Picomolar inhibition of cholera toxin by a pentavalent ganglioside GM1os-calix[5]arene

Article abstract of DOI:10.1039/c3ob40515j

Cholera toxin (CT), the causative agent of cholera, displays a pentavalent binding domain that targets the oligosaccharide of ganglioside GM1 (GM1os) on the periphery of human abdominal epithelial cells. Here, we report the first GM1os-based CT inhibitor that matches the valency of the CT binding domain (CTB). This pentavalent inhibitor contains five GM1os moieties linked to a calix[5]arene scaffold. When evaluated by an inhibition assay, it achieved a picomolar inhibition potency (IC₅₀ = 450 pM) for CTB. This represents a significant multivalency effect, with a relative inhibitory potency of 100000 compared to a monovalent GM1os derivative, making GM1os-calix[5]arene one of the most potent known CTB inhibitors. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob40515j

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Picomolar inhibition of cholera toxin by a pentavalent
ganglioside GM1os-calix[5]arene†
Cite this: DOI: 10.1039/c3ob40515j
Jaime Garcia-Hartjes,‡a Silvia Bernardi,‡b Carel A. G. M. Weijers,a Tom Wennekes,a
Michel Gilbert,c Francesco Sansone,b Alessandro Casnati*b and Han Zuilhof*a,d
Cholera toxin (CT), the causative agent of cholera, displays a pentavalent binding domain that targets the
oligosaccharide of ganglioside GM1 (GM1os) on the periphery of human abdominal epithelial cells.
Here, we report the first GM1os-based CT inhibitor that matches the valency of the CT binding domain
(CTB). This pentavalent inhibitor contains five GM1os moieties linked to a calix[5]arene scaffold. When
evaluated by an inhibition assay, it achieved a picomolar inhibition potency (IC50 = 450 pM) for CTB. This
represents a significant multivalency effect, with a relative inhibitory potency of 100 000 compared to a
monovalent GM1os derivative, making GM1os-calix[5]arene one of the most potent known CTB
inhibitors.
Received 13th March 2013,
Accepted 29th April 2013
DOI: 10.1039/c3ob40515j
www.rsc.org/obc
prerequisite for endocytosis of the toxic enzymatically active A
subunit of CT, and the ensuing severe clinical symptoms.8 One
Introduction
Cholera still represents a serious health problem in areas of avenue in cholera research is to study the binding of CTB to
the developing world where there is a lack of clean water and GM1 and to develop CTB inhibitors that might prevent CT
proper sanitation. In 2012, the World Health Organization esti- from binding the hosts’ cell surface and thereby also the deve-
mated that annually 3–5 million cholera cases occur that result lopment of cholera. Here, we present the second of two
in more than 100 000 deaths.1 Although several treatments examples of a pentavalent GM1os-based inhibitor for CTB,
exist for cholera,2 resistance development and mutations in GM1os-calix[5]arene (1; Fig. 1). In the previous paper in this
the causative pathogen mean that efforts made to better issue, we also reported on pentavalent inhibitors of CTB based
understand the disease pathogenesis and develop new treat- on a GM1os-presenting corannulene scaffold. In the past,
ments are crucial.3,4 The symptoms of cholera are caused by several studies have focused on the development of multi-
cholera toxin (CT), which is produced by the Vibrio cholerae valent glycosylated inhibitors for CTB based on ganglioside
bacterium. CT is a member of the AB5 toxin family that con- GM1.9 It is noteworthy that in none of these studies, inhibitors
tains a pentameric binding domain (CTB) for recognition and were investigated with a pentavalent structure that matches the
binding to cell surfaces.5 The natural target ligand for CTB is pentavalent structure of CTB. On the other hand, pentavalent
the glycosphingolipid ganglioside GM1, on cellular mem-
branes of the infected hosts’ intestinal epithelial surface. CTB
can bind five GM1 saccharide epitopes simultaneously with
the terminal Gal- and the Neu5Ac carbohydrate units of the
ganglioside as the major contributors to the binding.6,7
The adhesion of CTB to ganglioside GM1 on cell surfaces is the
aLaboratory of Organic Chemistry, Wageningen University, Dreijenplein 8,
6703 HB Wageningen, The Netherlands. E-mail: Han.Zuilhof@wur.nl
bUniversità degli Studi di Parma, Dipartimento di Chimica, Parco Area delle
Scienze 17/a, 43124 Parma, Italy. E-mail: Casnati@unipr.it
cInstitute for Biological Sciences, National Research Council Canada,
100 Sussex Drive, Ottawa, Ontario, Canada
dDepartment of Chemical and Materials Engineering, King Abdulaziz University,
Jeddah, Saudi Arabia
†Electronic supplementary information (ESI) available: Copies of NMR spectra
for all reported new compounds. See DOI: 10.1039/c3ob40515j
‡Both authors contributed equally to this work.
Fig. 1 Developed CTB inhibitor: penta-GM1os-calix[5]arene (1).
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cyclens10,11 and cyclic peptides12 have been described as CTB was made by coupling five GM1os units to a calix[5]arene
inhibitors, but those contained only the much simpler galac- scaffold.
tose epitope likely to get around the difficulty to obtain
sufficient tailor-made GM1os. Therefore, also GM1 mimics
have been used, e.g. Thompson and Schengrund described
poly(propylene) imine dendrimers that present the Galβ1–
Results and discussion
3GalNAcβ1–4(Neu5Acα2–3)Galβ-epitope of GM1,13 with IC50 We designed a 5-fold symmetric calix[5]arene as a pentavalent
values for the tetravalent and octavalent dendrimers of 7 and scaffold structure. This calix[5]arene (Fig. 1) presents small
3 nM, respectively. Bernardi et al. published a series of GM1- methoxy groups at the lower rim, which confer a high confor-
mimics (pseudo-GM1), in which the residues in the GM1os mational flexibility to the macrocyclic structure.23 The upper
that are not essential for binding were replaced by a confor- rim of the calixarene inhibitor is decorated with the GM1 penta-
mationally restricted cyclohexane-diol and the Neu5Ac-unit saccharide separated from the macrocyclic core by appropriate
was substituted by various α-hydroxy acids.14,15 When attached linkers. Fan and coworkers12 have demonstrated that an
to multivalent dendritic structures,16 the relative inhibitory optimal linker length is vital for the potency of a synthetic
potency (RIP) values per mimic unit of the tetravalent and multivalent inhibitor. For the calix[5]arene, described here, a
octavalent inhibitors were 111 and 55, respectively. Interest- 31 atom-containing linker was chosen. This should allow the
ingly, when the same mimic was linked to a divalent calix[4]- simultaneous interaction of the five GM1os units with the five
arene scaffold,17 a 4000-fold enhancement in binding efficiency B-subunits of a single toxin.5
was achieved compared to the monovalent pseudo-GM1. These
The route towards our target (1) started with the synthesis
data suggested to us that the calixarene macrocycle, from of the pentavalent scaffold that began with the preparation of
which the binding inhibitors are projected, could be a promis- the known p-tert-butyl-calix[5]arene.24 This product was con-
ing multivalent scaffold18–20 to design CTB inhibitors with verted into p-H-calix[5]arene 225 by following literature pro-
improved efficiency. In collaboration with the group of Pieters, cedures. Next, penta-aldehyde 3 was obtained in 57% yield
we previously published divalent, tetravalent, and octavalent from 2 by exploiting the Duff formylation reaction.26,27 Com-
dendritic structures decorated with GM1os.21,22 For the octa- pound 3 was subsequently methylated at the lower rim by
valent compound the unprecedentedly low IC50 value of 50 pM using CH3I and K2CO3 in acetonitrile affording the penta-
was observed with an RIP of 17 500 per arm compared to its methoxy-calix[5]arene 4 in 68% yield (Scheme 1).
