added
the unprotected azido sialic acid donor 1 (0.2 mmol, 70 mg), com-
pound 12 (0.032 mmol, 9 mg), CuSO4 (0.02 mmol, 3.2 mg) and so-
dium ascorbate (0.1 mmol, 20 mg). A mixture of H2O/tBuOH/
DCM, 1:2:1 (4 mL) was then added and the reaction flask was
covered with aluminum foil and warmed to 60 °C. After stirring at
this temperature overnight, the mixture was cooled to room tem-
perature and the solvents were evaporated. Pyridine (1.5 mL) and
acetic anhydride (1 mL) were added and the mixture was stirred for
5 h at room temperature under argon. After evaporation of the
volatiles, the mixture was loaded on a silica gel column and eluted
with a gradient of ethyl acetate/methanol, 1:0 to 9:1. The corre-
sponding 1,2,3-triazole dendrimer 13 was obtained as a colorless
Supporting Information (see also the footnote on the first page of
1
this article): Spectral data, H, 13C NMR of compounds 1, 2a–h,
3, 6–14 and IC50 curves of 2a–h, 11, 14, Neu5Ac2en.
Acknowledgments
1
film in 32% yield (24 mg). H NMR (500 MHz, CDCl3, 25 °C): δ
This work was supported by the National Institutes of Health
(NIH) (NIH AI065786). The authors would like to thank Dr. Dmi-
tri Zagorevski for ESIMS and HRMS analyses.
= 8.1 (s, 4 H, 4ϫ1Ј-H), 5.5–5.4 (m, 8 H, 4ϫH-4,6), 5.1 (td, J =
11.4, J = 5.1 Hz, 4 H, 4ϫ8-H), 4.6 (s, 8 H, 8ϫ3Ј-H), 4.4 (d, J =
10.7 Hz, 4 H, 4ϫ9-H), 4.3 (d, J = 11.4 Hz, 4 H, 4ϫ7-H), 4.2–4.0
(m, 8 H, 4ϫ5,9-H), 3.8 (s, 12 H, 4 COOMe), 3.6 (s, 8 H, 8ϫ2ЈЈ-
H), 3.4 (dd, J = 13.0, J = 4.3 Hz, 4 H, 4ϫ3eq-H), 2.6 (dd, J =
12.6, J = 12.3 Hz, 4 H, 4ϫ3ax-H), 2.2, 2.1, 2.07, 2.01 (4s, 48 H, 16
CH3COO), 1.9 (s, 12 H, 4 CH3CONH) ppm. 13C NMR (125 MHz,
CDCl3, 25 °C): δ = 174.3 (4 C-1), 173.1, 172.6, 172.5, 172.3 (16
CH3COO), 168.5 (4 CH3CONH), 148.1 (4 C-2Ј), 124.1 (4 C-1Ј),
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Received: February 3, 2009
Published Online: April 9, 2009
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