Synthesis of sialoclusters appended to calix[4]arene platforms via multiple azide-alkyne cycloaddition. New inhibitors of hemagglutination and cytopathic effect mediated by BK and influenza A viruses

Article abstract of DOI:10.1039/b800598b

Tetra- and octavalent sialoside clusters were prepared in good yields exploiting for the first time the multiple copper-catalyzed cycloaddition of a propargyl thiosialoside with calix[4]arene polyazides. The cycloadducts featured the hydrolytically stable

Full text of DOI:10.1039/b800598b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of sialoclusters appended to calix[4]arene platforms via multiple
azide-alkyne cycloaddition. New inhibitors of hemagglutination and
cytopathic effect mediated by BK and influenza A viruses
Alberto Marra,*a Lisa Moni,a Daniele Pazzi,a Alfredo Corallini,*b Deborah Bridib and Alessandro Dondoni*a
Received 14th January 2008, Accepted 7th February 2008
First published as an Advance Article on the web 28th February 2008
DOI: 10.1039/b800598b
Tetra- and octavalent sialoside clusters were prepared in good yields exploiting for the first time the
multiple copper-catalyzed cycloaddition of a propargyl thiosialoside with calix[4]arene polyazides. The
cycloadducts featured the hydrolytically stable carbon-sulfur bond at the anomeric position and the
1,4-disubstituted triazole ring as the spacer between the sialic acid moieties and the platform. It was
demonstrated that these unnatural motifs did not hamper the desired biological activity of the
sialoclusters. In fact, they were able to inhibit, at submillimolar concentrations, the
hemagglutination and the viral infectivity mediated both by BK and influenza A viruses.
a latent state. Virus isolation and Southern blot hybridization
Introduction
analysis established that the kidney is the main site of BKV
Naturally occurring sialic acids constitute a family of more than 50
latency in healthy individuals. By these technical approaches,
structurally distinct nine-carbon 3-deoxy-ulosonic acids,1 the most
BKV sequences were also detected in other organs, such as
liver, stomach, lungs, parathyroid glands, and lymph nodes.4 The
detection of BKV DNA in tonsils suggests that the oropharynx
may be the initial site of BKV infection.5 Besides the above
mentioned PVN disease, other inflammatory syndromes affecting
several organs were described after BKV infection or reactivation
in both immunocompetent6 and immunosuppressed6,7 individuals.
Moreover, an association between hemorrhagics cystitis and
BKV was shown in immunosuppressed bone marrow transplant
recipients8 as well as in immunocompetent people.9 It has been
recently demonstrated by one of us (A. C.) that whole BKV, its
complete DNA, and also subgenomic DNA fragments containing
the early region, are able to transform embryonic fibroblasts and
cells cultured from kidney and brain of hamster, mouse, rat, rabbit
and monkey.10 BKV is highly oncogenic in rodents10 since young
or newborn hamsters, mice and rats, inoculated with BKV through
different routes, developed tumors.11 However, the role of BKV in
human neoplasia is still uncertain.
widespread derivative being 5-N-acetyl-neuraminic acid (Neu5Ac,
1) (Fig. 1). These monosaccharides are found at the non-reducing
end of glycoconjugates, where they are invariably a-D-linked to a
hexopyranose, usually D-galactose or D-galactosamine, or other
sialic acid fragments. Due to their peculiar position within cell-
surface glycoproteins and glycolipids, sialic acids are exposed to
the external environment and involved in numerous physiological
and pathological recognition phenomena, including the adhesion
of bacteria and viruses to human cells.2
Fig. 1 The most common sialic acid found in Nature.
Examples of the latter pathogens are the BK and influenza
A viruses. BK virus (BKV) is a human polyomavirus known
to be the etiological agent of a severe form of nephropathy,
a complication observed in 5–8% of kidney transplants, which
often leads to the organ loss.3 The extensive immunosuppressive
treatment used to prevent kidney rejection enables a dramatic
enhancement of the BKV replication resulting in the polyomavirus
associated nephropathy (PVN). However, primary infection by
BKV is usually asymptomatic and only occasionally may be
accompanied by mild respiratory illness or urinary tract disease.
During primary infection, viremia occurs and the virus spreads
to several organs of the infected individual where it remains in
Since no reliable drugs against BKV are available to date,3b
there is a great need to develop new, non-conventional antiviral
molecules based on recent discoveries of the actual receptors of
BKV onto the host cell. Early experiments showed that BKV does
not bind to sialic acid-depleted erythrocytes12 and Vero cells.13
Then, further evidence14 of the Neu5Ac-mediated attachment of
the virus to the cell has proved the key role exerted by 5-N-acetyl-
neuraminic acid, linked to N-glycoproteines or gangliosides, as
ligand of the viral capsid proteins.15,16 Therefore, artificial Neu5Ac-
decorated structures able to interfere with this sugar-protein
recognition process may act as anti-BKV agents. It is worth noting
that the potential of sialic acid-based unnatural ligands of BKV, as
inhibitors of its binding to the host cell, has never been explored.17
A similar approach can be envisaged to design new antiviral
drugs against other pathogens such as the influenza A viruses,
which include the human, avian, swine, and equine influenza
aDipartimento di Chimica, Laboratorio di Chimica Organica, Universita` di
Ferrara, Via Borsari 46, I-44100, Ferrara, Italy
bDipartimento di Medicina Sperimentale e Diagnostica, Sezione di Microbi-
ologia, Universita` di Ferrara, Via Borsari 46, I-44100, Ferrara, Italy
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viruses. Although both influenza A and B type viruses are
responsible for annual flu epidemics in humans, only the A virus
can cause sporadic pandemics, characterized by excess mortality
and morbidity, since there is no animal reservoir of the influenza
B virus.18 Aquatic birds provide the natural reservoir for the
influenza A viruses, although avian flu is, in general, asymptomatic
in wild birds. Occasionally, highly pathogenic strains of the
influenza A viruses (e.g. H5N1) cause serious systemic infections
in domestic poultry and even cross the species barrier infecting
humans.19 These single-stranded RNA viruses carry two surface
glycoproteins, a trimeric lectin (hemagglutinin, HA)20 and a
tetrameric glycosidase (neuraminidase, NA),18c and are subtyped
according to the reactivity of HA (15 subtypes) and NA (9
subtypes). Both proteins are present in multiple copies on the
virus surface (ca. 500 units of HA and 100 units of NA per virion)
and recognize the same host cell sugar, 5-N-acetyl-neuraminic
acid. The hemagglutinin mediates the adhesion of the virus to the
cell, followed by the fusion of viral and cell membranes, whereas
the neuraminidase cleaves the terminal sialic acid linked to the
glycoconjugate receptors to allow progeny virus release and to
promote the spread of the infection facilitating the movement
of the viral particles through mucus.21,22 Therefore, designed
molecules that bind stronger than the natural ligand to either HA
or NA would be, in principle, good anti-flu drugs. During the past
two decades both issues have been actively addressed, but only
some neuraminidase inhibitors,23 zanamivir (2) and oseltamivir
(3), have reached the market (Fig. 2), whereas compounds targeted
against hemagglutinin24 failed to become drugs.25 This was mainly
due to the weak HA-binding properties shown by monomeric
sialic acid derivatives. In fact, compound 6, one of the best
monovalent inhibitors of the bromelain-released hemagglutinin
(a soluble form of HA that lacks the C-terminal anchoring
peptide), has a dissociation constant of 3.7 lM,26 only three
orders of magnitude lower than that found for methyl a-sialoside
5 (KD = 2.8 mM), the prototypal HA ligand (Fig. 3). On the other
hand, good monomeric neuraminidase inhibitors,27 including the
commercially available ones, have dissociation constants in the
picomolar range.28 However, NA inhibitors are effective only
after the onset of infection in the host and are not suitable for
prophylactic purposes. To this end, a great deal of work has been
devoted to the synthesis of multivalent HA ligands,29 non-natural
compounds displaying multiple copies of sialic acid moieties, for
which it was anticipated a strong HA affinity due to simultaneous
binding events.
Fig. 3 Natural and unnatural ligands of influenza A virus hemagglutinin.
(e.g. a virus) to multiple receptor sites on another one (e.g. a cell).30
The resulting affinity enhancement is orders of magnitude greater
than that expected on the basis of a mere multiplication factor
of valency. Indeed, multivalency is a means Nature has found
to increase weak interactions to biologically relevant levels. This
finding is especially important in carbohydrate-protein recognition
events, in which monovalent affinities are usually quite low. In the
case where a protein, which is bearing clustered binding sites,
links to a multivalent ligand displaying sugar units with proper
orientation and spacing, the phenomenon is termed31 glycoside
cluster effect.30b
Non-natural multivalent HA ligands that present a-sialoside
units tethered to proteins,32 homopeptides,33 polysaccharides,34
polymers,35 liposomes,36 nanostructures,37 dendrimers,38 and low
molecular weight scaffolds (to form clusters)39 have been prepared
and, in most cases, tested as inhibitors of the hemagglutination
and the cytopathic effects mediated by influenza A viruses.40
High molecular weight multivalent sialosides32–37 showed a po-
tent HA inhibitory activity, in some cases35h higher than that
observed for the most effective natural inhibitor (equine a2-
macroglobulin), since the minimum concentrations41 required
to inhibit hemagglutination by the virus were in the nano-
or picomolar range. However, serious concerns34,35l,37b,42 were
raised about the toxicity of polymeric sialosides having a poly-
acrylamide backbone.35a–35i,35m,35n,35p In addition, the polydisperse
nature of macromolecular materials, which prevents rigorous
purification and characterization, together with their potential
immunogenicity, may constitute an insurmountable obstacle to the
approval as antiviral therapeutics by national regulatory agencies.
Therefore, structurally well-defined multivalent sialosides, such as
dendrimer38 and cluster39 derivatives, are valuable lead compound
candidates.