monovalent counterpart. However, its mismatched valency
Oxidation of 4 with NaClO2 and NH2SO3H produced the
compared to CTB prompted us to investigate a pentavalent penta-carboxylic acid 5. The unsymmetrically substituted
scaffold as core structure that when decorated with GM1os azido-penta-(ethyleneglycol)-amine 7 was synthesized from
has the potential to form 1 : 1 inhibitor–CTB complexes. hexa-ethylene glycol by ditosylation, substitution to the diazide,
The current paper presents the convergent synthesis of the and finally selective Staudinger reduction of one azide.28,29
first, water-soluble, pentavalent CTB inhibitor (1), which Initial attempts to condense amine 7 with the carboxylic acids
Scheme 1 Synthesis of the penta-azido-calix[5]arene (8) scaffold. Reagents and conditions: (a) HMTA, CF3COOH, reflux, N2, 5 days, 57%; (b) CH3I, K2CO3 CH3CN,
reflux, N2, 20 h, 68%; (c) NaClO2, NH2SO3H, (CH3)2CO, CHCl3, H2O, rt, 24 h, 79%; (d) (COCl)2, dry CH2Cl2, N2, rt, 18 h, quant.; (e) Et3N, dry CH2Cl2, N2, rt, 20 h, 44%.
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in 5 using HBTU resulted in difficult purification and low production of 9 on gram scale. Compound 9 was subsequently
yields (∼20%) of 8. However, when this spacer (7) was attached “clicked” to scaffold 8 under standard CuAAC conditions in
to calix[5]arene 5 via penta-acyl chloride intermediate 6 it pro- H2O while exposed to microwave irradiation to successfully
vided penta-azido-calix[5]arene 8 in a 44% yield.
provide our crude target inhibitor 1. With our target penta-
With the calix[5]arene (8) scaffold in hand we proceeded to valent GM1os-calix[5]arene 1 in hand, in order to properly assess
the next stage, attaching five GM1 oligosaccharides. We chose the role of the GM1os in inhibitor 1, we also set out to syn-
the copper-catalyzed azide–alkyne cycloaddition (CuAAC) reac- thesize derivatives of 1 containing fragments of the GM1os to
tion to achieve this, which meant a GM1os derivative with a use for comparison in the biological assays. The first of these
terminal alkyne was required. This C11-alkyne-terminated was penta-GM2os-calix[5]arene (11) that lacks the terminal
GM1os 9 (Scheme 2) was made via a chemo-enzymatic galactose epitope compared to the GM1os. We synthesized 11
procedure previously reported by us,30 which allowed the using the same CuAAC reaction conditions from scaffold 8 and
Scheme 2 Synthesis of GM1os-calix[5]arene (1) and GM2os-calix[5]arene (11); (a) CuSO4·5H2O, sodium ascorbate, Triton X-100, CH3OH, H2O, MW (150 W), 80 °C,
1 h; 51% 1, 59% 11.
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azido-calixarene scaffold 8, but instead of using the depro-
tected carbohydrates, acetyl-protected galactoside 12, and lacto-
side 13 were reacted. The resulting products could now be
purified by normal phase silica gel chromatograpy. The acetyl-
protected 14 and 15 were deprotected by employing standard
Zemplén31 conditions to obtain galactoside-calix[5]arene 16,
and lactoside-calix[5]arene 17, respectively, which did not
require further purification after work-up.
Finally, in order to properly determine the multivalency
effect of the interaction of 1 with CTB in our biological assays,
we also synthesized the monovalent GM1os derivative 20
(Scheme 4). This was achieved by first in situ generation of the
acyl chloride of commercially available 4-methoxybenzoic acid
with oxalyl chloride and, subsequently, reacting this with
amino-azide 7, yielding azide 19 in 20% over two steps. Again,
by employing the microwave-assisted CuAAC reaction on
alkyne-terminated 9 and azide 19, GM1os-monomer 20 was
obtained in a reasonable yield (49%).
Fig. 2 LCMS trace of the purification of GM2-calix[5]arene 11. (left) Chromato-
gram of the purification on a reversed phase column (see experimental section
for details). (right) Mass spectra for two fractions, the pentavalent product 11 at
RT = 12 min, and the tetravalent byproduct at 21 min.
The inhibitory potency of the four pentavalent compounds
(1, 11, 16, and 17) was determined by ELISA experiments. In
the assays, the ability of 1, 11, 16 and 17 to inhibit the binding
of HRP-labeled CTB was measured in competition with the
natural ligand ganglioside GM1, which was adsorbed to the
well surface of the ELISA plate. GM1os-calix[5]arene 1 showed
a high inhibition potency, i.e., a very low IC50 value of 450 pM
(Fig. 3, Table 1). Comparing the IC50 value (44 μM) of the
monovalent control compound (20) to that of 1 revealed a 100-
thousand increase in inhibitory potency, and an RIP of 20-
thousand per arm. Pentavalent inhibitors based on a more
rigid corannulene scaffold, which we also report in this issue,
inhibited CTB in the nanomolar range.32 Assay results for the
GM2os-calix[5]arene 11 confirmed the importance of using
GM1os. Lacking only the terminal galactose compared to 1, it
produced an IC50 of 9 μM, which is 20-thousand fold worse
compared to 1. The galactose-terminated (16) and lactose-ter-
minated (17) calix[5]arenes displayed a higher inhibition con-
centration than their solubility in the assay medium, and their
IC50 could therefore only be determined as being >1 mM
(Table 1).
a chemo-enzymatically produced alkyne-terminated C11-linked
GM2os sugar (10).
With both our target 1 and its derivative 11 in hand as
crude products, we investigated what purification method
would be suitable for these large complex molecules. An initial
purification by size exclusion chromatography (SEC) efficiently
removed an excess of alkyne-terminated GM1os 9 and GM2os
10, respectively. However, the crude products both also con-
tained a minor amount of tetravalent byproducts as could be
clearly seen with mass spectrometry and their separation
proved to be quite challenging. Initial attempts to separate
these by aqueous HPLC GPC failed, but after extensive opti-
mization, reversed phase HPLC purification proved the most suc-
cessful for this final purification step (see experimental
section for details). Fig. 2 shows a typical HPLC chromatogram
for the separation of both the GM1os- and GM2os-calix[5]-
arenes. Despite attempts to improve the moderate resolution,
the purification remained quite complicated because of long
elution times (up to 40 minutes per run) and high affinity of
the products with the column material. Collection of small
fractions in a specific retention time window over multiple
HPLC injections and subsequent lyophilization resulted
indeed in pure pentavalent GM1os- and GM2os-calix[5]arenes
(1 and 11) as shown by HR-MS and NMR analyses. The other
impure fractions that also contained 1 or 11 were collected,
pooled, lyophilized and re-injected to achieve optimal yields.
Attempts were made to isolate the tetravalent byproducts, but
insufficient amounts could be obtained for further analyses.
