Selectivity among two lectins: Probing the effect of topology, multivalency and flexibility of "clicked" multivalent glycoclusters

Article abstract of DOI:10.1002/chem.201002635

The design of multivalent glycoconjugates has been developed over the past decades to obtain high-affinity ligands for lectin receptors. While multivalency frequently increases the affinity of a ligand for its lectin through the so-called "glycoside clust

Full text of DOI:10.1002/chem.201002635

DOI: 10.1002/chem.201002635
Selectivity among Two Lectins: Probing the Effect of Topology, Multivalency
and Flexibility of “Clicked” Multivalent Glycoclusters
Samy Cecioni,[a, b] Sophie Faure,[c] Ulrich Darbost,[d] Isabelle Bonnamour,[d]
Hꢀlꢁne Parrot-Lopez,[d] Olivier Roy,[c] Claude Taillefumier,[c] Michaela Wimmerovꢂ,[e]
Jean-Pierre Praly,[a] Anne Imberty,[b] and Sꢀbastien Vidal*[a]
Abstract: The design of multivalent
glycoconjugates has been developed
over the past decades to obtain high-af-
finity ligands for lectin receptors.
While multivalency frequently increas-
es the affinity of a ligand for its lectin
through the so-called “glycoside cluster
effect”, the binding profiles towards
different lectins have been much less
investigated. We have designed a series
of multivalent galactosylated glycocon-
jugates and studied their binding prop-
erties towards two lectins, from plant
and bacterial origins, to determine
their potential selectivity. The synthesis
was achieved through copper(I)-cata-
tions of two galactose-binding lectins
from Pseudomonas aeruginosa (PA-IL)
and Erythrina cristagalli (ECA) with
the synthesized glycoclusters were
studied by hemagglutination inhibition
assays (HIA), surface plasmon reso-
nance (SPR) and isothermal titration
microcalorimetry (ITC). The results
obtained illustrate the influence of the
scaffoldꢀs geometry on the affinity to-
wards the lectin and also on the rela-
tive potency in comparison with a mon-
ovalent galactoside reference probe.
lysed
azide–alkyne
cycloaddition
(CuAAC) under microwave activation
between propargylated multivalent
scaffolds and an azido-functionalised
carbohydrate derivative. The interac-
Keywords: calixarenes
· carbohy-
drates · glycoclusters · lectins · mul-
tivalency
Introduction
duction, intracellular trafficking of proteins, inflammation,
metastasis and development of the neuronal network. The
affinity of lectins for their substrate is usually relatively
weak with typical dissociation constants in the millimolar
range.[1] The clustering of receptors at the surface of the cell
and/or the multivalent display of carbohydrate epitopes am-
The interactions between lectins and carbohydrates play a
central role in a large number of biological processes, such
as cell–cell communication, cell adhesion, cell recognition,
cell differentiation, host–pathogen interactions, signal trans-
[a] S. Cecioni, Dr. J.-P. Praly, Dr. S. Vidal
Institut de Chimie et Biochimie Molꢁculaires
et Supramolꢁculaires, Laboratoire de Chimie
Organique 2 - Glycochimie, UMR5246
Universitꢁ Claude Bernard Lyon 1 and CNRS
43 Boulevard du 11 Novembre 1918
69622, Villeurbanne (France)
[d] Dr. U. Darbost, Dr. I. Bonnamour, Dr. H. Parrot-Lopez
Institut de Chimie et Biochimie Molꢁculaires
et Supramolꢁculaires, Chimie Supramolꢁculaire
Appliquꢁe (CSAp), UMR 5246
Universitꢁ Claude Bernard Lyon 1 and CNRS
43 Boulevard du 11 Novembre 1918
69622, Villeurbanne (France)
Fax : (+33)472-431-184
E-mail: sebastien.vidal@univ-lyon1.fr
[e] Dr. M. Wimmerovꢃ
National Centre for Biomolecular Research and
Department of Biochemistry, Faculty of Science
[b] S. Cecioni, Dr. A. Imberty
ˇ
CERMAV - CNRS, affiliated with Universitꢁ
Joseph Fourier and ICMG, BP 53, 38041
Grenoble Cedex 9 (France)
Masaryk University, Kotlꢃrskꢃ 2
611 37 Brno (Czech Republic)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201002635.