Our interest in sugar clusters dates back to the mid-1990s, when
we reported the first synthesis43 of glycosylated calixarenes (calix-
sugars) such as the water-soluble tetra-O-galactosyl-calix[4]arene
7 (Fig. 4). Then, various calixsugars were prepared by us44 and
others,39e,45 while the synthesis of hydrolytically stable, carbon-
linked glycosyl-calixarenes turned out to be a quite difficult
task. In fact, the Wittig olefination of calixarene aldehydes by
anomeric sugar phosphoranes gave poor results, since only a
bis-C-glycosylated calix[4]arene was obtained in modest yield.44c
Also the approach based on the Pd(0)-mediated Sonogashira-
Heck-Cassar cross-coupling between poly-iodoarenes and ethynyl
C-glycosides (C-glycosyl acetylenes), was inefficient for the
Fig. 2 Commercially available neuraminidase inhibitors.
Results and discussion
Synthesis of multivalent sialosides
Many recognition processes take place through the attachment of
multiple binding sites on one molecule (e.g. a protein) or organism
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synthesis of C-calixsugars, while it was successfully applied to the
preparation of a different class of C-glycoside clusters.46 Finally,
taking advantage of the potential of the Cu(I)-mediated azide-
alkyne cycloaddition,47,48 ethynyl C-glycosides were coupled with
various scaffolds bearing multiple azide functions to give in high
yields C-glycoclusters, including the C-calixsugar 8 (Fig. 4).49
These molecules displayed a 1,4-disubstituted 1,2,3-triazole ring
as the linker, a formidable keystone,48e connecting the sugar
ligand to the scaffold. Recently, following a two-step procedure
developed by Sharpless and Demko,50 the hitherto unreported 1,5-
disubstituted tetrazole-linked glycoclusters were made available as
well.51 Specifically, the facile cycloaddition between azidomethyl
C-glycosides and tosyl cyanide gave the corresponding 1-glycosyl-
5-sulfonyl-tetrazole derivatives, which were submitted to multiple
nucleophilic substitution by calixarene tetrols to afford tetravalent
C-glycoclusters,51 e.g. 9 (Fig. 4).
known39g propargyl thiosialoside 10 was chosen as dipolarophile
in the planned Cu-assisted cycloaddition reactions (Scheme 1). As
the 1,3-dipole counterpart, we used the calixarene tetra-azide 11,
a stable compound adopting the fixed cone conformation that has
been recently synthesized in our laboratory.49 Hence, the tetra-
azide 11 was allowed to react at room temperature overnight
with the sugar alkyne 10 (1.2 equiv. per azide group) in the
presence of freshly distilled N,N-diisopropylethylamine (Hu¨nig’s
base) and 25 mol% of commercially available copper(I) iodide.
To achieve complete solubility of the cycloaddition partners,
anhydrous DMF, instead of toluene, was used as the reaction
solvent. As partially deacetylated sialoclusters were formed due
to the presence of adventitious water, the crude reaction mixture
was acetylated (Ac2O, Py, r.t., 3 h). The tetravalent sialocluster
12 was isolated in 65% yield by column chromatography on
silica gel. The trivalent cycloadduct was recovered in 20% yield,
although contaminated by 12 and other byproducts. The MS and
1H NMR analyses readily confirmed the C4-symmetric structure
of 12, while the 1,4-disubstitution pattern of the 1,2,3-triazole
spacers was firmly established by 13C NMR spectroscopy. In fact,
_
a large positive D(dC4 dC5) value (20.7 ppm) was found in the
13C spectrum of 12, as expected for 1,4-disubstituted triazole
derivatives,55 including sugar clusters.49 On the other hand, it has
been demonstrated that 1,5-disubstituted triazole rings display
small negative values (ca. −5 ppm) for the chemical shift difference
of the same carbon atoms.49,55 To provide suitable material
for biological studies, the sialocluster 12 was first deacetylated
by transesterification (MeONa, MeOH, r.t., 3 h) to give the
corresponding methyl ester derivative. Then, the saponification
(aqueous NaOH, r.t., 18 h) of the crude product afforded the water
soluble tetra-acid 13 in 76% overall yield after filtration through a
short column of C18 silica gel.
Aiming at investigating the effect of the chain length on the
molecular recognition properties of the sialoside clusters, we
targeted the calix[4]arene 15 in which the azide functions are
linked to the aromatic ring through single methylene spacers
(Scheme 2). Hence, the known44b upper rim calix[4]arene tetrol
14 was converted into 15 upon treatment with diphenyl phos-
phoryl azide (DPPA, 1.5 equiv. per hydroxy group) and 1,8-
Fig. 4 Calixarene-based glycoside clusters prepared in the Authors’
laboratory.
Unfortunately, the latter methodology cannot be exploited for
the synthesis of sialoside clusters, because the acyl functions of the
Neu5Ac units do not tolerate the conditions of the nucleophilic
substitution step. Hence, we envisaged the copper-catalyzed azide-
alkyne cycloaddition to be a convenient entry to multivalent
sialosides. Calix[4]arenes, the most versatile members of this class
of cyclic oligophenols,52 were chosen as scaffolds due to the
easy derivatization at both the upper (wide) and lower (narrow)
rims and their three-dimensional preorganized architecture. In
particular, among the four possible conformations (cone, partial
cone, 1,2-alternate, 1,3-alternate) adopted by calix[4]arenes, the
cone conformer allows a spatially close arrangement of up to
four sugar ligands at either sides of the hydrophobic cavity. In
order to prepare glycoclusters resistant to enzymatic hydrolysis for
potential in vivo applications, the native anomeric oxygen atom of
the sialic acid moieties had to be replaced by a carbon or sulfur53
atom. The resulting isosteres would retain the hemagglutinin
binding affinity because the functional groups involved in this
sugar-protein recognition,54 i.e. axial carboxylate, amide function,
and glycerol side chain, are not altered.
◦
diazabicyclo[5.4.0.]undec-7-ene (DBU) at 120 C for 15 h. After
column chromatography on silica gel, the short-arm calix[4]arene
tetra-azide 15 was isolated in 55% yield as a white crystalline solid.
The cycloaddition between 15 and the propargyl thiosialoside 10
was carried out as described above to afford the peracetylated
tetravalent sialocluster 16 in 75% isolated yield. Also in this case,
the 1,4-regiochemistry of the triazole ring was established by 13C-
_
NMR analyses (D(dC4 dC5) = 20.8 ppm). Then, the removal of the
acetyl protecting groups and the hydrolysis of the methyl esters,
followed by C18 silica gel purification, gave the tetra-acid 17 in
80% yield.
The approach to sialoclusters displaying the sugar moieties close
to each other was carried out using a cone-configured calix[4]arene
azidated at the lower rim. This side of the macrocycle is signifi-
cantly narrower than the upper side. Therefore, the known56 tetra-
allyl-calixarene 18 (Scheme 3) was transformed into the tetrol 19
by one-pot multiple hydroboration and oxidation (73%). Then,
compound 19 was submitted to the azidation using DPPA and
DBU (120 ◦C, 18 h) to give, after column chromatography on silica
gel, the lower rim tetra-azide 20 in 67% yield as a white crystalline
Since a-D-linked C-sialosides bearing a terminal triple bond
in their aglycon chain appeared to us to be difficult access, the
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Scheme 1
Scheme 2
solid. The Cu-catalyzed cycloaddition of 20 with thiosialoside 10
(4.8 equiv.) under standard conditions afforded crystalline 21 in
53% isolated yield. The main reason for the moderate yield of
isolated 21 was due to the loss of the product in the course of
the chromatographic purification because of the low solubility
of the cycloadduct in the eluent employed. Unfortunately, direct
crystallization of the crude mixture, as well as size-exclusion
chromatography were not suitable means for the purification of
21. The C4-symmetric structure and the regioisomeric assignment
of the disubstituted triazole residues were confirmed by MS and
NMR analyses (see Experimental Section). Moreover, the fixed
cone conformation of the calix[4]arene scaffold was substantiated
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Scheme 3
by the presence of signals for the equatorial and axial protons of
the methylene bridges between the phenyl rings as large doublets
at 3.15 and 4.30 ppm, respectively.57 Finally, the acetyl and
methyl protecting groups were removed under basic conditions
as described for the above sialoclusters at the upper rim to give the
tetra-acid 22 in 50% yield as a white crystalline solid.
with copper salts. A colorless, analytical sample of 27 was obtained
by deacetylation, chromatography on C18 silica gel column, and
acetylation (see Experimental Section). This compound showed
very broadened signals in its 1H NMR spectrum recorded at room
temperature, probably due to a slow equilibrium between the
conformations adopted by the glycosylated chains linked to the
metacyclophane core. Fortunately, the sharp spectrum acquired
at 120 ◦C (in DMSO-d6) allowed to assign the C4-symmetric
structure and the cone conformation to 27, while the formation
of 1,4-disubstituted triazole rings was confirmed by 13C NMR
Since sialylation at either the upper or lower rim of calix[4]arene
scaffolds was quite efficient, we planned to prepare a cluster
constituted of sialic acid residues at both sides of the calixarene
cavity. In principle, such a densely sialylated molecule can bind
simultaneously to a couple of hemagglutinin trimers located onto
a single virion or two distinct viral particles. In both cases the HA
inhibitory activity was expected to be stronger than that displayed
by the tetravalent sialoclusters 13, 17, and 22. To this end, the
tetra-phenol 23 (Scheme 4) was further allylated at the lower rim
to give the octa-allyl derivative 24 in nearly quantitative yield.
The fixed cone conformation of this product was proved57 by the
_
analysis (D(dC4 dC5) = ∼19 ppm). Although slightly contaminated
by copper salts, compound 27 was transformed into the target
octavalent sialocluster 28 by standard transesterification of the
acetate functions and methyl ester saponification, followed by
column chromatography on C18 silica gel. By this procedure, 28
was isolated in a pure form and 61% yield. Also the octavalent
sialocluster 28 showed a complex H NMR spectrum at 25 ◦C,
1
presence in its H NMR spectrum of two doublets at 3.10 and
while raising the temperature to 120 ◦C caused the coalescence of
the signals.