Besides the GM2os containing calixarene (11), we also pre-
pared two further derivatives of 1 that contained fragments of
GM1os, a pentavalent β-galactoside- (16) and β-lactoside-calix-
[5]arene (17) (Scheme 3). These more simple carbohydrates
enabled a modified synthesis procedure that circumvented the
potentially challenging HPLC purification, as encountered for
compounds 1 and 11. The coupling was also performed by
employing the microwave-assisted CuAAC reaction on penta-
Conclusions
In summary, we here report the synthesis and initial biological
evaluation of the first known example of a pentavalent GM1os-
based inhibitor of cholera toxin that matches the valency of
the cholera toxin B-subunit. With an IC50 of 450 pM, the penta-
valent GM1os-calix[5]arene (1) also displays the highest relative
inhibitory potency, 20-thousand per arm (compared to 20),
documented thus far for CT inhibitors. We are currently using
the here reported convergent synthetic route to further explore
the structure–activity-relationship of
1 and improve its
potency. Among other issues we are interested in investigating
the effect of the length, rigidity and hydrophobicity of the used
spacer and restricting the flexibility of the calix[5]arene
scaffold to a fixed cone structure.
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Scheme 3 Synthesis of galactoside-calix[5]arene (16), and lactoside-calix[5]arene (17); (a) CuSO4·5H2O, sodium ascorbate, DMF, H2O, MW (150 W), 80 °C, 1 h;
67% 14, 57% 15; (b) NaOMe–MeOH, 4 h – 18 h, H+-resin; 90% 16, 72% 17.
Scheme 4 Synthesis of GM1-monomer 20; (a) (COCl)2, dry CH2Cl2, N2, rt, 18 h; (b) 7, Et3N, dry CH2Cl2, N2, rt, 20 h; 20% in two steps; (c) 9, CuSO4·5H2O, sodium
ascorbate, Triton X-100, CH3OH, H2O, MW (150 W), 80 °C, 1 h; 49% 20.
using staining reagents: FeCl3 (1% in H2O–CH3OH 1 : 1),
H2SO4 (5% in EtOH), ninhydrin (5% in EtOH), basic solution
of KMnO4 (0.75% in H2O). Merck silica gel 60 (70–230 mesh)
Experimental section
General experimental information
All moisture sensitive reactions were carried out under a nitro- was used for flash chromatography and for preparative TLC
gen atmosphere, using previously oven-dried glassware. All dry plates. 1H NMR and 13C NMR spectra were recorded on Bruker
solvents were prepared according to standard procedures, dis- AV300, Bruker AV400, Bruker DPX400, and Bruker AV600
tilled before use and stored over 3 Å or 4 Å molecular sieves. equipped with cryoprobe spectrometers (observation of 1H
Reagents were obtained from commercial sources and used nucleus at 300 MHz, 400 MHz, 600 MHz, respectively, and of
without further purification unless stated otherwise. Analytical 13C nucleus at 75 MHz, 100 MHz, and 151 MHz, respectively).
TLC was performed using prepared plates of silica gel (Merck Chemical shifts are reported in parts per million (ppm), cali-
60 F-254 on aluminium) and then, according to the functional brated on the residual peak of the solvent, whose values are
groups present on the molecules, revealed with UV light or referred to tetramethylsilane (TMS, δTMS = 0), as the internal
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Compounds 31,32,33,34,35-pentahydroxycalix[5]arene 2,25
17-azide-3,6,9,12,15-pentaoxaheptadecane-1-amine 7,29 undec-
10-ynyl-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside 12,30 undec-
10-ynyl-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-
tri-O-acetyl-β-D-glucopyranoside 1330 were prepared according
to literature procedures.
GM1-calix[5]arene (1)
Calix[5]arene 8 (15.7 mg, 6.93 μmol) was dissolved in 0.5 mL
of CH3OH in a microwave tube. Then the GM1os derivative 9
(59.8 mg, 52.1 μmol), previously synthesized by chemo-enzy-
matic procedures,30,34 was combined with CuSO4·5H2O
(0.52 mg, 2.1 μmol), sodium ascorbate (0.82 mg, 4.2 μmol),
4 mL of H2O and a drop of Triton X-100. The mixture was
heated at 80 °C by microwave irradiation (150 W) for 60 min.
The reaction progression was monitored via TLC (eluent:
AcOEt–CH3OH–H2O–AcOH 4 : 2 : 1 : 0.1) and ESI-MS analyses.
The crude mixture was purified via size exclusion column
chromatography (Sephadex G-15, eluent: H2O 100%) and
HPLC purification (see General information) giving product 1
as a white solid. Yield: 51%. 1H NMR (600 MHz, D2O): δ (ppm)
7.61 (s, 5H, H5 triazole); 7.50 (s, 10H, Ar); 4.69 (bs, 5H, H1-
GalNAc); 4.43 (d, 5H, J = 8.0 Hz, H1-Gal′); 4.44–4.41 (m, 5H,
H1-Gal); 4.38–4.37 (m, 5H); 4.32–4.31 (5H, d, J = 7.9 Hz, H1-
Glc); 4.05–4.02 (m, 15H); 3.94–3.92 (m, 5H, H2-GalNAc);
3.86–3.60 (m, 105H); 3.60–3.45 (m, 80H); 3.46–3.33 (m, 75H);
3.26–3.24 (m, 5H, H2-Gal); 3.18–3.15 (m, 5H, H2-Glc); 3.10 (bs,
10H); 2.56–2.54 (m, 5H, H3a-Neu5Ac); 2.45–2.42 (m, 10H, tria-
zole-CH2CH2CH2); 1.92 (s, 15H, NC(O)CH3-Neu5Ac); 1.89 (s,
15H, NC(O)CH3-GalNAc); 1.85–1.80 (m, 5H, H3b-Neu5Ac);
1.44–1.35 (m, 20H, CH2 aliphatic chain); 1.14–1.08 (m, 10H,
CH2CH2OC1-Glc), 1.03 (m, 40H, CH2 aliphatic chain). 13C
NMR (151 MHz, D2O): δ (ppm) 175.4, 175.1, 174.5 (C(O)); 169.1
(ArC(O)NH); 159.7 (Ar-ipso); 134.8 (Ar-ortho); 129.2 (Ar-para);
128.9 (Ar-meta); 123.7 (C5 triazole); 105.1 (C1-Gal′); 103.0 (C1-
Gal); 102.8 (C1-GalNAc); 102.5 (C1-Glc); 102.0 (C2-Neu5Ac);
80.7 (C3-GalNAc); 79.0 (C4-Glc); 77.6 (C4-Gal); 75.2 (C5-Gal′);
75.1 (C5-Glc); 74.9 (C3-Gal); 74.7; 74.4 (C5-Gal); 73.4 (C6-
Neu5Ac); 73.1 (C2-Glc); 72.8 (C3-Gal′); 72.6 (C7-Neu5Ac); 71.0
(C2-Gal′); 70.9 (β-COCH2); 70.4 (C2-Gal); 70.1–69.8; 69.4; 69.2;
69.0; 68.9; 68.4; 68.3 (C4-GalNAc); 63.1; 61.4; 61.3; 60.9; 60.5;
52.0; 51.5 (C2-GalNAc); 50.3; 40.0; 37.2; 31.1 (ArCH2Ar); 29.3,
29.1, 29.0, 28.9, 28.8 (CH2 aliphatic chain); 25.6 (CH2CH2OC1-
Glc); 25.0 (triazole-CH2CH2CH2); 23.0 (NHC(O)CH3-GalNAc);
22.4 (NHC(O)CH3-Neu5Ac). HR-ESI-MS(−): m/z 1600.5013
[100% (M − 5H)5−] calcd: 1600.7112.
Fig. 3
GM1os-calix[5]arene
GM2os-calix[5]arene
GM1os-monomer;
Fitted curves of the experimental ELISA inhibition data. For details of the inhi-
bition assays see experimental section.