[c] Dr. S. Faure, Dr. O. Roy, Dr. C. Taillefumier
Clermont Universitꢁ, Universitꢁ Blaise Pascal
Laboratoire SEESIB, BP 10448, F-63000 Clermont-Ferrand
and CNRS, UMR 6504, Laboratoire SEESIB
63177 Aubiꢂre cedex (France)
2146
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Chem. Eur. J. 2011, 17, 2146 – 2159

FULL PAPER
plifies drastically the strength of the interaction between the
partners to reach very selective and stable interactions
through the so-called “glycoside cluster effect”.[2–4]
onance (SPR) and isothermal titration microcalorimetry
(ITC).
The design of multivalent glycoconjugates,[5] such as gly-
coclusters,[6–11] glycodendrimers,[12–14] glycopolymers,[15–17] and
glyconanoparticles[18–22] has brought a large set of data on
the binding properties of each class of conjugates with lec-
tins. While the positive influence of multivalency on the af-
finity to lectins has been clearly demonstrated, the influence
of the glycoconjugatesꢀ topology on the affinity has been
scarcely investigated. Two recent reports demonstrated that
calix[4]arene-based glycoconjugates bind differently to a gal-
actose-binding lectin based on their topological pat-
terns.[23,24]
Another approach would be to compare the binding prop-
erties of a series of multivalent glycoconjugates to several
lectins with different architectures and binding-site topolo-
gies.[25–27] Two galactose-binding lectins, from bacteria and
plant origin, have been selected for such a comparison. PA-
IL is a soluble lectin from Pseudomonas aeruginosa that
binds to galactose and a-galactose-containing oligosacchar-
ides with medium-range affinity (Kd ~50 mm).[28,29] The crys-
tal structure reveals a tetrameric arrangement with a general
rectangular shape.[30] Agglutinin from Erythrina cristagalli
(ECA) displays weaker affinity for galactose (Kd =1.25 mm)
and lactose (Kd =300 mm) and assembles in the canonical
legume–lectin dimer presenting binding sites at its extremi-
ties.[31] Comparison of the lectin architectures and distances
between binding sites is displayed in Figure 1.
Results and Discussion
Synthesis of multivalent glycoclusters: The assembly of the
multivalent glycoconjugates was performed by using a con-
vergent approach. The copper(I)-catalysed azide–alkyne cy-
cloaddition (CuAAC; “click” chemistry) between propargy-
lated scaffolds and an azido-functionalised carbohydrate
probe afforded the corresponding triazolyl cycloadducts.[32–42]
The use of microwave irradiation accelerated the 1,3-dipolar
cycloaddition process. This methodology is probably among
the most efficient in terms of yields, reaction time and che-
moselectivity.
b-Peptoids are surrogates of b-peptides in which the side
chains are moved from the backbone carbon atoms to the
amide nitrogen atoms.[43] We have recently developed a
method for the preparation of b-peptoids functionalised
with propargyl residues.[44] The efficient solution-phase
methodology allowed for the rapid gram-scale synthesis of
linear oligomers of valencies up to six. The corresponding
cyclic b-peptoids were also synthesized with appended prop-
argyl groups and their functionalisation with azido-deriva-
tised molecules was investigated. We focused here on the
synthesis of the tetravalent linear and cyclic oligomers and
their conjugation to a carbohydrate probe through “click”
chemistry.[45–47] The secondary amine of the linear tetrapro-
pargylated b-peptoid 1[44] was acetylated and the corre-
sponding acetamide 2 was then conjugated to 1-azido-3,6-di-
oxaoct-8-yl-2,3,4,6-tetra-O-acetyl-b-d-galactopyranoside
A[40,48] through CuAAC under microwave irradiation to
afford the peracetylated glycoconjugate 3 (Scheme 1). Sapo-
nification of both the tert-butyl ester and acetates afforded
the hydroxylated glycocluster 4. In a similar fashion, the
cyclic tetravalent b-peptoid 5 was subjected to our standard
“click” chemistry conditions to afford the corresponding gly-
coconjugate 6 and subsequent solvolysis of the ester groups
provided the cyclic glycocluster 7.