1
4.38 ppm (J = 13.0 Hz) corresponding to the methylene bridges
protons. Then 24 was submitted to the hydroboration-oxidation
one-pot sequence to afford the octaol 25 (87%), which in turn was
azidated using DPPA and DBU at high temperature. The syrupy
calix[4]arene octa-azide 26 was isolated in pure form (52%) by
column chromatography on Sephadex LH-20. The purification of
this polyazide on a silica gel column was unsuccessful very likely
because of the strong interactions with this stationary phase. The
cycloaddition of 26 with propargyl thiosialoside 10 (1.2 equiv.
per azide group) was performed using our standard procedure to
give the octavalent cluster 27 in 72% yield after Sephadex LH-20
column chromatography. By means of this technique the sialoclus-
ter 27 was recovered as a brown solid, probably due to a complex
A rigorous evaluation of the enhancement of hemagglutinin
inhibitory activity shown by glycoclusters with respect to a
monovalent ligand, i.e. the glycoside cluster effect, required a
monomeric sugar featuring the same aglycon structure.58 Hence,
the thiosialoside 30 (Scheme 5) appeared to be more appropriate
than methyl O-sialoside 5 (Fig. 3) as the reference monovalent
ligand. Actually, compound 30 embodied all the functional groups
present in the above multivalent sialosides, such as the equatorial
thioglycoside linkage, the 1,4-disubstituted triazole moiety, and
the phenyl ring. Its protected derivative, the acetylated methyl
ester 29, was readily prepared in 78% yield by Cu-promoted
cycloaddition of 10 with crude benzyl azide (ca. 5 equiv.) prepared
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Scheme 4
Table 1 HI of BKV by multivalent sialosides
Entry
Compound
HI activitya (mM)
Relative potencyb
1
2
3
4
5
30
13
17
22
28
0.28
1
0.17 (0.68)c
0.25 (1.00)c
0.11 (0.44)c
0.20 (1.60)c
1.65 (0.41)d
1.12 (0.28)d
2.55 (0.64)d
1.40 (0.17)d
a Minimum concentration required for complete HI. b All potencies
normalized to that of monovalent sialoside 30. c Actual concentration
of sialic acid units. d Based on the sialic acid contents.
Scheme 5
in situ immediately before use (Scheme 5). Then, standard
deprotection reactions allowed the conversion of 29 into water
soluble 30 in almost quantitative yield.
of the sugar ligand. Although each multivalent sialoside was
more active than the monovalent derivative 30 (relative potencies:
from 1.12 to 2.55), the same was no longer true when the actual
concentration of sialic acid moieties was taken into consideration
(relative potencies: from 0.17 to 0.64). Indeed, the installation
of multiple copies of neuraminic acid onto a platform led to
a decrease of the inhibition activity per sugar unit, this being
in contrast to the occurrence of the glycoside cluster effect.
These disappointing results may be ascribed to a low surface
concentration of viral hemagglutinin, which hampers multiple
Biological assays
The capability of the synthesized multivalent sialosides to bind
BKV particles was tested using the hemagglutination inhibition
(HI) assay (Table 1). All compounds were able to inactivate BKV
at submillimolar concentrations, the lower rim tetrasialoside 22
being the most potent of this set of inhibitors (0.11 mM, entry
4). Therefore, the use of the non-natural calixarene scaffold and
triazole linker did not prevent the molecular recognition properties
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concomitant interactions to take place. Unfortunately, no data
are available in the literature about the number and spatial
arrangement of hemagglutinin on the BKV surface.
Preliminary tests demonstrated that compounds 13, 17, 22, 28,
and 30 did not manifest cytotoxicity against Vero cells. Hence, to
further assess the ability of these sialosides to bind BKV virions,
experiments of neutralisation of infection were carried out. Eight
to ten days after infection in Vero cells, an evident cytopathic effect
(CPE) was present in cultures infected with the control positive,
whereas all other cultures, infected with BKV but pretreated
with the mono- and multivalent sialosides (at the concentrations
indicated in Table 1) or anti-BKV antibodies, did not show CPE. In
order to confirm these results, all media obtained from the infected
cultures were frozen and thawed three times, then submitted
to the hemagglutination assay. Only the medium derived from
cultures infected with the control positive gave hemagglutination,
indicating the formation of new viral particles due to a cycle
of BKV infection. Finally, the presence of structural antigens
was analyzed by immunofluorescence (IF) in mock-infected cells,
infected cells, and in cells infected with the artificial inhibitor-
preincubated BKV (Fig. 5). When BKV was preincubated with
the mono- and multivalent sialosides, the adsorption step was
inhibited and the nuclei of the cells did not show the presence of
viral proteins (Fig. 5c–g). The same result was obtained when the
virus was preincubated with specific antibodies (Fig. 5h), whereas
in cells infected with BKV, not pretreated, viral proteins were
expressed and the nuclei of the cells appeared fluorescent (Fig. 5b).
The biological activity of the four sialoside clusters prepared
was also tested against the influenza A virus. The standard HI
assay (Table 2) demonstrated that 13, 22, and 28 were able to
inhibit the virus-induced hemagglutination, whereas the upper rim
tetrasialoside 17 failed to show HI activity at concentrations up to
50 mM (entry 3). In striking contrast to that observed with BKV,
the monovalent sialoside 30 was a very weak hemagglutination
inhibitor (0.28 vs. 100 mM, entry 1 in Table 1 and 2). On
the other hand, the upper and lower rim polysialosides 13, 22,
and 28 were active at submillimolar concentrations showing
similar potencies (0.25–0.37 mM). These results indicate that a
moderate glycoside cluster effect was operative since, considering
the inhibition activity per sialic acid unit, the above multivalent
sialosides were 50–83 times more active than the monovalent
derivative 30 (Table 2). Moreover, it appears from the HI assay
that the reduced length and flexibility of the methylene spacers
at the upper rim of 17 did not allow an efficient sugar-protein
recognition. Finally, theHI activityofthe octasialoside 28 (Table 2,
entry 5) was close to that of the tetrasialosides 13 and 22, indicating
that only one of the two sets of sialic acid units linked at both
Fig. 5 Inhibition of the cytopathic effect in Vero cell cultures infected by
BKV. The fluorescent nuclei indicate the expression of viral proteins. a)
Mock-infected cells. b) Cells infected with BKV. c-g) Cells infected with
BKV pretreated with monovalent sialoside 30, upper rim tetrasialoside 13,
upper rim tetrasialoside 17, lower rim tetrasialoside 22, octasialoside 28,
respectively. h) Cells infected with BKV, pretreated with specific antibodies.
calixarene rims was involved in the interaction with the viral
hemagglutinin. We suspect that this behaviour is due to the
unsuitable distance between the two sets of sugar ligands, very
likely too short to allow the simultaneous binding to two distinct
virions.
The multivalent sialosides 13, 22, and 28 were also submitted to
a neutralisation assay of influenza A virus (H3N2 strain) infectivity
performed as described for the BKV (see Experimental Section).
Ten days after infection of MDCK cells, the cytopathic effect
(CPE) was present in cells infected with the virus, not pretreated,
whereas the same cultures, infected with the virus and pretreated
with the above inhibitors, did not show CPE.
Table 2 HI of influenza A virus by multivalent sialosides
Entry
Compound
HI activitya (mM)
Relative potencyb
1
2
3
4
5
30
13
17
22
28
100
1
0.37 (1.48)c
>50 (>200)c
0.30 (1.20)c
0.25 (2.00)c
270 (68)d
—
333 (83)d
400 (50)d
a Minimum concentration required for complete HI. b All potencies
normalized to that of monovalent sialoside 30. c Actual concentration
of sialic acid units. d Based on the sialic acid contents.
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2.1 Hz, 4 H-7), 4.90 (ddd, 4H, J3ax,4 = 11.4, J3eq,4 = 4.6, J4,5
=
10.5 Hz, 4 H-4), 4.41 and 3.07 (2d, 8H, J = 13.3 Hz, 4 ArCH2Ar),
4.34 (dd, 4H, J9a,9b = 12.5 Hz, 4 H-9a), 4.23 (t, 8H, J = 7.0 Hz,
Conclusions
While non-polymeric multivalent sialosides have been already
described38,39 and, in some cases, submitted to biological assays,
our results pave the way for the synthesis of viral hemagglutinin
inhibitors via multiple azide-alkyne cycloaddition. This powerful
ligation method allowed us to prepare both inhibitors of the
influenza A virus showing a moderate glycoside cluster effect and
the first artificial inhibitors of the BKV.
4 ArCH2CH2CH2), 4.10 (dd, 4H, 4 H-9b), 4.09 (ddd, 4H, J5,6
=
10.6 Hz, 4 H-5), 4.03 and 3.98 (2d, 8H, J = 14.0 Hz, 4 SCH2), 3.91
(dd, 4H, 4 H-6), 3.83 (t, 8H, J = 7.5 Hz, 4 CH3CH2CH2O), 3.68
(s, 12H, 4 OMe), 2.76 (dd, 4H, J3ax,3eq = 12.5 Hz, 4 H-3eq), 2.32 (t,
8H, J = 7.2 Hz, 4 ArCH2CH2CH2), 2.20, 2.19, 2.16, 2.05, and 1.90
(5 s, 60H, 20 Ac), 2.04–1.91 (m, 20H, 4 H-3ax, 4 ArCH2CH2CH2,
4 CH3CH2CH2O), 1.00 (t, 12H, J = 7.5 Hz, 4 CH3CH2CH2O).
13C NMR (100 MHz): d170.8, 170.2, 170.1, and 168.2 (CO), 155.0
(C Ar), 143.2 (C-4 Tr.), 134.9 (C Ar), 133.2 (C Ar), 127.9 (CH
Ar), 122.5 (C-5 Tr.), 82.8 (C-2), 76.6 (CH3CH2CH2O), 74.1 (C-6),
69.5 (C-4), 68.1 (C-8), 67.2 (C-7), 62.4 (C-9), 53.0 (OMe), 49.6
(ArCH2CH2CH2), 49.3 (C-5), 37.6 (C-3), 32.0 (ArCH2CH2CH2,
ArCH2CH2CH2), 31.0 (ArCH2Ar), 23.3 (SCH2, CH3CH2CH2O),
23.2, 21.3, and 20.9 (CH3CO), 10.3 (CH3CH2CH2O); MALDI-
TOF MS (3107.41): 3130.3 (M+ + Na), 3146.1 (M+ + K). Anal.