Table 1 CTB inhibition potency for reported compounds
Entry
Saccharide
Valency
#
IC50
1
2
3
4
5
GM1os
GM1os
GM2os
Galactose
Lactose
5
1
5
5
5
1
450 pM
44 μM
9 μM
>1 mM
>1 mM
20
11
16
17
standard. 13C NMR spectra were performed with proton decou-
pling. Electrospray ionization (ESI) mass analyses were per-
formed with a Waters spectrometer, while high resolution ESI
mass analyses were recorded on a Thermo Scientific Q Exactive
spectrometer. Melting points were determined on an Electro-
thermal apparatus in closed capillaries. Microwave reactions
were performed on a CEM Discovery System reactor running
on Discover Application Chemdriver Software v3.6.0. HPLC
was performed on an HP 1100 series with a DAP 190–600 nM
detector, equipped with a Waters Xterra 100 × 4.6 mm C18
column eluted with isocratic iPrOH–H2O 35 : 65, and a flow of
0.4 mL min−1, unless stated otherwise. Materials for the ELISA
experiments i.e. bovine serum albumin (BSA), bovine brain
GM1, ortho-phenylenediamine (dihydrochloride salt) (OPD),
cholera toxin horseradish peroxide (CTB-HRP) conjugate,
Tween-20, 30% H2O2 solution, sodium citrate, and citric acid
were purchased at Sigma Aldrich and used without further
modification, phosphate-buffered saline (PBS) 10× concentrate
was diluted ten times with demineralized water prior to use.
Nunc F96 MaxisorpTM 96-well microtiter plates were used as
purchased at Thermo Scientific. The microtiter plates were
washed using an automated Denville® 2 Microplate Washer.
5,11,17,23,29-Pentaformyl-31,32,33,34,35-pentahydroxycalix[5]-
arene (3)
Optical density (OD) was measured between 1.5 and 0.5 units Calix[5]arene 225 (0.41 g, 0.78 mmol) was added to a solution
on a Thermo Labsystems Multiskan Spectrum Reader running of HMTA (2.5 g, 17.8 mmol) in 50 mL TFA and the mixture was
on Skanit software version 2.4.2. Data analysis and curve refluxed for 5 days under N2. The solvent was then removed
fitting of inhibition experiments were performed on Prism under reduced pressure and the residue dissolved in 12 mL of
Graphpad software v5.04. Simplified nomenclature proposed a 1 : 1 CH2Cl2–HCl 1 M solution. The mixture was stirred at
by Gutsche33 is used to name the calix[5]arene compounds.
room temperature for 24 h. The aqueous phase was extracted
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with CH2Cl2 (5 × 5 mL). The combined organic phases were 5,11,17,23,29-Pentakis[(17-azide-3,6,9,12,15-pentaoxahepta-
washed with water (2 × 10 mL), dried over anhydrous Na2SO4, decane-1-amino)carbonyl]-31,32,33,34,35-pentamethoxy-calix-
filtered and the solvent removed under vacuum. The residue [5]arene (8)
was purified by trituration in CHCl3–hexane 1 : 1 to give the
product 3 as a brownish solid. Yield: 57%. H NMR (300 MHz,
In
a round-bottomed flask, 0.12 g of calix[5]arene 5
1
(0.14 mmol) and 0.51 mL of oxalyl chloride (5.82 mmol) were
solubilized in 15 mL of dry CH2Cl2 under a nitrogen atmos-
phere. The solution was stirred for 18 h at room temperature
and then the solvent evaporated to dryness. The residual com-
pound 6 was dissolved again in 5 mL of dry CH2Cl2 and then
added dropwise to a solution of amine compound 7 (0.29 g,
0.87 mmol) and NEt3 (0.12 mL, 0.87 mmol) in 5 mL of dry
CH2Cl2. The mixture was stirred for 20 h at room temperature
under a nitrogen atmosphere. The mixture was then washed
with 1 M HCl, an aqueous solution of Na2CO3 and water till
neutral pH was reached. The solvent was removed under
vacuum and the crude purified by flash chromatography
(eluent: CHCl3–CH3OH 95 : 5) to give the product 8 as a yellow
oil. Yield: 44%. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.50 (s,
10H, ArH); 7.07 (bs, 5H, CONH); 3.89 (s, 10H, ArCH2Ar);
3.65–3.50 (m, 110H, OCH2, CH2NHCO); 3.34 (t, 10H, J = 4.8
Hz, CH2N3); 3.28 (s, 15H, OCH3). 13C NMR (100 MHz, CDCl3):
δ (ppm) 167.3 (CO); 159.2 (Ar-ipso); 134.2 (Ar-para); 129.8 (Ar-
ortho); 128.2 (Ar-meta); 70.6, 70.5, 70.2, 70.0, (OCH2); 60.8
(OCH3); 50.6 (CH2N3); 39.7 (CH2NHCO); 30.9 (ArCH2Ar).
HR-ESI-MS(+): m/z 1131.5698 [100% (M + 2H)2+]; calcd: 1131.5756.
CDCl3–CD3OD 9 : 1): δ (ppm) 9.75 (s, 5H, CHO); 7.70 (s, 5H,
ArOH); 7.22 (s, 10H, ArH); 3.45 (s, 10H, ArCH2Ar). 13C NMR
(75 MHz, CDCl3–CD3OD 9 : 1): δ (ppm) 192.1 (CHO); 150.6 (Ar-
ipso); 131.9 (Ar-para); 128.1 (Ar-meta); 127.2 (Ar-ortho); 31.4
(ArCH2Ar). HR-ESI-MS(+): m/z 671.1921 [100% (M + H)+] calcd:
671.1917. M.p. > 300 °C.
5,11,17,23,29-Pentaformyl-31,32,33,34,35-pentamethoxy-calix-
[5]arene (4)
In a two-neck round-bottomed flask, pentaformylcalix[5]arene
3 (0.7 g, 1.1 mmol) was dissolved in 150 mL of dry CH3CN,
then K2CO3 (4.5 g, 32 mmol) and CH3I (2 mL, 32 mmol) were
added and the mixture was refluxed for 20 h under a nitrogen
atmosphere. The solvent was removed under reduced pressure
and the residue dissolved in 150 mL of a 1 : 1 solution CH2Cl2–
HCl 1 M. The mixture was stirred for 2 h at room temperature.
The organic layer was separated, and the aqueous phase
extracted with CH2Cl2 (2 × 50 mL). The combined organic
phases were washed with water (2 × 100 mL), dried over an-
hydrous Na2SO4, filtered and the solvent removed under reduced
pressure. The product 4 was obtained as a brown solid.
Yield: 68%. 1H NMR (300 MHz, CDCl3): δ (ppm) 9.72 (s, 5H,
CHO); 7.50 (s, 10H, ArH); 3.92 (s, 10H, ArCH2Ar); 3.25 (s, 15H,
OCH3). 13C NMR (75 MHz, CDCl3–CD3OD 9 : 1): δ (ppm)
191.4 (CHO); 161.8 (Ar-ipso); 134.8 (Ar-para); 132.0 (Ar-ortho);
130.8 (Ar-meta); 60.6 (OCH3); 30.7 (ArCH2Ar). HR-ESI-MS(+):
GM2-calix[5]arene (11)
Calix[5]arene 8 (15.7 mg, 6.93 μmol) was dissolved in 0.5 mL
of CH3OH in a microwave tube. Then the GM2os derivative 10
(50.5 mg, 52.1 μmol), previously synthesized by chemo-enzy-
matic procedures,30,34 was added together with samples of
CuSO4·5H2O (0.52 mg, 2.1 μmol), sodium ascorbate (0.82 mg,
4.2 μmol), 4 mL of H2O and a drop of Triton X-100. The
mixture was heated at 80 °C by microwave irradiation (150 W)
for 60 min. The reaction progress was monitored via TLC
(eluent: AcOEt–CH3OH–H2O–AcOH 4 : 2 : 1 : 0.1) and ESI-MS
analyses. The crude material was purified via size exclusion
column chromatography (Sephadex G-15, eluent: H2O 100%),
followed by HPLC purification (see General information)
m/z 741.2700 [100% (M
217–219 °C.