Calix[n]arenes are typical scaffolds used for the design of
glycoconjugates[23,40,49–55] with, in particular, recent examples
of construction of such glycoconjugates through CuAAC.[47]
Calix[6]arenes provide interesting scaffolds in which the car-
bohydrate epitopes will be condensed in a narrow space (al-
though the conformational rigidity of calix[6]arenes is much
weaker than for calix[4]arenes) and will create a high-densi-
ty volume of ligands available for binding to a lectin. To ex-
ploit the particularly dense functionalisation of these scaf-
folds, we have prepared two calix[6]arenes with either three
of six propargyl residues and their subsequent condensation
with the azido-functionalised carbohydrate probe was ach-
ieved by “click” chemistry.
Figure 1. Structures of A) PA-IL in complex with galactose (PDB code
1OKO) and B) ECA in complex with lactose (PDB code 1GZC). The
peptide chain is represented as ribbons, metal ions as spheres and carbo-
hydrate ligands as sticks. The distances have been measured from the gly-
cosidic oxygen of bound galactose.
We present here the synthesis of a series of multivalent
glycoclusters and their binding properties with lectins pre-
senting different quaternary structures as determined by he-
magglutination inhibition assay (HIA), surface plasmon res-
The regioselective trimethylation of calix[6]arene
8
[56]provided 9[57] and the propargylation of the remaining
phenolic positions was achieved with propargyl bromide and
sodium hydride in refluxing THF to afford the tripropargy-
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2147

S. Vidal et al.
scaffold 13 with A[40,48] provided
the desired glycoconjugate 14
and deprotection of the ace-
tates afforded the hydroxylated
hexavalent glycocluster 15. In
the case of the hexapropargy-
lated calixarene 13, only one
signal is observed for tBu (d=
0.81 ppm), ArH (d=7.15 ppm)
and the bridging methylene
protons (dꢀ4 ppm). This data
clearly indicates that compound
13 is flexible on the NMR time
scale and also that the major
conformation possesses
a C6
symmetry axis. Conversely, due
to an increase of the bulkiness
of the grafted arms, a loss of
symmetry is observed for com-
pounds 14 and 15 on the NMR
time scale. Nevertheless, due to
the high flexibility of those ar-
chitectures, 1H NMR spectra
are broad and a mixture of sev-
eral conformations is observed
in both the cases.
Porphyrins are attractive
scaffolds for the design of mul-
tivalent glycoconjugates partic-
ularly based on the potential
applications of their intrinsic
properties.[60–65] The synthesis of
the tetrapropargylated porphy-
rin 17[66,67] was performed from
4-propargyloxy-benzaldehyde
(16)[68] (Scheme 3).
Scheme 1. a) Ac2O, CH2Cl2, RT, 5 h, 97%; b) A, CuI, iPr2NEt, DMF, mW, 1108C, 15 min, 86% for 3, 77% for
6; c) NaOH, THF, H2O, RT, 16 h, 77%; d) MeOH/Et3N/H2O (4:1:1), RT, 4 days, 87%.