Calcd. for C144H192N16O52S4: C, 55.66; H, 6.23; N, 7.21. Found: C,
55.91; H, 6.35; N, 7.40.
Experimental section
All moisture-sensitive reactions were performed under a nitro-
gen atmosphere using oven-dried glassware. Anhydrous solvents
were dried over standard drying agents59 and freshly distilled
prior to use. Reactions were monitored by TLC on silica gel
60 F254 with detection by charring with sulfuric acid. Flash
column chromatography60 was performed on silica gel 60 (230–
400 mesh). Melting points were determined with a capillary
apparatus. Optical rotations were measured at 20
2
◦C in
the stated solvent; [a]D values are given in deg cm3.g−1.dm−1.
1H NMR (300 and 400 MHz) and 13C NMR spectra (75 and
100 MHz) were recorded for CDCl3 solutions at room temperature
unless otherwise specified. Peak assignments were aided by 1H-1H
COSY and gradient-HMQC experiments. In the 1H NMR spectra
reported below, the n and m values quoted in geminal or vicinal
proton-proton coupling constants Jn,m refer to the number of the
corresponding sugar protons. MALDI-TOF mass spectra were
acquired using 2,5-dihydroxy-benzoic acid as the matrix. ESI mass
spectra were recorded for 6:4 CH3CN-H2O solutions containing
0.1% of trifluoroacetic acid.
5,11,17,23-Tetrakis{{3-{4-[(5-acetamido-3,5-dideoxy-2-thio-a-D-
glycero-D-galacto-2-nonulopyranosylonic acid)methyl]-1H-1,2,3-
triazol-1-yl}propyl}}-25,26,27,28-tetrapropoxy-calix[4]arene (13)
A solution of 12 (62 mg, 0.02 mmol) in a 0.2 M solution
of NaOMe in MeOH (4 cm3, prepared from Na and MeOH
immediately before use) was stirred at room temperature for
3 h in a nitrogen atmosphere, then neutralized with Dowex
50 × 2–400 resin (H+ form, activated and washed with H2O
and MeOH immediately before the use), and filtered through
a sintered glass filter. The resin was washed with MeOH, and
the solution was concentrated to give crude 5,11,17,23-tetra-
kis{{3-{4-[(methyl 5-acetamido-3,5-dideoxy-2-thio-a-D-glycero-
D-galacto-2-nonulopyranosylonate)methyl]-1H-1,2,3-triazol-1-yl}-
5,11,17,23-Tetrakis{{3-{4-[(methyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-2-thio-a-D-glycero-D-galacto-2-nonulopyrano-
sylonate)methyl]-1H-1,2,3-triazol-1-yl}propyl}}-25,26,27,28-
tetrapropoxy-calix[4]arene (12)
propyl}}-25,26,27,28-tetrapropoxy-calix[4]arene.
1H
NMR
(300 MHz, CD3OD) selected data: d 7.92 (s, 4H, 4 H-5 Tr.), 6.56
(s, 8H, Ar), 4.44 and 3.11 (2d, 8H, J = 12.8 Hz, 4 ArCH2Ar),
4.21 (t, 8H, J = 6.8 Hz, 4 ArCH2CH2CH2), 4.13 and 4.01 (2d,
8H, J = 14.1 Hz, 4 SCH2), 3.79 (s, 12H, 4 OMe), 2.79 (dd, 4H,
J3eq,4 = 4.5, J3ax,3eq = 12.7 Hz, 4 H-3eq), 2.02 (s, 12H, 4 Ac), 1.84
(dd, 4H, J3ax,4 = 11.3 Hz, 4 H-3ax), 1.04 (t, 12H, J = 7.4 Hz,
4 CH3CH2CH2O). A solution of the methyl ester tetra-adduct
in 0.2 M aqueous NaOH (2 cm3) was kept at room temperature
for 18 h in a nitrogen atmosphere, then neutralized with Dowex
50 × 2–400 resin, and filtered through a sintered glass filter. The
resin was washed with H2O, and the solution was concentrated.
The residue was eluted from a C18 silica gel cartridge with 1:1
H2O-MeOH, then MeOH, and dried under high vacuum to give
13 (36 mg, 76%) as an amorphous solid; [a]D = +24.7 (c 0.7,
MeOH). 1H NMR (300 MHz, CD3OD) selected data: d 7.90
(s, 4H, 4 H-5 Tr.), 6.55 (s, 8H, Ar), 4.44 and 3.11 (2d, 8H, J =
12.8 Hz, 4 ArCH2Ar), 4.20 (t, 8H, J = 7.0 Hz, 4 ArCH2CH2CH2),
4.15 and 4.02 (2d, 8H, J = 14.0 Hz, 4 SCH2), 2.82 (dd, 4H,
J3eq,4 = 3.6, J3ax,3eq = 12.5 Hz, 4 H-3eq), 2.03 (s, 12H, 4 Ac), 1.84
(dd, 4H, J3ax,4 = 10.5 Hz, 4 H-3ax), 0.94 (t, 12H, J = 7.5 Hz, 4
CH3CH2CH2O). 13C NMR (75 MHz, CD3OD): d 175.3 (C), 173.2
(C), 156.1 (C), 145.4 (C), 136.1 (C), 135.0 (C), 129.5 (CH), 124.9
(CH), 84.4 (C), 78.0 (CH2), 77.2 (CH), 72.7 (CH), 70.2 (CH), 69.2
A mixture of calix[4]arene tetra-azide 11 (92 mg, 0.10 mmol),
propargyl thiosialoside 10 (262 mg, 0.48 mmol), freshly distilled
N,N-diisopropylethylamine (418 lL, 2.40 mmol), CuI (23 mg,
0.12 mmol), and anhydrous DMF (5 cm3) was sonicated in an
ultrasound cleaning bath for 1 min, then magnetically stirred in the
dark at room temperature for 16 h, and concentrated. A solution
of the residue in pyridine (2 cm3) and acetic anhydride (2 cm3)
was kept at room temperature for 3 h, then concentrated, diluted
with AcOEt (100 cm3), washed with phosphate buffer at pH 7 (2 ×
10 cm3), dried (Na2SO4), and concentrated. The residue was eluted
from a column of silica gel with acetone to give first a tris-adduct
1
(51 mg, 20%) slightly contaminated by the tetra-adduct 12. H
NMR (400 MHz) selected data: d 7.53 (s, 1H, H-5 Tr.), 7.52 (s,
2H, 2 H-5 Tr.), 6.44 (d, 4H, J = 3.3 Hz, Ar), 6.39 (s, 4H, Ar),
4.87 (ddd, 3H, J3ax,4 = J4,5 = 10.9, J3eq,4 = 4.7 Hz, 3 H-4), 4.38 and
3.04 (2d, 8H, J = 13.0 Hz, 4 ArCH2Ar), 3.69, 3.68, and 3.66 (3 s,
9H, 3 OMe), 2.73 (dd, 3H, J3ax,3eq = 12.8 Hz, 3 H-3eq). MALDI-
TOF MS (2561.88): 2585.9 (M+ + H + Na), 2600.9 (M+ + K).
Eluted second was 12 (202 mg, 65%) as a syrup; [a]D = +40.0 (c
0.4, CHCl3). 1H NMR (300 MHz): d 7.56 (s, 4H, 4 H-5 Tr.), 6.44
(s, 8H, Ar), 5.52 (d, 4H, J5,NH = 10.0 Hz, 4 NH), 5.48 (ddd, 4H,
J7,8 = 8.5, J8,9a = 2.7, J8,9b = 5.5 Hz, 4 H-8), 5.36 (dd, 4H, J6,7
This journal is The Royal Society of Chemistry 2008
=
Org. Biomol. Chem., 2008, 6, 1396–1409 | 1403
©

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(CH), 64.5 (CH2), 53.7 (CH), 50.7 (CH2), 42.1 (CH2), 33.0 (CH2),
32.8 (CH2), 31.8 (CH2), 24.5 (CH2), 22.7 (CH3), 10.9 (CH3). ESI
MS (2378.71): 1190.5 (M + 2)/2, 793.9 (M + 3)/3. Anal. Calcd.
for C108H152N16O36S4: C, 54.53; H, 6.44; N, 9.42. Found: C, 54.38;
H, 6.51; N, 9.33.