+
H)+] calcd: 741.2700. M.p.:
5,11,17,23,29-Pentacarboxy-31,32,33,34,35-pentamethoxycalix-
[5]arene (5)
Pentaformyl-pentamethoxycalix[5]arene 4 (0.25 g, 0.34 mmol)
was dissolved in a two-neck round-bottomed flask in 100 mL
of a mixture acetone–CHCl3 1 : 1, and cooled to 0 °C with an
ice-water bath. In another flask, a solution of NaClO2 80%
pure (0.47 g, 4.20 mmol) was dissolved in the minimum
amount of water. Subsequently, sulfamic acid (0.49 mg,
5.04 mmol) was added. This solution was slowly poured into
the reaction flask. The mixture was stirred at 0 °C for 15 min
and gradually warmed to room temperature while it remained
stirred for 24 h. The solvent was then removed under reduced
pressure and the residue triturated with 1 M HCl. After fil-
tration on a Büchner funnel, product 5 was obtained as a
solid. Yield: 79%. 1H NMR (300 MHz, CD3OD): δ (ppm) 7.75 (s,
10H, ArH); 3.93 (s, 10H, ArCH2Ar); 3.30 (s, 15H, OCH3). 13C
NMR (25 MHz, CD3OD): δ (ppm) 169.6 (CO); 162.1 (Ar-ipso);
1
giving pure product 11 as a white solid. Yield: 59%. H NMR
(400 MHz, D2O/CD3OD): δ (ppm) 7.65 (s, 5H, H5 triazole); 7.54
(s, 10H, ArH); 4.70 (m, 5H, H1-Gal (determined by HSQC));
4.47–4.42 (m, 15H, H1-GalNAc); 4.36–4.35 (d, 5H, J = 7.6 Hz,
H1-Glc); 4.08–4.06 (m, 10H); 3.89–3.66 (m, 95H, ArCH2Ar);
3.58–3.43 (m, 150H); 3.32–3.30 (m, 5H, H2-Gal); 3.23–3.19 (m,
5H, H2-Glc); 3.14 (bs, 10H); 2.61–2.58 (m, 5H, H3a-Neu5Ac);
2.48 (bs, 10H); 1.97 (s, 15H, NHC(O)CH3-Neu5Ac); 1.95 (s, 15H,
NHC(O)CH3-GalNAc); 1.89–1.84 (m, 5H, H3b-Neu5Ac);
1.51–1.36 (bs, 20H, CH2 aliphatic chain); 1.23–0.95 (50H, m,
CH2 aliphatic chain). HR-ESI-MS(−): m/z 1440.4670 [100%
(M − 5H)5−] calcd: 1438.6588.
Peracetylated-galactosylcalix[5]arene (14)
135.8 (Ar-ortho); 132.0 (Ar-meta); 126.8 (Ar-para); 61.3 (–OCH3); Calix[5]arene 8 (32.0 mg, 14.1 μmol) and the β-galactoside
31.9 (ArCH2Ar). HR-ESI-MS(+): m/z 843.2269 [100% (M + Na)+] derivative 12 (52.9 mg, 106 μmol) were dissolved in 2.5 mL of
calcd: 843.2265.
DMF in a microwave tube. CuSO4·5H2O (2.0 mg, 8.5 μmol),
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sodium ascorbate (3.3 mg, 16.9 μmol) and 0.5 mL H2O were 1.65–1.55 (m, 10H, triazole-CH2CH2CH2); 1.55–1.44 (m, 10H,
then added. The mixture was heated at 80 °C by microwave β-COCH2CH2); 1.39–1.15 (m, 50H, CH2 aliphatic chain). 13C
irradiation (150 W) for 60 min. When the reaction was com- NMR (100 MHz, CDCl3): δ ppm 170.4, 170.3, 170.2, 170.1,
pleted (checked via TLC, eluent: CH2Cl2–CH3OH 20 : 1), it was 169.8, 169.6, 169.1 (Ac); 167.3 (C(O)NH); 159.2 (Ar-ipso); 148.2
quenched by addition of water (15 mL) and extracted with (C4 triazole); 134.2 (Ar-ortho); 129.9 (Ar-para); 128.2 (Ar-meta);
AcOEt (5 × 15 mL). The combined organic layers were dried 121.7 (C5 triazole); 101.1 (C1′); 100.6 (C1); 76.3 (C4); 72.8 (C3);
over anhydrous Na2SO4, filtered and the solvent removed 72.5 (C5); 71.7 (C2); 71.0 (C3′); 70.6 (C5′); 70.5 (OCH2); 70.2
under vacuum. The crude material was purified on preparative (β-COCH2); 69.8 (OCH2); 69.6 (OCH2CH2-triazole); 69.1 (C2′);
TLC plates (eluent: CH2Cl2–CH3OH 9 : 1) giving product 14 as 66.6 (C4′); 62.1 (C6); 60.8 (C6′); 60.7 (ArOCH3); 50.0 (OCH2CH2-
1
a yellow oil. Yield: 67%. H NMR (300 MHz, CD3OD): δ (ppm) triazole); 39.7 (C(O)NHCH2); 31.0 (ArCH2Ar); 29.5, 29.3, 25.8,
8.21 (bs, 5H, C(O)NH); 7.77 (s, 5H, H5 triazole); 7.61 (bs, 10H, 25.7 (CH2 aliphatic chain); 20.9, 20.8, 20.6, 20.5 (CH3C(O)).
ArH); 5.38 (d, 5H, J = 2.7 Hz, H4); 5.16–5.02 (m, 10H, H3, H2); HR-ESI-MS(+): m/z 1571.9445 [100% (M + 4Na)4+] calcd: 1571.940.