lated scaffold 10[58] (Scheme 2). The acetylated glycoconju-
gate 11 was then obtained by conjugation with the carbohy-
drate derivative A[40,48] and unmasking of the hydroxyl
groups gave the desired glycocluster 12. As attested by the
1H and 13C NMR spectra, calixarenes 11 and 12 adopt the
same flattened cone conformation. The two sets of signals
observed for tBu and ArH confirm that 11 and 12 display a
C3 symmetry axis.[59] Moreover, the two well-defined dou-
blets for the bridging methylene protons (ArCH2Ar) indi-
cate that the cone inversion does not occur on the NMR
time scale. Finally in both cases, the methoxy protons dis-
play a drastic downfield shift with dOMe =2.15 and 2.19 ppm
for 11 and 12, respectively; this indicates that the three me-
thoxy groups are oriented toward the centre of the cavity,
while the three glycosylated arms are pointing outside of the
calixarene core. In parallel, the per-propargylation of cal-
ix[6]arene 8 was also performed by using propargylbromide
with sodium hydride as a base but the isolated yield of the
hexapropargyl calix[6]arene 13 was only modest and not op-
timized. A better protocol for this process is now under in-
vestigation in our group. The CuAAC of the propargylated
The subsequent CuAAC process systematically incorpo-
rated CuI in the non-metallated porphyrin 17 during the cy-
cloaddition reaction leading to poor yields of cycloadducts
or even complex mixtures of decomposed molecules. The
NMR spectroscopic analyses of the mixtures obtained pro-
vided non-exploitable data due to the presence of copper
ions in the porphyrin. We then tried a series of “click”
chemistry conditions by using different sources of CuI for
the conjugation of the non-metallated porphyrin 17 with the
carbohydrate probe A[40,48] (Table 1). Since the metallation
of the porphyrin was the main problem observed, we have
selected triphenylphosphine–CuI complexes, which have
been described recently to be more selective in CuAAC.[69]
Both the bromide (available from commercial sources) and
iodide[70] complexes were used, with or without microwave
irradiation. Unfortunately, the partial incorporation of
copper ions in the porphyrinꢀs core was always observed
and the non-metallated porphyrin could not be isolated
under these conditions. The introduction of Zn2+ was, there-
fore, required prior to the “click” conjugation to obtain the
desired Zn-metallated porphyrin 18.[66,67] This metallation
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Selectivity among Two Lectins
FULL PAPER
patible with the use of micro-
wave heating. The acetylated
glycoconjugate 19 was then de-
protected to afford the desired
glycocluster 20.
Methyl
b-d-galactopyrano-
side 22 and the triethylenegly-
col-triazolyl 21[24] were used as
the monovalent references to
determine the influence of the
linker attached at the anomeric
position, but also to study the
importance of both valency and
topology for each scaffold
(Scheme 4). We have also re-
cently synthesized two other
tetravalent
glycoclusters[24]
based on a calix[4]arene core
with two different locked topol-
ogies. The cone conformer 24
presents its four carbohydrate
epitopes on one side of the cal-
ixarene rim (i.e. lower rim),
whereas the 1,3-alternate con-
former 23 displays two residues
on each side of the ring. We
have then studied the binding
properties of the monovalent
reference galactosides 21 and
22, the five tetravalent glyco-
clusters 4, 7, 20, 23 and 24 and
also the hexavalent derivative
15 towards two galactose-bind-
ing lectins: PA-IL from Pseudo-
monas aeruginosa (PA-IL) and
Erythrina cristagalli (ECA).
The trivalent glycocluster 12
could not be included in this
study due to its very poor solu-
bility in water even with the ad-
dition of 5% DMSO.
Scheme 2. a) MeI, K2CO3, acetone, reflux, 24 h, 35%; b) HCꢁCCH2Br, NaH, THF, reflux, 16 h, 87% for 9,
18% for 13; c) A, CuI, iPr2NEt, DMF, mW, 1108C, 15 min, 65% for 11, 84% for 14; d) MeOH/Et3N/H2O
(5:1:1), RT, 4 days, 86% for 12, 79% for 15.
Table 1. Optimization of CuAAC conjugation of the propargylated non-
Hemagglutination inhibition assays (HIA): A major interest
of lectin-induced erythrocytes agglutination is probably that
it reproduces the mode of binding of glycoconjugates to lec-
tins in biological systems and is usually used as a rapid
screening tool for multivalent glycoconjugates. Rabbit red
cells hemagglutination by PA-IL and ECA was inhibited by
the different glycoclusters synthesized above. The monova-
lent ligands 21 and 22 require millimolar range concentra-
tion for both lectins (Table 2). There is, therefore, almost no
measurable influence of the linker arm based on HIA. The
data measured for the multivalent glycoclusters will, there-
fore, account only for the valency and topology of these
macromolecules.