5,11,17,23-Tetrakis{{4-[(5-acetamido-3,5-dideoxy-2-thio-a-D-
glycero-D-galacto-2-nonulopyranosylonic acid)methyl]-1H-1,2,3-
triazol-1-yl}methyl}-25,26,27,28-tetrapropoxy-calix[4]arene (17)
The sialocluster 16 (30 mg, 0.01 mmol) was deacetylated and
demethylated as described for the preparation of 13. The crude
product was eluted from a C18 silica gel cartridge with MeOH,
concentrated, and dried under high vacuum to give 17 (18 mg,
5,11,17,23-Tetrakis(3-azidomethyl)-25,26,27,28-tetrapropoxy-
calix[4]arene (15)
1
80%) as an amorphous solid; [a]D = +16.0 (c 0.5, MeOH). H
NMR (300 MHz, CD3OD): d 7.86 (s, 4H, 4 H-5 Tr.), 6.69 (s, 8H,
Ar), 5.32 (s, 8H, 4 ArCH2N), 4.45 and 3.20 (2d, 8H, J = 13.3 Hz,
4 ArCH2Ar), 4.15 and 4.05 (2d, 8H, J = 14.5 Hz, 4 SCH2), 3.87
(t, 8H, J = 7.5 Hz, 4 CH3CH2CH2O), 3.81–3.72 (m, 16H), 3.67–
3.59 (m, 4H), 3.58–3.50 (m, 8H), 2.86 (bd, 4H, J3ax,3eq = 12.0 Hz,
4 H-3eq), 2.03 (s, 12H, 4 Ac), 1.95 (tq, 8H, J = 7.5, 7.5 Hz, 4
CH3CH2CH2O), 1.76–1.68 (m, 4H, 4 H-3ax), 1.02 (t, 12H, J =
7.5 Hz, 4 CH3CH2CH2O). 13C NMR (75 MHz, CD3OD): d 175.4
(C), 174.3 (C), 158.0 (C), 146.4 (C), 136.7 (C), 130.2 (C), 129.5
(CH), 124.8 (CH), 86.1 (C), 78.1 (CH2), 76.9 (CH), 70.3 (CH),
69.5 (CH), 64.5 (CH2), 54.9 (CH2), 53.9 (CH), 42.5 (CH2), 31.7
(CH2), 24.6 (CH2), 24.4 (CH2), 22.7 (CH3), 10.7 (CH3). ESI MS
(2266.50): 1134.4 (M + 2)/2, 756.7 (M + 3)/3. Anal. Calcd. for
C100H136N16O36S4: C, 52.99; H, 6.05; N, 9.89. Found: C, 52.88; H,
6.11; N, 9.80.
A mixture of calixarene tetrol 14 (285 mg, 0.40 mmol),
sodium azide (208 mg, 3.20 mmol), diphenyl phosphoryl azide
(520 lL, 2.40 mmol), 1,8-diazabicyclo[5.4.0.]undec-7-ene (240 lL,
◦
1.60 mmol), and anhydrous DMF (4 cm3) was stirred at 120 C
for 15 h then partially concentrated, diluted with Et2O (100 cm3),
washed with H2O (2 × 10 cm3), dried (Na2SO4), and concentrated.
The residue was eluted from a column of silica gel with 15:1
cyclohexane-AcOEt to give 15 (179 mg, 55%) as a white solid.
◦
1
Mp 149–150 C (cyclohexane). H NMR (300 MHz): d 6.64 (s,
8H, Ar), 4.48 and 3.18 (2d, 8H, J = 13.4 Hz, 4 ArCH2Ar), 3.98 (s,
8H, 4 CH2N3), 3.88 (t, 8H, J = 7.5 Hz, 4 CH3CH2CH2O), 1.96 (tq,
8H, J = 7.5, 7.5 Hz, 4 CH3CH2CH2O), 1.02 (t, 12H, J = 7.5 Hz,
4 CH3CH2CH2O). 13C NMR (75 MHz): d 156.5 (C), 135.2 (C),
128.6 (C), 128.5 (CH), 76.9 (CH2), 54.2 (CH2), 30.8 (CH2), 23.2
(CH2), 10.2 (CH3); MALDI-TOF MS (812.96): 835.8 (M+ + Na),
851.8 (M+ + K). Anal. Calcd. for C44H52N12O4: C, 65.01; H, 6.45;
N, 20.68. Found: C, 65.12; H, 6.52; N, 20.81.
25,26,27,28-Tetrakis(3-hydroxypropoxy)-calix[4]arene (19)
To
a
cooled (0 ◦C), stirred solution of 18 (1.17 g,
2.0 mmol) in anhydrous THF (10 cm3) was added dropwise 9-
boracyclo[3.3.1]nonane (48 cm3, 24.0 mmol, of a 0.5 M solution
in hexane). The solution was allowed to reach room temperature
in 1.5 h, then cooled to 0 ◦C and slowly diluted with 10 M
NaOH (1.5 cm3) and 35% H2O2 (3.8 cm3). The mixture was
stirred at room temperature for 30 min and then warmed to
60 ◦C. Stirring was continued for an additional 1.5 h, then
the mixture was cooled to room temperature, diluted with 1 M
phosphate buffer at pH 7 (50 cm3), concentrated to remove the
organic solvents, and extracted with CH2Cl2 (2 × 100 cm3). The
combined organic phases were dried (Na2SO4) and concentrated.
The residue was eluted from a short column (5 × 5 cm) of silica gel
with AcOEt, then 1:1 AcOEt-acetone, acetone, and 9:1 acetone-
MeOH to give 19 (0.96 g, 73%) as a white solid. Mp 256–257 ◦C
(Acetone). 1H NMR (300 MHz, CDCl3 + D2O): d 6.67–6.58 (m,
12H, Ar), 4.42 and 3.23 (2d, 8H, J = 13.5 Hz, 4 ArCH2Ar),
4.08 (t, 8H, J = 7.0 Hz, 4 HOCH2CH2CH2O), 3.90 (t, 8H,
J = 6.3 Hz, 4 HOCH2CH2CH2O), 2.20 (s, 3H, 0.5 (CH3)2CO),
2.18 (tt, 8H, J = 6.3, 7.0 Hz, 4 HOCH2CH2CH2O). 13C NMR
(75 MHz): d 155.8 (C Ar), 134.9 (C Ar), 128.3 (CH Ar), 122.3
(CH Ar), 72.4 (HOCH2CH2CH2O), 60.2 (HOCH2CH2CH2O),
32.9 (HOCH2CH2CH2O), 30.9 (ArCH2Ar); MALDI-TOF MS
(656.80): 679.8 (M+ + Na), 695.8 (M+ + K). Anal. Calcd. for
C40H48O8·0.5(CH3)2CO C, 72.68; H, 7.49. Found: C, 72.76; H, 7.54.
5,11,17,23-Tetrakis{{4-[(methyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-2-thio-a-D-glycero-D-galacto-2-nonulopyrano-
sylonate)methyl]-1H-1,2,3-triazol-1-yl}methyl}-25,26,27,28-tetra-
propoxy-calix[4]arene (16)
The cycloaddition between the tetra-azide 15 (24 mg, 0.03 mmol)
and propargyl thiosialoside 10 (79 mg, 0.14 mmol) was carried
out as described for the preparation of 12 to give, after acetylation
of the crude mixture and column chromatography on silica gel
(acetone), 16 (68 mg, 75%) as a syrup; [a]D = +45.5 (c 1.0, CHCl3).
1H NMR (400 MHz): d 7.40 (s, 4H, 4 H-5 Tr.), 6.58 (bs, 8H, Ar),
5.45 (ddd, 4H, J7,8 = 8.5, J8,9a = 2.6, J8,9b = 5.3 Hz, 4 H-8), 5.33
(dd, 4H, J6,7 = 2.3 Hz, 4 H-7), 5.32 (d, 4H, J5,NH = 10.0 Hz, 4
NH), 5.23 and 5.18 (2d, 8H, J = 14.6 Hz, 4 ArCH2N), 4.89 (ddd,
4H, J3ax,4 = 11.2, J3eq,4 = 4.7, J4,5 = 10.5 Hz, 4 H-4), 4.40 and
3.09 (2d, 8H, J = 13.6 Hz, 4 ArCH2Ar), 4.29 (dd, 4H, J9a,9b
=
=
12.5 Hz, 4 H-9a), 4.08 (dd, 4H, 4 H-9b), 4.06 (ddd, 4H, J5,6
10.7 Hz, 4 H-5), 4.00 and 3.94 (2d, 8H, J = 14.2 Hz, 4 SCH2),
3.88 (dd, 4H, 4 H-6), 3.81 (t, 8H, J = 7.5 Hz, 4 CH3CH2CH2O),
3.57 (s, 12H, 4 OMe), 2.70 (dd, 4H, J3ax,3eq = 12.6 Hz, 4 H-3eq),
2.16, 2.14, 2.02, 2.01, and 1.88 (5 s, 60H, 20 Ac), 1.96 (dd, 4H, 4
H-3ax), 1.89 (tq, 8H, J = 7.4, 7.5 Hz, 4 CH3CH2CH2O), 0.98 (t,
12H, J = 7.4 Hz, 4 CH3CH2CH2O). 13C NMR (75 MHz): d 170.8,
170.7, 170.2, 170.1, and 168.2 (CO), 156.9 (C Ar), 143.2 (C-4 Tr.),
135.4 (C Ar), 128.3 (CH Ar), 128.2 (C Ar), 122.4 (C-5 Tr.), 82.9
(C-2), 77.0 (CH3CH2CH2O), 74.0 (C-6), 69.4 (C-4), 68.2 (C-8),
67.2 (C-7), 62.3 (C-9), 53.8 (ArCH2N), 52.9 (OMe), 49.3 (C-5),
37.6 (C-3), 30.8 (ArCH2Ar), 23.4 (SCH2), 23.15, 21.3, 20.80, and
20.76 (CH3CO), 23.10 (CH3CH2CH2O), 10.2 (CH3CH2CH2O);
25,26,27,28-Tetrakis(3-azidopropoxy)-calix[4]arene (20)
The calixarene tetrol 19 (328 mg, 0.50 mmol) was azidated as
described for the preparation of 15 to give, after column chro-
matography on silica gel (3:1 CH2Cl2-cyclohexane), 20 (253 mg,
67%) as a white solid. Mp 108–110 ◦C (MeOH). 1H NMR
(300 MHz): d6.63 (s, 12H, Ar), 4.36 and 3.24 (2d, 8H, J = 13.5 Hz,
MALDI-TOF MS (2995.19): 3018.7 (M+ + Na), 3034.7 (M+
+
K). Anal. Calcd. for C136H176N16O52S4: C, 54.54; H, 5.92; N, 7.48.
Found: C, 54.73; H, 6.01; N, 7.62.