4.61 (d, 5H, J = 7.3 Hz, H1); 4.51 (t, 10H, J = 5.0 Hz, OCH2CH2-
Galactosylcalix[5]arene (16)
triazole); 4.20–4.05 (m, 15H, H5, H6a, H6b); 3.94 (bs, 10H,
ArCH2Ar); 3.88–3.77 (m, 15H, OCH2CH2-triazole, β-COCHa); The peracetylated-galactosylcalix[5]arene 14 (45.0 mg, 9.46 μmol)
3.70–3.44 (m, 105H, OCH2, β-COCHb, C(O)NHCH2); 3.29 (s, was dissolved in 5 mL of CH3OH, drops of a freshly prepared
15H, ArOCH3); 2.67 (t, 10H, J = 7.6 Hz, triazole-CH2CH2CH2); MeONa in methanol solution were added till pH 8–9. The
2.13 (s, 15H, Ac); 2.02 (s, 15H, Ac); 2.01 (s, 15H, Ac); 1.94 (s, mixture was stirred at room temperature for 4 h. The progress
15H, Ac); 1.71–1.59 (m, 10H, triazole-CH2CH2CH2), 1.59–1.47 of the reaction was monitored via ESI-MS analysis. Amberlite
(m, 10H, β-COCH2CH2), 1.40–1.23 (m, 50H, CH2 aliphatic resin IR 120/H+ was subsequently added to quench the reac-
chain). 13C NMR (75 MHz, CD3OD) δ ppm: 172.0, 171.5, 171.2 tion, and the mixture was gently stirred for 30 min. until
(Ac); 169.6 (C(O)NH); 160.9 (Ar-ipso); 149.0 (C4 triazole); 135.8 neutral pH was reached. The resin was then filtered off and
(Ar-ortho); 130.7 (Ar-para); 129.7 (Ar-meta); 123.9 (C5 triazole); the solvent removed under vacuum to give pure product 16 as
1
102.2 (C1); 72.4 (C3); 71.7 (C5); 71.5, 71.4, 71.3, 70.9 (OCH2); a yellow oil. Yield. 90%. H NMR (300 MHz, CD3OD): δ (ppm)
70.6 (OCH2CH2-triazole); 70.5 (C2); 68.8 (C4); 62.6 (C6); 61.5 8.23 (bs, 5H, C(O)NH); 7.77 (s, 5H, H5 triazole); 7.61 (s, 10H,
(ArOCH3); 51.3 (OCH2CH2-triazole); 41.0 (C(O)NHCH2); 32.0 ArH); 4.51 (t, 10H, J = 5.0 Hz, OCH2CH2-triazole); 4.20 (d, 5H,
(ArCH2Ar); 30.6, 30.4, 30.2 (CH2 aliphatic chain); 27.0 (triazole- J = 7.1 Hz, H1); 3.94 (bs, 10H, ArCH2Ar); 3.92–3.80 (m, 20H, H4,
CH2CH2CH2); 26.3 (CH2 aliphatic chain); 20.8, 20.6, 20.5 β-COCHa, OCH2CH2-triazole); 7.77–7.69 (m, 10H, H6a, H6b);
(CH3C(O)). HR-ESI-MS(+): m/z 1607.7872 [100% (M + 3Na)3+] 3.66–3.40 (m, 120H, β-COCHb, H2, H3, H5, OCH2, C(O)-
calcd: 1607.7813.
NHCH2); 3.29 (s, 15H, OCH3); 2.67 (t, 10H, J = 7.6 Hz, triazole-
CH2CH2CH2); 1.72–1.53 (m, 20H, triazole-CH2CH2CH2,
β-COCH2CH2); 1.44–1.24 (m, 50H, CH2 aliphatic chain). 13C
Peracetylated-lactosylcalix[5]arene (15)
Calix[5]arene 8 (27.5 mg, 12.0 μmol) and the β-lactoside com- NMR (75 MHz, CD3OD): δ ppm 169.6 (C(O)NH); 160.9 (Ar-ipso);
pound 13 (70.0 mg, 89.0 μmol) were dissolved in 2.5 mL DMF 149.0 (C4 triazole); 135.8 (Ar-ortho); 130.7 (Ar-para); 129.7 (Ar-
in a microwave tube. CuSO4·5H2O (2.6 mg, 10.4 μmol), sodium meta); 124.0 (C5 triazole); 105.0 (C1); 76.6, 75.1, 72.6 (C2, C3,
ascorbate (4.4 mg, 22.2 μmol) and 0.5 mL H2O were then C5); 71.5, 71.4, 71.3, 70.8, 70.6, 70.4, 70.3 (OCH2, β-COCH2,
added. The mixture was heated at 80 °C by microwave C4); 62.5 (C6); 61.5 (ArOCH3); 51.3 (OCH2CH2-triazole); 41.0
irradiation (150 W) for 60 min. When the reaction was com- (C(O)NHCH2); 32.0 (ArCH2Ar); 30.8, 30.6, 30.5, 30.4, 30.3, 27.1
pleted (checked via TLC, eluent: CH2Cl2–CH3OH 94 : 6), it was (CH2 aliphatic chain); 26.3 (triazole-CH2CH2CH2). HR-ESI-MS
quenched by addition of water (15 mL) and extracted with (+): m/z 1305.0569 [100% (M + 3H)3+] calcd: 1305.0601
AcOEt (5 × 15 mL). The combined organic layers were dried
Lactosylcalix[5]arene (17)
over anhydrous Na2SO4, filtered and the solvent removed
in vacuo. The crude was purified by flash chromatography The peracetylated-lactosylcalix[5]arene 15 (42.0 mg, 6.8 μmol)
(elution in gradient: CH2Cl2–CH3OH 96 : 4→95 : 5) giving was dissolved in 5 mL of CH3OH, and drops of a freshly pre-
product 15 as a yellow oil. Yield: 57%. 1H NMR (300 MHz, pared methanol solution of MeONa were added till pH 8–9.
CDCl3): δ (ppm) 7.46 (bs, 10H, ArH); 7.40 (s, 5H, H5 triazole); The mixture was stirred at room temperature for 18 h. The pro-
6.83 (bs, 5H, C(O)NH); 5.33 (d, 5H, J = 3.3 Hz, H4′); 5.17 (t, 5H, gress of the reaction was monitored via ESI-MS analysis.
J = 9.3 Hz, H3); 5.09 (dd, 5H, J1′–2′ = 7.9 Hz, J2′–3′ = 10.4 Hz, Amberlite resin IR 120/H+ was subsequently added for quench-
H2′); 4.93 (dd, 5H, J2′–3′ = 10.4 Hz, J3′–4′ = 3.3 Hz, H3′); 4.86 (dd, ing, and the mixture was gently stirred for 30 min till neutral
5H, J2–3 = 9.3 Hz, J1–2 = 8.1 Hz, H2); 4.50–4.38 (m, 25H, H1′, pH. The resin was then filtered off and the solvent removed
H1, H6a, OCH2CH2-triazole); 4.15–4.00 (m, 15H, H6b, H6a′, under vacuum to give pure product 17 as a yellow oil. Yield:
H6b′); 3.90–3.71 (m, 35H, H5′, H4, β-COCHa, ArCH2Ar, 72%. 1H NMR (300 MHz, CD3OD): δ (ppm) 7.79 (s, 5H, H5 tria-
OCH2CH2-triazole); 3.65–3.47 (m, 105H, OCH2, H5, C(O)- zole); 7.60 (s, 10H, ArH); 4.51 (t, 10H, J = 5.0 Hz, OCH2CH2-tria-
NHCH2); 3.42 (m, 5H, β-COCHb); 3.21 (s, 15H, ArOCH3); 2.65 zole); 4.35 (d, 5H, J = 7.3 Hz, H1′); 4.26 (d, 5H, J = 7.8 Hz, H1);
(t, 10H, J = 7.7 Hz, triazole-CH2CH2CH2); 2.12 (s, 15H, Ac); 2.09 3.93 (bs, 10H, ArCH2Ar); 3.90–3.65 (m, 40H, H4′, H6ab′, H6ab,
(s, 15H, Ac); 2.04–1.98 (m, 60H, Ac); 1.94 (s, 15H, Ac); Glc β-COCHa, OCH2CH2-triazole); 3.65–3.35 (m, 135H, H3, H4,
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H5, H2′, H3′, H5′, Glc β-COCHb, OCH2, C(O)NHCH2); 3.28 (s, (O)CH3-GalNAc), 1.75 (1H, m, H3b-Neu5Ac), 1.40 (4H, m), 1.11
15H, OCH3); 3.23 (t, 5H, J = 8.4 Hz, H2); 2.66 (t, 10H, J = 7.6 (2H, m, Glc β-COCH2CH2), 1.03 (8H, m, –CH2CH2CH2–); 13C
Hz, triazole-CH2CH2CH2); 1.69–1.53 (m, 20H, CH2 aliphatic NMR (75 MHz, D2O): δ (ppm) 174.8, 174.5, 173.9, 168.5, 159.2,
chain); 1.42–1.23 (m, 50H, CH2 aliphatic chain). 13C NMR 128.4 (2 × CH Ar), 123.1 (C5 triazole), 113.2 (2 × CH Ar) 104.6
(75 MHz, CD3OD): δ ppm 169.6 (C(O)NH); 160.9 (Ar-ipso); (C1-Gal′), 102.7 (C1-Gal), 102.4 (C1-GalNAc), 102.0 (C1-Glc),
148.8 (C4 triazole); 135.7 (Ar-ortho); 130.7 (Ar-para); 129.7 (Ar- 101.5 (C2-Neu5Ac), 80.2 (C3-GalNAc), 78.5 (C4-Glc), 77.0 (C4-
meta); 124.2 (C5 triazole); 105.1 (C1′); 104.2 (C1); 80.7 (C4); Gal), 74.7, 74.6, 74.4, 74.2, 74.1, 74.0, 73.9 (C2 Glc), 72.6 (C3
77.0, 76.5, 76.4, 74.8, 74.7, 72.5 (C2, C3, C5, C2′, C3′, C5′); 71.5, Gal′), 72.3, 72.1 (C2-Gal′), 70.6 (Glc β-COCH2), 70.4 (C2-Gal),
71.4, 71.3, 70.9, 70.6, 70.4 (OCH2, β-COCH2,); 70.3 (C4′); 62.5, 70.4–68.5 (–OCH2–), 68.4 (C4-GalNAc), 67.8, 62.7, 60.9, 60.8,
61.9 (C6, C6′); 61.5 (ArOCH3); 51.4 (OCH2CH2-triazole); 41.0 60.4, 60.0, 51.4, 51.0 (C2 GalNAc), 49.8, 39.5, 36.7, 28.8–28.3
(C(O)NHCH2); 32.0 (ArCH2Ar); 30.8, 30.6, 30.5, 30.4, 30.2, 27.1 (CH2CH2CH2), 25.0 (Glc β-COCH2CH2), 24.5 (CH2-triazole),
(CH2 aliphatic chain); 26.2 (triazole-CH2CH2CH2). HR-ESI-MS 22.5 (NHC(O)CH3-GalNAc), 21.9 (NH(O)CH3-Neu5Ac); HR-ESI-
(+) m/z: 1575.1475 [100% (M + 3H)3+] calcd: 1575.1481.