metallated porphyrin 17 with the azido-functionalized carbohydrate de-
rivative A.[a]
CuI catalyst
CuI
T [8C]
Activation
Observations
110
110
RT
RT
mW
mW
none
none
metallation
metallation
metallation
metallation
A
U
N
R
A
ACHTUNGTRENNUNG
[a] Conditions used: 17 (40 mg, 1 equiv),
A
(6 equiv), CuI catalyst
(0.5 equiv), iPr2NEt (5 equiv) in DMF (~5 mL).
process was optimised under microwave heating to reach
fast and complete Zn2+ insertion. The CuAAC conjugation
of the carbohydrate moiety then proceeded without trans-
metallation to afford the glycocluster 19 and was even com-
The linear peptoid glycocluster 4 is not inhibiting hemag-
glutination with either ECA or PA-IL under the experimen-
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S. Vidal et al.
On the opposite side, the por-
phyrin-based glycocluster 20
displays a clear preference for
PA-IL versus ECA with
a
strong inhibition effect on the
bacterial lectin. The relative po-
tency observed for ECA is
again very poor, but a much
larger enhancement could be
measured for PA-IL. The geo-
metrical arrangement of the
porphyrin scaffold is, therefore,
much more appropriate to fit
the spatial distribution of the
multiple binding sites of PA-IL.
The tetravalent calix[4]arene-
based glycoclusters 23 and 24
display only poor affinity to-
wards ECA, at least in the
same range as for the monova-
lent probes. Although their top-
ology is very different from the
porphyrin glycocluster 20, the
relative potency observed is
still in the same range. When
considering the binding proper-
ties of the glycoclusters 23 and
24 towards PA-IL, an improve-
ment could be observed with
inhibition in the micromolar
range and a relative potency of
roughly 30 for both molecules
with a slight preference for the
cone conformer 24. While the
topology of the calix[4]arene
scaffold does not play a major
Scheme 3. a) EtCO2H, pyrrole, 1208C, 1 h, 23%; b) ZnCl2, Et3N, mW, 1208C, 15 min, 87%; c) A, CuI, iPr2NEt,
DMF, mW, 1108C, 15 min, 64%; d) MeOH/CH2Cl2/Et3N/H2O (5:1:1:1), RT, 4 days, 99%.
role between 1,3-alternate and cone conformers, the square-
planar topology of the porphyrin core is again much more
suitable for a proper inhibition of PA-IL-induced hemagglu-
tination.
Table 2. Hemagglutination inhibition assays for ECA and PA-IL.
Ligand
Valency
Minimum concentration[a]
Relative potency[b]
[mm]
[b]
ECA
PA-IL
ECA
PA-IL
The hexavalent glycocluster 15 displays the same inhibi-
tion properties as the tetravalent porphyrin-based molecule
20, with a strong inhibition effect on PA-IL, and a weaker
one on ECA. The inhibition is better than the one obtained
with the calix[4]arenes 23 and 24, which indicates that the
higher valency is effective in increasing the affinity. Howev-
er, this hexavalent compound is not better than the tetra-
meric one 20. The square-planar topology of 20 is, therefore,
more appropriate than the circular organization of the cal-
ix[6]arene derivative 15 in solution and a better affinity is
measured towards PA-IL.
21
22
4
1
1
4
4
4
4
4
6
2500
5000
>2500
625
1250
1160
2500
1250
10000
5000
>2000
>2000
63
1
1
2
0.5
nr[c]
4
2
2.1
1
nr[c]
nr[c]
159
20
7
20
23
24
15
500
290
63
35
159
2
[a] Minimum concentration required to inhibit the agglutination of eryth-
rocytes. [b] Calculated as the ratio of the monovalent reference galacto-
side 21 value to the compoundꢀs value. [c] Not relevant.
The HIA measurements display a selective binding profile
for ECA and PA-IL. None of the compounds are really ef-
fective for binding ECA and few differences were observed
between monovalent and multivalent ligand. While ECA
does not select a strong ligand among the six glycoclusters
studied, PA-IL displayed a much more contrasted selectivity
tal conditions used. Nevertheless, its cyclic counterpart 7 dis-
plays a strong effect towards ECA while almost no binding
was observed for PA-IL, which demonstrates selectivity re-
lated to the topology. The resulting potency is, however, too
low to be described as an efficient “cluster effect”.