1404 | Org. Biomol. Chem., 2008, 6, 1396–1409
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The Royal Society of Chemistry 2008
©

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4 ArCH2Ar), 4.00 (t, 8H, J = 7.0 Hz, 4 N3CH2CH2CH2O), 3.55
(t, 8H, J = 6.7 Hz, 4 N3CH2CH2CH2O), 2.18 (tt, 8H, J = 6.7,
7.0 Hz, 4 N3CH2CH2CH2O). 13C NMR (75 MHz): d 155.8 (C),
134.7 (C), 128.4 (CH), 122.6 (CH), 71.8 (CH2), 48.7 (CH2), 30.9
(CH2), 29.5 (CH2); MALDI-TOF MS (756.86): 780.0 (M+ + Na).
Anal. Calcd. for C40H44N12O4: C, 63.48; H, 5.86; N, 22.21. Found:
C, 63.61; H, 5.94; N, 22.30.
129.6 (CH), 125.2 (CH), 123.6 (CH), 84.2 (C), 77.2 (CH), 72.7
(CH), 72.7 (CH2), 70.2 (CH), 69.1 (CH), 64.5 (CH2), 53.7 (CH),
48.9 (CH2), 42.0 (CH2), 32.0 (CH2), 24.5 (CH2), 22.7 (CH3). ESI
MS (2210.39): 1106.2 (M + 2)/2, 738.0 (M + 3)/3. Anal. Calcd.
for C96H128N16O36S4: C, 52.16; H, 5.84; N, 10.14. Found: C, 52.10;
H, 5.89; N, 10.03.
5,11,17,23-Tetra-allyl-25,26,27,28-allyloxy-calix[4]arene (24)
To a cooled (0 ◦C), stirred solution of tetrol 23 (1.05 g, 1.8 mmol)
in DMF (18 cm3) was added NaH (0.43 g, 10.8 mmol, of a 60%
dispersion in oil) and, after 15 min, allyl bromide (0.75 cm3,
8.6 mmol). The mixture was stirred at room temperature for 2 h,
then diluted with CH3OH (1 cm3) and, after 10 min, diluted with
1 M phosphate buffer at pH 7 (50 cm3) and extracted with Et2O
(2 × 100 cm3). The combined organic phases were dried (Na2SO4)
and concentrated. The residue was eluted from a column of silica
gel with 2:1 cyclohexane-CH2Cl2 to give 24 (1.31 g, 98%) as a
25,26,27,28-Tetrakis{3-{4-[(methyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-2-thio-a-D-glycero-D-galacto-2-nonulopyrano-
sylonate)methyl]-1H-1,2,3-triazol-1-yl}propoxy}-
calix[4]arene (21)
The cycloaddition between the tetra-azide 20 (76 mg, 0.10 mmol)
and propargyl thiosialoside 10 (262 mg, 0.48 mmol) was carried
out as described for the preparation of 12 to give, after acetylation
of the crude mixture and column chromatography on silica gel
(2:1 acetone-AcOEt, then acetone), 21 (156 mg, 53%) as a white
solid. Mp 144–146 (dec.) (AcOEt-cyclohexane); [a]D = +41.0 (c
1.5, CHCl3). 1H NMR (400 MHz): d 7.55 (s, 4H, 4 H-5 Tr.), 6.58
(s, 12H, Ar), 5.44 (ddd, 4H, J7,8 = 8.7, J8,9a = 2.9, J8,9b = 5.3 Hz, 4 H-
8), 5.35 (d, 4H, J5,NH = 10.0 Hz, 4 NH), 5.34 (dd, 4H, J6,7 = 2.3 Hz,
4 H-7), 4.87 (ddd, 4H, J3ax,4 = 11.5, J3eq,4 = 4.5, J4,5 = 10.6 Hz, 4 H-
1
syrup. H NMR (300 MHz): d 6.50 (s, 8H, Ar), 6.38 (ddt, 4H,
J = 6.4, 10.3, 17.2 Hz, 4 CH2=CHCH2O), 5.82 (ddt, 4H, J =
6.5, 10.2, 17.0 Hz, 4 ArCH2CH=CH2), 5.27 (ddt, 4H, J = 1.5,
1.5, 17.2 Hz, Hcis of 4 CH2=CHCH2O), 5.19 (ddt, 4H, J = 1.1,
1.5, 10.3 Hz, Htrans of 4 CH2=CHCH2O), 4.98 (ddt, 4H, J =
=
1.5, 1.8, 10.2 Hz, Htrans of 4 ArCH2CH CH2), 4.90 (ddt, 4H,
4), 4.50 (t, 8H, J = 6.8 Hz, 4 CH2CH2CH2O), 4.30 (dd, 4H, J9a,9b
=
=
J = 1.8, 1.8, 17.0 Hz, Hcis of 4 ArCH2CH CH2), 4.46 (ddd, 8H,
12.5 Hz, 4 H-9a), 4.30 and 3.15 (2d, 8H, J = 13.6 Hz, 4 ArCH2Ar),
4.09 (dd, 4H, 4 H-9b), 4.07 (ddd, 4H, J5,6 = 10.8 Hz, 4 H-5), 4.02
and 3.94 (2d, 8H, J = 14.2 Hz, 4 SCH2), 3.93 (t, 8H, J = 7.0 Hz, 4
CH2CH2CH2O), 3.86 (dd, 4H, 4 H-6), 3.68 (s, 12H, 4 OMe), 2.72
(dd, 4H, J3ax,3eq = 12.8 Hz, 4 H-3eq), 2.42 (tt, 8H, J = 6.8, 7.0 Hz, 4
CH2CH2CH2O), 2.16, 2.14, 2.02, 2.01, and 1.88 (5 s, 60H, 20 Ac),
1.99 (dd, 4H, 4 H-3ax). 13C NMR (100 MHz): d170.8, 170.3, 170.1,
and 168.2 (CO), 155.7 (C Ar), 143.2 (C-4 Tr.), 134.6 (C Ar), 128.5
(CH Ar), 122.9 (CH Ar), 122.6 (C-5 Tr.), 82.9 (C-2), 74.0 (C-6),
71.3 (CH2CH2CH2O), 69.5 (C-4), 68.2 (C-8), 67.2 (C-7), 62.3 (C-
9), 53.1 (OMe), 49.2 (C-5), 47.4 (CH2CH2CH2O), 37.6 (C-3), 30.9
(ArCH2Ar, CH2CH2CH2O), 23.3 (SCH2, CH3CH2CH2O), 23.2,
21.3, and 20.8 (CH3CO); MALDI-TOF MS (2939.09): 2961.1
(M+ + Na), 2977.1 (M+ + K). Anal. Calcd. for C132H168N16O52S4:
C, 53.94; H, 5.76; N, 7.63. Found: C, 54.08; H, 5.82; N, 7.80.
J = 1.1, 1.5, 6.4 Hz, 4 CH2=CHCH2O), 4.38 and 3.10 (2d, 8H,
J = 13.0 Hz, 4 ArCH2Ar), 3.09 (ddd, 8H, J = 1.5, 1.8, 6.5 Hz, 4
ArCH2CH CH2). 13C NMR (75 MHz): d 154.1 (C), 138.2 (CH),
=
135.9 (CH), 134.9 (C), 133.2 (C), 128.3 (CH), 116.7 (CH2), 114.9
(CH2), 75.9 (CH2), 39.4 (CH2), 31.3 (CH2), 26.9 (CH2). MALDI-
TOF MS (745.00): 767.9 (M+ + Na), 783.9 (M+ + K). Anal. Calcd.
for C52H56O4: C, 83.83; H, 7.58. Found: C, 84.02; H, 7.70.
5,11,17,23-Tetrakis(3-hydroxypropyl)-25,26,27,28-tetrakis(3-
hydroxypropoxy)-calix[4]arene (25)
The octa-allyl-calixarene 24 (372 mg, 0.50 mmol) was submitted
to the hydroboration-oxidation as described for the preparation of
19. The crude mixture was triturated with cyclohexane, then Et2O,
and finally wit◦h AcOEt to give 25 (386 mg, 87%) as a white solid.
1
Mp. 213–215 C (Acetone). H NMR (300 MHz, DMSO-d6 +
D2O): d 6.47 (s, 8H, Ar), 4.28 and 3.05 (2d, 8H, J = 12.7 Hz,
4 ArCH2Ar), 3.86 (t, 8H, J = 7.3 Hz, 4 HOCH2CH2CH2O),
3.57 (t, 8H, J = 6.3 Hz, 4 HOCH2CH2CH2O), 3.30 (t,
8H, J = 6.3 Hz, 4 ArCH2CH2CH2OH), 2.24 (t, 8H, J =
7.5 Hz, 4 ArCH2CH2CH2OH), 2.03 (tt, 8H, J = 6.3, 7.3 Hz,
4 HOCH2CH2CH2O), 1.48 (tt, 8H, J = 6.3, 7.5 Hz, 4
ArCH2CH2CH2OH). 13C NMR (75 MHz, DMSO-d6): d 154.0
(C), 134.9 (C), 134.0 (C), 127.7 (CH), 72.3 (CH2), 60.0 (CH2), 58.1
(CH2), 34.3 (CH2), 33.0 (CH2), 31.0 (CH2). MALDI-TOF MS
(889.12): 912.1 (M+ + Na). Anal. Calcd. for C52H72O12: C, 70.24;
H, 8.16. Found: C, 70.40; H, 8.27.
25,26,27,28-Tetrakis{3-{4-[(5-acetamido-3,5-dideoxy-2-thio-a-D-
glycero-D-galacto-2-nonulopyranosylonic acid)methyl]-1H-1,2,3-
triazol-1-yl}propoxy}-calix[4]arene (22)
The sialocluster 21 (59 mg, 0.02 mmol) was deacetylated and
demethylated as described for the preparation of 13. The crude
product was eluted from a C18 silica gel cartridge with 1:1 H2O-
MeOH, then MeOH, concentrated, and dried under high vacuum
to give 22 (22 mg, 50%) as a white solid. Mp 192–194 (dec.)