MS(−) m/z: 1587.7009 [100% (M − H)−] calcd: 1587.7034.
CTB5 inhibition assays
17-Azide-3,6,9,12,15-pentaoxaheptadecane-1-aminocarbonyl-p-
methoxybenzene (19)
Each well of a 96-well microtiter plate was coated with a 100 μL
native GM1 solution (1.3 μM in ethanol) after which the
solvent was evaporated. Unattached GM1 was removed by
washing with PBS (3 × 450 μL), the remaining free binding
sites were blocked by incubation with 100 μL of a 1% (w/v) BSA
solution in PBS for 30 min at 37 °C. Detection limits were
determined by placing a CT-horseradish peroxidase conjugate
(CT-HRP), without inhibitor, on the plate, which gives the
highest response, and the lowest response was determined by
the optical density of the blank, i.e. the native GM1-coated well
with all components except the inhibitor and the toxin. These
two values represent the minimum and the maximum values
of optical density, 0% and 100% of binding of the CT to the
GM1-coating of the wells. Subsequently, the wells were washed
with PBS (3 × 450 μL). In separate vials, a logarithmic serial
dilution was performed that started from 2.0 mM of 150 μL
saccharide-calixarenes in 0.1% BSA and 0.05% Tween-20 in
PBS. Next, each vial was mixed and incubated with 150 μL of a
50 ng mL−1 CTB-HRP solution in the same buffer. This gave
an initial inhibitor concentration of 1.0 mM. In the case of
potent inhibitors, based on the logarithmic experiments, a
more accurate, serial dilution of a factor two was performed
around the expected IC50-values. The inhibitor–toxin mixtures
were incubated at room temperature for 2 h and then trans-
ferred to the coated wells. After 30 min of incubation at room
temperature, unbound CTB-HRP-calixarene complexes were
removed from the wells by washing with 0.1% BSA, 0.05%
Tween-20 in PBS (3 × 500 μL). 100 μL of a freshly prepared OPD
solution (25 mg OPD·2HCl, 7.5 mL 0.1 M citric acid, 7.5 mL
0.1 M sodium citrate and 6 μL of a 30% H2O2 solution, pH was
adjusted to 6.0 with NaOH) was added to each well and
allowed to react with HRP in the absence of light, at room
temperature, for 15 minutes. The oxidation reaction was
quenched by addition of 50 μL 1 M H2SO4. Within 5 min, the
absorbance was measured at 490 nm.
Oxalyl chloride (1.5 mL, 16.0 mmol) was added to a solution of
4-methoxybenzoic acid (0.30 g, 2.0 mmol) in 15 mL of dry
CH2Cl2 and the mixture was stirred at room temperature under
N2 for 18 h. The solvent was then removed under vacuum and
the residue dissolved again in 5 mL of dry CH2Cl2. This solu-
tion was added dropwise to a round bottomed flask containing
the azidoamine compound 7 (0.91 g, 3.0 mmol) and NEt3
(0.5 mL, 3.0 mmol) in 10 mL of dry CH2Cl2. The mixture was
let to react for 20 h at room temperature under an N2 atmos-
phere. The reaction was monitored via TLC (eluent: AcOEt). A
1 M HCl solution (20 mL) was then added to quench the reac-
tion, and the product extracted with CH2Cl2 (2 × 20 mL). The
combined organic phases were washed with NaHCO3 saturated
aqueous solution (15 mL), brine (15 mL), water (15 mL), dried
over anhydrous Na2SO4, filtered and the solvent evaporated
under reduced pressure. The crude was purified by flash
chromatography (eluent: AcOEt–acetone 9 : 1). Product 19 was
1
obtained pure as a yellow oil. Yield: 20%. H NMR (300 MHz,
CDCl3): δ (ppm) 7.74 (d, 2H, J = 8.9 Hz, Ar-meta); 6.87 (d, 2H,
J = 8.9 Hz, Ar-ortho); 6.78 (bs, 1H, C(O)NH); 3.80 (s, 3H, OCH3);
3.65–3.54 (m, 22H, OCH2, C(O)NHCH2); 3.32 (t, 2H, J = 5.0 Hz,
CH2N3). 13C NMR (75 MHz, CDCl3): δ ppm 167.0 (Ac); 162.0
(Ar-ipso); 128.8 (Ar-meta); 126.9 (Ar-para); 113.6 (Ar-ortho); 70.6,
70.5, 70.2, 70.0, 69.9 (OCH2); 55.4 (OCH3); 50.6 (CH2N3); 39.7
(C(O)NHCH2). ESI-MS(+) m/z: 463.0 [100% (M + Na)+]; 435.0
[60% (M − N2 + Na)+].