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Selectivity among Two Lectins
FULL PAPER
Scheme 4. Structure of the glycoclusters and their two monovalent reference ligands studied for their binding properties towards two galactose-binding
lectins.
profile with a large preference for the porphyrin-based gly-
cocluster 20 in comparison with the other tetravalent li-
gands.
Surface plasmon resonance (SPR): The SPR experiments
were designed to mimic the inhibition of the lectin adhesion
to a surface cell. A poly[N-(2-hydroxyethyl)acrylamide]
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S. Vidal et al.
polymer (PAA) presenting multiple a-galactose residues is
attached by its biotin substituents to streptavidin-coated
chips. The binding of PA-IL and ECA lectins is measured in
the presence of increasing concentrations of glycoclusters,
providing IC50 values (Table 3). The sensorgrams obtained
monovalent compound is the galactoside 21 displaying IC50
values in the micromolar range for both lectins.
The cyclic peptoid glycocluster 7 displays weaker inhibi-
tion potency towards ECA in comparison to its linear ana-
logue 4. In contrast, no significant differences are observed
for the binding of both compounds to PA-IL. These results
are rather different from the one obtained by HIA experi-
ments.
Table 3. Inhibition of the adhesion of ECA and PA-IL to a surface cov-
ered with galactosides determined by SPR.
The porphyrin glycocluster 20 displays only a modest in-
crease in binding towards ECA relative to the monovalent
reference, whereas PA-IL displays a stronger preference for
this topology. This observation confirms the HIA results,
which indicates that the square-planar topology of this scaf-
fold is very selective for the architecture of PA-IL.
The tetravalent calixarene glycoclusters were previously
assayed[24] with SPR and the results reported here are in
good agreement with the published ones considering varia-
tions in experimental conditions (e.g. equipment used, batch
of lectin). Glycoclusters 23 and 24 display affinities in the
low micromolar range towards PA-IL and the topology of
the macromolecules is influential for the binding to the
lectin, with the highest affinity observed for the 1,3-alternate
conformer 23. The best inhibitor for both lectins is the hexa-
valent calixarene glycocluster 15 displaying the highest rela-
tive potencies in comparison to the monovalent reference
galactoside 21.
[a]
Ligand
Valency
IC50 [mm]
Relative potency[b] [b]
ECA
PA-IL
ECA
PA-IL
21
4
7
20
23
24
15
1
4
4
4
4
4
6
210
63.5
3.5
2.5
1.4
0.9
1.4
0.8
1
18
5
1
18
25
45
70
45
80
11.5
41.6
26.6
8
nd[c]
nd[c]
nd[c]
40
nd[c]
5.2
[a] Minimum concentration of the inhibitor required to prevent 50% of
the adhesion of the lectin onto the galactosylated surface of the SPR
chip. [b] Calculated as the ratio of the monovalent reference galactoside
21 value to the compoundꢀs value. [c] Not determined.
with compound 15 and the inhibition curve are displayed in
Figure 2. Data for all other compounds are provided in the
Supporting Information (Figures S35–S43). The reference
The glycoclusters evaluated here by SPR display IC50
values in the micromolar to submicromolar range for both
lectins (i.e. ECA and PA-IL). When considering the tetrava-
lent glycoclusters, the influence of the topology of the mole-
cules could always be detected and selectivity in binding be-
tween both lectins was also observed.
Isothermal titration microcalorimetry (ITC): Both HIA and
SPR techniques demonstrated that the multivalent glyco-
clusters could result in important affinity improvements for
PA-IL in comparison with ECA. Since PA-IL has been dem-
onstrated to be a therapeutic target against lung infections
by Pseudomonas aeruginosa,[71] an in-depth investigation has
been performed by ITC to further characterize the interac-
tion of PA-IL with the synthesized glycoclusters (Table 4).