(AcOEt); [a]D = +28.9 (c 0.9, MeOH). 1H NMR (400 MHz,
CD3OD): d 7.95 (s, 4H, 4 H-5 Tr.), 6.63–6.55 (m, 12H, Ar), 4.58
(t, 8H, J = 6.7 Hz, 4 CH2CH2CH2O), 4.36 and 3.15 (2d, 8H,
J = 13.6 Hz, 4 ArCH2Ar), 4.12 and 4.01 (2d, 8H, J = 14.5 Hz, 4
SCH2), 3.92 (t, 8H, J = 6.6 Hz, 4 CH2CH2CH2O), 3.84–3.71 (m,
16H), 3.61 (dd, 4H, J8,9b = 5.4, J9a,9b = 11.3 Hz, 4 H-9b), 3.54–3.48
(m, 8H), 2.79 (dd, 4H, J3eq,4 = 4.5, J3ax,3eq = 12.8 Hz, 4 H-3eq),
2.47 (tt, 8H, J = 6.6, 6.7 Hz, 4 CH2CH2CH2O), 2.00 (s, 12H, 4
Ac), 1.79 (dd, 4H, J3ax,4 = 10.8, 4 H-3ax). 13C NMR (75 MHz,
CD3OD): d 175.2 (C), 173.0 (C), 157.1 (C), 145.6 (C), 136.1 (C),
5,11,17,23-Tetrakis(3-azidopropyl)-25,26,27,28-tetrakis(3-
azidopropoxy)-calix[4]arene (26)
The calixarene octaol 25 (178 mg, 0.20 mmol) was azidated
as described for the preparation of 15 to give, after column
chromatography on Sephadex LH-20 (CH2Cl2), 26 (113 mg, 52%)
as a dark yellow syrup. An analytical sample was obtained by
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chromatography on preparative TLC (silica gel 60, 0.5 mm, 3.5:1
CH2Cl2-cyclohexane). 1H NMR (300 MHz): d6.48 (s, 8H, Ar), 4.30
and3.15 (2d, 8H, J = 13.4 Hz, 4 ArCH2Ar), 3.97 (t, 8H, J = 7.2Hz,
4 CH2CH2CH2O), 3.54 (t, 8H, J = 6.7 Hz, 4 ArCH2CH2CH2),
3.19 (t, 8H, J = 6.8 Hz, 4 CH2CH2CH2O), 2.40 (t, 8H, J =
7.5 Hz, 4 ArCH2CH2CH2), 2.19 (tt, 8H, J = 6.8, 7.2 Hz, 4
CH2CH2CH2O), 1.73 (tt, 8H, J = 6.7, 7.5 Hz, 4 ArCH2CH2CH2).
13C NMR (75 MHz): d 154.0 (C Ar), 134.6 (C Ar), 134.4 (C
Ar), 128.3 (CH Ar), 72.0 (CH2CH2CH2O), 50.5 (CH2CH2CH2O),
48.7 (ArCH2CH2CH2), 32.0 (ArCH2CH2CH2), 30.9 (ArCH2Ar),
30.5 (ArCH2CH2CH2), 29.5 (CH2CH2CH2O). MALDI-TOF MS
(1089.22): 1112.4 (M+ + Na). Anal. Calcd. for C52H64N24O4: C,
57.34; H, 5.92; N, 30.86. Found: C, 57.56; H, 6.08; N, 31.01.
dideoxy-2-thio-a-D-glycero-D-galacto-2-nonulopyranosylonic
acid)methyl]-1H-1,2,3-triazol-1-yl}propoxy}-calix[4]arene (28)
A solution of 27 (54 mg, 0.01 mmol) in a 0.2 M solution of NaOMe
in MeOH (3 cm3, prepared from Na and MeOH immediately
before the use) was stirred at room temperature in a nitrogen
atmosphere. After 1.5 h the solution turned turbid, therefore
was diluted with H2O (ca. 1 cm3) and stirred for an additional
2 h, then neutralized with Dowex 50 × 2–400 resin (H+ form,
activated and washed with H2O and MeOH immediately before
the use), and filtered through a sintered glass filter. The resin was
washed with H2O and MeOH, and the solution was concentrated.
A solution of the methyl ester octa-adduct in 0.2 M aqueous
NaOH (3 cm3) was kept at room temperature for 24 h in a nitrogen
atmosphere, then neutralized with Dowex 50 × 2–400 resin, and
filtered through a sintered glass filter. The resin was washed with
H2O, and the solution was concentrated. The residue was eluted
from a C18 silica gel cartridge with H2O-MeOH (from 2:1 to 1:1),
then MeOH, and dried under high vacuum to give 28 (24 mg,
5,11,17,23-Tetrakis{3-{4-[(methyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-2-thio-a-D-glycero-D-galacto-2-nonulopyrano-
sylonate)methyl]-1H-1,2,3-triazol-1-yl}propyl}-25,26,27,28-
tetrakis{3-{4-[(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-2-thio-a-D-glycero-D-galacto-2-nonulopyranosylonate)-
methyl]-1H-1,2,3-triazol-1-yl}propoxy}-calix[4]arene (27)
1
61%) an amorphous solid; [a]D = +6.7 (c 0.2, H2O). H NMR
(300 MHz, DMSO-d6 + D2O, 120 ◦C) selected data: d 7.88 (bs,
4H, 4 H-5 Tr.), 7.81 (bs, 4H, 4 H-5 Tr.), 6.45 (s, 8H, Ar), 1.90 (s,
24H, 8 Ac). 13C NMR (75 MHz, DMSO-d6 + D2O) selected data:
d 173.5 (C), 172.0 (C), 144.9 (C), 134.8 (C), 128.7 (CH), 124.0
(CH), 85.8 (C), 75.5 (CH), 71.9 (CH), 69.3 (CH), 68.1 (CH), 63.4
(CH2), 53.1 (CH), 49.5 (CH2), 47.5 (CH2), 42.2 (CH2), 31.7 (CH2),
30.5 (CH2), 23.9 (CH2), 23.0 (CH3). ESI MS (3996.30): 1332.9
(M + 3)/3, 1000.6 (M + 4)/4. Anal. Calcd. for C164H232N32O68S8:
C, 49.29; H, 5.85; N, 11.22. Found: C, 49.15; H, 5.92; N, 11.13.
The cycloaddition between the octa-azide 26 (44 mg, 0.04 mmol)
and propargyl thiosialoside 10 (209 mg, 0.38 mmol) was carried
out as described for the preparation of 12. The crude mixture was
acetylated (1:1 pyridine-acetic anhydride) at room temperature
for 3 h and concentrated. A solution of the residue in AcOEt
(40 cm3) was washed with phosphate buffer at pH 7 (10 cm3)
and water (10 cm3), then concentrated. The residue was eluted
from a column of Sephadex LH-20 with 1:1 CH2Cl2-MeOH to
give 27 (157 mg, 72%) as a light brown solid. In order to obtain
an analytical sample, compound 27 was deacetylated under basic
conditions (MeOH, Dowex 1 × 8 resin, r.t., 48 h), eluted from a
C18 silica gel cartridge with MeOH, and acetylated (1:1 pyridine-
Ac2O, r.t., 14 h). A solution of the residue in CH2Cl2 was washed
with water and concentrated to give 27 as a colorless amorphous
solid; [a]D = +43.8 (c 0.5, CHCl3). 1H NMR (300 MHz, DMSO-
d6, 120 ◦C) selected data: d 7.84 (s, 4H, 4 H-5 Tr.), 7.79 (s, 4H, 4
H-5 Tr.), 7.38 (bd, 8H, J5,NH = 9.0 Hz, 8 NH), 6.53 (s, 8H, Ar),
5.37–5.30 (m, 8H, 8 H-8), 5.21 (bd, 8H, J7,8 = 7.0 Hz, 8 H-7), 4.82
(ddd, 8H, J3ax,4 = 11.0, J3eq,4 = 4.5, J4,5 = 10.0 Hz, 8 H-4), 4.50
(t, 8H, J = 6.8 Hz, 4 CH2CH2CH2O), 4.20 (t, 8H, J = 6.6 Hz, 4
ArCH2CH2CH2), 3.77 (s, 12H, 4 OMe), 3.75 (s, 12H, 4 OMe), 3.12
1-Benzyl-4-[(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-2-thio-a-D-glycero-D-galacto-2-nonulopyranosylonate)-
methyl]-1H-1,2,3-triazole (29)
A mixture of benzyl bromide (150 lL, 1.26 mmol), sodium
azide (246 mg, 3.78 mmol), and MeOH (2.5 cm3) was stirred
at room temperature for 16 h, then partially concentrated using
a stream of nitrogen, diluted with H2O (10 cm3), and extracted
with Et2O (2 × 20 cm3). The combined organic phases were
dried (Na2SO4), the solvent was partially removed under vacuum,
and the solution concentrated to dryness using a stream of
nitrogen to give benzyl azide as a colorless oil, which was used
in the next step without further purification. A mixture of crude
benzyl azide (ca. 1.25 mmol), propargyl thiosialoside 10 (138 mg,
0.25 mmol), freshly distilled N,N-diisopropylethylamine (220 lL,
1.26 mmol), CuI (12 mg, 0.06 mmol), and anhydrous DMF
(2.5 cm3) was sonicated in an ultrasound cleaning bath for 1 min,
then magnetically stirred in the dark at room temperature for 6 h,
and concentrated. A solution of the residue in pyridine (1 cm3)
and acetic anhydride (1 cm3) was kept at room temperature for
14 h, then concentrated. The residue was eluted from a column
of silica gel with AcOEt to give 29 (134 mg, 78%) as a colorless
(d, 4H, J = 13.0 Hz, 4 Heq of 4 ArCH2Ar), 2.71 (dd, 8H, J3ax,3eq
=
12.8 Hz, 8 H-3eq), 2.08 and 2.07 (2 s, 24H, 8 Ac), 2.04 (s, 24H, 8
Ac), 1.96 and 1.95 (2 s, 24H, 8 Ac), 1.94 (s, 24H, 8 Ac), 1.84 (dd,
8H, 8 H-3ax), 1.72 (s, 24H, 8 Ac). 13C NMR (75 MHz, DMSO-d6)
selected data: d 170.0, 169.6, 169.4, 169.2, 169.0, and 168.0 (CO),
153.7 (C Ar), 141.8 (C-4 Tr.), 141.7 (C-4 Tr.), 134.0 (C Ar), 128.0
(CH Ar), 123.2 (C-5 Tr.), 123.0 (C-5 Tr.), 82.6 (C-2), 73.5 (C-6),
69.4 (C-4), 67.5 (C-8), 66.9 (C-7), 61.8 (C-9), 53.0 (OMe), 47.6
(C-5), 37.2 (C-3), 22.5, 20.9, and 20.5 (CH3CO); MALDI-TOF
MS (5453.69): 5477.8 (M+ + H + Na), 5492.7 (M+ + K). Anal.