GM1os-monomer (20)
Starting from compounds 19 and 9, following the same pro-
cedure as for compound 1, and using reversed phase column
chromatography for the purification (gradient MeOH–H2O–
AcOH), monomer 20 was obtained as a white solid in 49%
1
yield. H NMR (400 MHz, D2O): δ (ppm) 7.81 (3H, m, triazole,
Ar), 6.98 (2H, d, J = 8.4 Hz, Ar), 4.71 (1H, s, H1-GalNAc), 4.39
(1H, d, J = 8.4 Hz, H1-Gal′), 4.42 (1H, m, H1-Gal), 4.38 (1H, m),
4.31, (1H, d, J = 8.2 Hz, H1-Glc), 4.05–4.01 (3H, m), 3.93 (1H,
m, H2-GalNAc), 3.75–3.40 (21H, m) , 3.60–3.47 (16H, m),
Acknowledgements
3.36–3.28 (14H, m), 3.25 (1H, m, H2-Gal), 3.18 (1H, m, H2- This work is co-financed by the INTERREG IV A Germany-
Glc), 3.10 (2H, s), 2.54 (1H, m, H3a-Neu5Ac), 2.35 (2H, m, CH2- Netherlands programme through the EU funding from the
triazole), 1.92 (3H, s, NHC(O)CH3-Neu5Ac), 1.85 (3H, s, NHC- European Regional Development Fund (ERDF), the Ministry for
This journal is © The Royal Society of Chemistry 2013
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Economic Affairs, Energy, Building, Housing and Transport of 15 A. Bernardi, D. Arosio, D. Potenza, I. Sànchez-Medina,
the State of North-Rhine Westphalia, the Dutch Ministry of
Economic Affairs, and the Province of Gelderland; it is
S. Mari, F. J. Cañada and J. Jiménez-Barbero, Chem.–Eur. J.,
2004, 10, 4395.
accompanied by the program management Euregio Rhein- 16 D. Arosio, I. Vrasidas, P. Valentini, R. M. J. Liskamp,
Waal. Additional funding was provided by Italian MIUR pro-
jects PRIN 200858SA98 and 2010JMAZML, and the EU COST
R. J. Pieters and A. Bernardi, Org. Biomol. Chem., 2004, 2,
2113.
Action CM1102 ‘MultiGlycoNano’. The CIM (University of 17 D. Arosio, M. Fontanella, L. Baldini, L. Mauri, A. Bernardi,
Parma) is acknowledged for NMR and mass measurements.
Truus Posthuma-Trumpie and Aart van Amerongen (FBR) are
A. Casnati, F. Sansone and R. Ungaro, J. Am. Chem. Soc.,
2005, 127, 3660.
acknowledged for their contribution in the ELISA experiments. 18 F. Sansone and A. Casnati, Chem. Soc. Rev., 2013, 42,
4623–4639.
19 F. Sansone, L. Baldini, A. Casnati and R. Ungaro, New
J. Chem., 2010, 34, 2715.
20 A. Dondoni and A. Marra, Chem. Rev., 2010, 110, 4949.
21 A. V. Pukin, H. M. Branderhorst, C. Sisu, C. A. G. M.
References
1 World Health Organisation, Fact sheet No107, 2011, 2012.
Weijers, M. Gilbert, R. M. J. Liskamp, G. M. Visser,
H. Zuilhof and R. J. Pieters, ChemBioChem, 2007, 8,
1500.
2 World Health Organisation, Weekly epidemiological record,
2011, vol. 86, p. 325.
3 N. Lycke, Nat. Immunol., 2012, 12, 592.
22 C. Sisu, A. J. Baron, H. M. Branderhorst, S. D. Connell,
C. A. G. M. Weijers, R. de Vries, E. D. Hayes, A. V. Pukin,
M. Gilbert, R. J. Pieters, H. Zuilhof, G. M. Visser and
W. B. Turnbull, ChemBioChem, 2009, 10, 329.
23 D. R. Stewart, M. Krawiec, R. P. Kashyap, W. H. Watson and
C. D. Gutsche, J. Am. Chem. Soc., 1995, 117, 586.
24 D. R. Stewart and C. D. Gutsche, Org. Prep. Proced. Int.,
1993, 25, 137.
4 J. Holmgren and A.-M. Svennerholm, Curr. Opin. Immunol.,
2012, 24, 343.
5 E. A. Merritt, S. Sarfaty, F. v. d. Akker, C. l’Hoir,
J. A. Martial and W. G. J. Hol, Protein Sci., 1994, 3, 166.
6 W. E. Minke, C. Roach, W. G. J. Hol and C. L. M. J.
Verlinde, Biochemistry, 1999, 38, 5684.
7 W. B. Turnbull, B. L. Precious and S. W. Homans, J. Am.
Chem. Soc., 2004, 126, 1047.
25 S. E. J. Bell, J. K. Browne, V. McKee, M. A. McKervey,
J. F. Malone, M. O’Leary and A. Walker, J. Org. Chem., 1998,
63, 489.
8 N. Sahyoun and P. Cuatrecasas, Proc. Natl. Acad.
Sci. U. S. A., 1975, 72, 3438; M. D. Hollenberg,
P. H. Fishman, V. Bennett and P. Cuatrecasas, Proc. Natl.
Acad. Sci. U. S. A., 1974, 71, 4224.
26 J. C. Duff, J. Chem. Soc., 1941, 547.
27 W. E. Smith, J. Org. Chem., 1972, 37, 3972.
28 A. W. Schwabacher, J. W. Lane, M. W. Scheisher,
K. M. Leigh and C. W. Johnson, J. Org. Chem., 1998, 63,
1727.
29 S. S. Iyer, A. S. Anderson, S. Reed, B. Swanson and
J. G. Schmidt, Tetrahedron Lett., 2004, 45, 4285.
30 A. V. Pukin, C. A. G. M. Weijers, B. van Lagen,
R. Wechselberger, B. Sun, M. Gilbert, M.-F. Karwaski,
D. E. A. Florack, B. C. Jacobs, A. P. Tio-Gillen, A. van
Belkum, H. P. Endtz, G. M. Visser and H. Zuilhof, Carbo-
hydr. Res., 2008, 343, 636.
31 G. Zemplén and E. Pascu, Ber. Dtsch. Chem. Ges., 1929, 62,
1613.
32 M. Matarella, J. Garcia-Hartjes, T. Wennekes, H. Zuilhof
and J. S. Siegel, Org. Biomol. Chem., 2013, DOI: 10.1039/
c3ob40438b, in press.
9 A. Bernardi, J. Jiménez-Barbero, A. Casnati, C. D. Castro,
T. Darbre, F. Fieschi, J. Finne, H. Funken, K.-E. Jaeger,
M. Lahmann, T. K. Lindhorst, M. Marradi, P. Messner,
A. Molinaro, P. Murphy, C. Nativi, S. Oscarson, S. Penadés,
F. Peri, R. J. Pieters, O. Renaudet, J.-L. Reymond,
B. Richichi, J. Rojo, F. Sansone, C. Schäffer, W. B. Turnbull,
T. Velasco-Torrijos, S. Vidal, S. Vincent, T. Wennekes,
H. Zuilhof and A. Imberty, Chem. Soc. Rev., 2013, 42,
4709–4727; T. R. Branson and W. B. Turnbull, Chem. Soc.
Rev., 2013, 42, 4613–4622.
10 E. Fan, Z. Zhang, W. E. Minke, Z. Hou, C. L. M. J. Verlinde
and W. G. J. Hol, J. Am. Chem. Soc., 2000, 122, 2663.
11 Z. Zhang, J. C. Pickens, W. G. J. Hol and E. Fan, Org. Lett.,
2004, 6, 1377.
12 Z. Zhang, J. Liu, C. L. M. J. Verlinde, W. G. J. Hol and
E. Fan, J. Org. Chem., 2004, 69, 7737.
33 C. D. Gutsche, Calixarenes Revisited, Royal Society of Chem-
istry, Cambridge, 1998.
13 J. P. Thompson and C. L. Schengrund, Glycoconjugate J.,
1997, 14, 837.
34 C. A. G. M. Weijers, M. C. R. Franssen and G. M. Visser,
Biotechnol. Adv., 2008, 26, 436.
14 A. Bernardi, L. Carrettoni, A. Grosso Ciponte, D. Monti and
S. Sonnino, Bioorg. Med. Chem. Lett., 2000, 10, 2197.
Org. Biomol. Chem.
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