The thermogram and titration curve obtained with the hexa-
valent glycocluster 15 are displayed in Figure 3. Data for all
other compounds are provided in the Supporting Informa-
tion (Figures S30–S34).
The observed dissociation constants (Kd) of 70 mm for b-
methyl-galactoside 22 and 150 mm for the compound with
longer aglycon 21 are in the same range than the previously
observed value of 50 mm for a-methyl-galactoside.[72]
The Kd value measured for the peptoid-based tetravalent
glycoclusters 4 is in the low micromolar range. The change
in scaffold from the linear compound 4 to the cyclic glyco-
cluster 7 improves markedly the affinity towards PA-IL,
with a resulting Kd of 296 nm. The cyclic spatial arrangement
provides access to a fourth lectin monomer as depicted by
the binding stoichiometry (n) value, which indicates a
change from a 1:3 to a 1:4 glycocluster/lectin ratio for 4 and
Figure 2. A) Sensorgrams obtained by injection of PA-IL incubated with
concentrations of compound 15 varying from 0 mm (top curve) to 25 mm
(bottom curve) on the PAA-a-d-galactose surface. B) Corresponding in-
hibition curve.
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Selectivity among Two Lectins
FULL PAPER
Table 4. Isothermal titration microcalorimetry (ITC) measurements for the glycoclusters and the monovalent
therefore, to be more sterically
constrained than its cyclic ana-
logue, which can bind a lectin
monomer on each of its four
galactose residues. The effective
“glycoside cluster effect” is also
apparent in the thermodynamic
of binding. Both compounds
display strong enthalpy of bind-
ing, roughly proportional to the
number of possible binding
sites (as determined by stoichi-
ometry n), with strong entropy
barrier.
probes 21 and 22 binding to PA-IL.[a]
Ligand
n[b]
ÀDH8
ÀTDS8
[kJmolÀ1
ÀDG8
[kJmolÀ1
Kd
[mm]
Relative potency[c] [b]
G
]
G
]
G
]
21[d]
22[f]
4
1[e]
36Æ1
39
14
15
54
139
23
65
34
47
22
24
33
37
37
39
37
39
150Æ1
70
1.8Æ0.4
1
2
83
500
454
852
357
1071
0.8[e]
0.33Æ0.02
0.18Æ0.01
0.46Æ0.04
0.24Æ0.03
0.33Æ0.01
0.32Æ0.01
87Æ11
176Æ4
60Æ7
104Æ1
71Æ6
7
20
0.30Æ0.03
0.33Æ0.06
0.176Æ0.006
0.42Æ0.16
0.14Æ0.01
23[d]
24[d]
15
85.9Æ0.6
[a] Experiments have been duplicated and standard deviations are lower than 10% on stoichiometry and ther-
modynamic data and lower than 20% on dissociation constants. [b] Binding stoichiometry defined as the
number of glycoclusters per monomer of PA-IL. [c] Calculated as the ratio of the monovalent reference galac-
toside 21 Kd value to the compoundꢀs Kd value. [d] Data from previous report.[24] [e] Value fixed during the fit-
ting procedure. [f] Only one measurement performed.
The porphyrin-based glyco-
cluster 20 displays a high affini-
ty with a Kd value of 332 nm as-
sociated to a stoichiometry value corresponding to the for-
mation of a 1:2 complex with the lectin. The rigidity and the
planar topology of this scaffold appears to be very favoura-
ble for lowering the entropy cost of binding, but this topolo-
gy does not allow the attachment of one lectin monomer on
each of the four galactose residues.
The hexavalent glycocluster 15 displays a Kd value of
136 nm, which makes it the highest affinity ligand identified
to date for PA-IL. The binding process could be achieved
through the formation of a chelate in which two binding
sites of the lectinꢀs tetramer are occupied by the carbohy-
drate epitopes and an additional “bind and jump” mode[73]
(statistical rebinding) might also be implicated as at least
one of the remaining four epitopes is still bound to a lectin
monomer either from the same initial tetramer or from an-
other lectin tetramer (Scheme 5). This process is a hypothe-
sis based on the average 1:3 stoichiometry (n) of the com-
plex measured by ITC and reflects the possible equilibrium
between the different modes of binding implicating three
Figure 3. Raw ITC data obtained by injections of 15 (120 mm) in a solu-
tion containing PA-IL (50 mm; top). Corresponding integrated titration
curve (bottom).