Calcd. for C236H312N32O100S8: C, 51.97; H, 5.77; N, 8.22. Found: C,
52.26; H, 5.89; N, 8.40.
1
syrup; [a]D = +37.0 (c 1.3, CHCl3). H NMR (300 MHz): d 7.48
(s, 1H, H-5 Tr.), 7.42–7.28 (m, 5H, Ar), 5.56 and 5.47 (2d, 2H,
J = 14.8 Hz, PhCH2), 5.45 (ddd, 1H, J7,8 = 8.9, J8,9a = 2.7, J8,9b
5.5 Hz, H-8), 5.32 (dd, 1H, J6,7 = 2.1 Hz, H-7), 5.17 (d, 1H, J5,NH
10.0 Hz, NH), 4.87 (ddd, 1H, J3ax,4 = 11.6, J3eq,4 = 4.5, J4,5
=
=
=
5,11,17,23-Tetrakis{3-{4-[(5-acetamido-3,5-dideoxy-2-thio-a-D-
glycero-D-galacto-2-nonulopyranosylonic acid)methyl]-1H-1,2,3-
triazol-1-yl}propyl}-25,26,27,28-tetrakis{3-{4-[(5-acetamido-3,5-
10.6 Hz, H-4), 4.30 (dd, 1H, J9a,9b = 12.5 Hz, H-9a), 4.07 (dd, 1H,
H-9b), 4.06 (ddd, 1H, J5,6 = 10.8 Hz, H-5), 4.01 and 3.97 (2d, 2H,
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J = 13.5 Hz, SCH2), 3.85 (dd, 1H, H-6), 3.58 (s, 3H, OMe), 2.72
(dd, 1H, J3ax,3eq = 12.7 Hz, H-3eq), 2.16, 2.14, 2.04, 2.02, and 1.89
(5 s, 15H, 5 Ac), 1.99 (dd, 1H, H-3ax). 13C NMR (75 MHz): d170.8,
170.7, 170.2, 170.1, and 168.1 (CO), 143.9 (C-4 Tr.), 134.7 (C Ar),
129.0 (CH Ar), 128.7 (CH Ar), 128.1 (CH Ar), 122.4 (C-5 Tr.), 82.7
(C-2), 73.9 (C-6), 69.4 (C-4), 68.1 (C-8), 67.2 (C-7), 62.3 (C-9), 54.0
(PhCH2), 52.8 (OMe), 49.3 (C-5), 37.6 (C-3), 23.14 (SCH2), 23.10,
21.1, 20.8, and 20.7 (CH3CO); MALDI-TOF MS (678.71): 701.2
(M+ + Na), 717.8 (M+ + K). Anal. Calcd. for C30H38N4O12S: C,
53.09; H, 5.64; N, 8.25. Found: C, 53.26; H, 5.77; N, 8.40.
for 5 h. The hemagglutination titer was calculated on the basis of
the highest virus dilution that gave a complete hemagglutination.
For HI titrations, the compounds were diluted serially with
25 lL of PBS on a microtiter plate. A 25 lL of viral suspension
containing eight hemagglutination doses were added to each well
◦
and the mixture was kept at 37 C for 1 h. Then 50 lL of a 1%
suspension of red cells in PBS were added to each well. The results
were read after 5 h of incubation at 4 ◦C. The HI titer was defined
as the maximum dilution of each compound that caused complete
inhibition of viral hemagglutination.
1-Benzyl-4-[(5-acetamido-3,5-dideoxy-2-thio-a-D-glycero-D-
galacto-2-nonulopyranosylonic acid)methyl]-1H-1,2,3-
triazole (30)
Neutralisation assay of viral infectivity
Neutralisation of viral infectivity was carried out by incubating
different amounts of each compound with 10–50 TCID50 (50%
tissue-culture infectious dose) of BKV or influenza A virus at
37 ◦C for 1 h. Then, the suspensions were added to Vero or MDCK
The thioglycoside 29 (68 mg, 0.10 mmol) was deacetylated as
described for the preparation of 13 to give crude 1-benzyl-
4-[(methyl 5-acetamido-3,5-dideoxy-2-thio-a-D-glycero-D-galacto-
2-nonulopyranosylonate)methyl]-1H-1,2,3-triazole (52 mg). 1H
NMR (300 MHz, D2O) selected data: d 7.78 (s, 1H, H-5 Tr.),
7.33–7.22 (m, 5H, Ar), 5.43 (s, 2H, PhCH2), 3.95 and 3.80 (2d,
◦
monolayers and, after 2 h at 37 C, the inoculum was removed,
the monolayers were washed three times with DMEM, and then
incubated in the medium containing 2% FCS. The cultures were
observed by light microscope for the presence of viral cytopathic
effects (CPE) for 8–10 days. As a positive control, two cultures
were infected with the virus alone and as a negative control, two
monolayers were inoculated with the virus pre-treated with specific
antibodies. Then, when the positive controls developed CPE, the
medium of each well was frozen awaiting determination of the
presence of virions by hemagglutination.
2H, J = 11.0 Hz, SCH2), 3.24 (s, 3H, OMe), 2.62 (dd, 1H, J3eq,4
=
4.6, J3ax,3eq = 12.9 Hz, H-3eq), 1.88 (s, 3H, Ac), 1.70 (dd, 1H,
J3ax,4 = 11.6 Hz, H-3ax). The crude methyl ester derivative was
hydrolyzed as described for the preparation of 13 to give, after
chromatography (C18 silica gel cartridge, 2:1 MeOH-H2O) 30
(48 mg, 96%) as an amorphous solid; [a]D = +1.8 (c 0.2, MeOH).
1H NMR (300 MHz, D2O) selected data: d 7.78 (s, 1H, H-5 Tr.),
7.32–7.26 (m, 3H, Ar), 7.24–7.18 (m, 2H, Ar), 5.46 (s, 2H, PhCH2),
Immunofluorescence
3.88 and 3.78 (2d, 2H, J = 15.0 Hz, SCH2), 3.66 (dd, 1H, J4,5
=
J5,6 = 10.3 Hz, H-5), 3.52 (ddd, 1H, J3ax,4 = 11.2, J3eq,4 = 4.8 Hz,
H-4), 2.64 (dd, 1H, J3ax,3eq = 12.8 Hz, H-3eq), 1.88 (s, 3H, Ac), 1.68
(dd, 1H, H-3ax). 13C NMR (75 MHz, D2O): d175.2 (C), 174.0 (C),
145.6 (C), 135.0 (C), 129.3 (CH), 128.9 (CH), 128.2 (CH), 124.4
(CH), 86.4 (C), 74.9 (CH), 71.9 (CH), 68.7 (CH), 68.1 (CH), 62.5
(CH2), 54.0 (CH2), 51.8 (CH), 40.8 (CH2), 23.7 (CH2), 22.2 (CH3).
Anal. Calcd. for C21H28N4O8S: C, 50.80; H, 5.68; N, 11.28. Found:
C, 50.68; H, 5.75; N, 11.20.
Viral coat protein antigens were detected by indirect immunoflu-
orescence. Subconfluent cell monolayers, cultured on cover slips,
were infected with BK or influenza A viruses, pre-treated or not
with the sugar derivatives under examination. Four to six days
after infection, cultures were fixed for 10 min in cold acetone,
◦
incubated at 37 C for 30 min with specific antibodies anti-coat
antigens, washed three times in PBS, and incubated at 37 ◦C
for 30 min with fluorescein-conjugated goat anti-rabbit IgGs
(Antibodies Incorporated, Davis, CA). After extensive washing
in PBS, the preparations were mounted in buffered glycerol and
observed with a fluorescence microscope.
Cells and viruses
Vero and MDCK cells were grown and propagated in Dul-
becco’s modified Eagle’s minimal essential medium (DMEM)
supplemented with 10% foetal calf serum (FCS) at 37 ◦C in
a humidified atmosphere with 5% CO2. Prototype BKV was
grown in Vero cells as described previously.61 BKV was titrated
by hemagglutination of type 0 human erythrocytes62 and by the
fluorescent antibody (FA) focus assay in Vero cells.63 Influenza A
virus (H3N2 strain) was grown in MDCK cell monolayers and
viral hemagglutination units (HAU) were determined as described
previously.64
Acknowledgements
The influenza A virus was kindly provided by Dr. R. Cusinato
(Dipartimento di Microbiologia e Virologia, Azienda Ospedaliera,
University of Padova, Italy). We are grateful to Codexis Inc.
(USA) for supplying 5-N-acetyl-neuraminic acid. This work was
financially supported by the University of Ferrara.
References
Hemagglutination and hemagglutination-inhibition assays
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26 E. G. Weinhold and J. R. Knowles, J. Am. Chem. Soc., 1992, 114,
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