7, respectively. The stoichiometry of the complex formed be-
tween the tetravalent glycocluster 7 and the lectin could
also be interpreted as a 1:5 complex, but this is rather im-
probable due to the four epitopes present on the glycoclus-
ter. Therefore, the n value of 0.18 measured for 7 is a little
bit lower than the expected value of 0.25 for a typical 1:4
complex but can still be considered as a 1:4 complex due to
structural features of the partners. The linear scaffold seems,
Scheme 5. Schematic representation of the possible binding modes be-
tween the hexavalent glycocluster 15 and PA-IL on the basis of a 1:3 stoi-
chiometric ratio of glycocluster to lectinꢀs monomer as determined by
ITC. A) “Bind and jump” mechanism. B) Dissociation of one chelate and
formation of another one.
Chem. Eur. J. 2011, 17, 2146 – 2159
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2153

S. Vidal et al.
carbohydrate epitopes out of six of glycocluster 15. The en-
thalpic contribution observed for the binding of 15 to PA-IL
corresponds approximately to three monovalent binding
events but the entropic cost is less important in comparison
with other multivalent high affinity glycoclusters (e.g. 7 and
23). The existence of multiple microstates and the “bind and
jump” process[73] illustrated in Scheme 5 rationalize the fact
that the entropy term does not vary proportionally with the
number of binding events.
The data collected by ITC provide an interesting insight
into the biomolecular interactions involved in solution be-
tween synthetic glycoclusters and PA-IL. The geometry of
the macromolecules influences the binding to the lectin not
only in terms of dissociation constant values and thermody-
namic patterns but also in the stoichiometric ratio with the
protein.
scaffolds, linker arms and carbohydrate epitopes. The syn-
thesis of five tetravalent glycoclusters incorporating a linear
and a cyclic peptoid scaffold, calix[4]arenes and a porphyrin
was achieved by 1,3-dipolar cycloaddition between the prop-
argylated cores and an azido-functionalized galactoside.
Similarly, an hexavalent glycocluster was prepared from the
corresponding hexapropargylated calix[6]arene. The detailed
analysis of the binding properties of each multivalent ligand
with two lectins (ECA and PA-IL) was performed by HIA,
SPR and ITC. The influence of the geometry of the central
scaffold could be observed particularly from the linear to
cyclic glycoclusters. The HIA and SPR analyses could illus-
trate the selectivity of each ligand for either ECA or PA-IL
with sometimes large differences in the affinities measured.
This selectivity observed in vitro implies that topology-in-
duced selectivity in natural multivalent binding in vivo
might be of prime importance. The calix[6]arene-based hex-
avalent glycocluster displayed the best binding properties.
These molecules are, therefore, candidates for the design of
anti-infective agents and more particularly for the develop-
ment of antibacterial treatments against infection by Pseu-
domonas aeruginosa.
Comparison of HIA, SPR and ITC results: This work also
illustrates the differences that can be observed when com-
paring binding data obtained from solution experiments
(ITC), surface adhesion (SPR) or aggregation (HIA). Some
of the high affinity compounds in solution (i.e. the peptoid
compounds 4 and 7) remain medium-range inhibitors of sur-
face binding and very poor ones against hemagglutination.
In this case, the conjugated effect of multivalency and high
flexibility can be the cause of this failure. Indeed, multiva-
lent compounds, instead of inhibiting the binding of the
lectin to the surface of chips, have the properties to establish
a complex tridimensional network cross-linking surface–
lectin–ligand–lectin. This would result in recruiting more
lectins to the surface, which is actually the opposite of the
desired effect. Interestingly, not all multivalent molecules
presented this bias, and two compounds, namely 15 and 20,
are excellent inhibitors in all experimental setups. Multiva-
lent interactions can proceed through several mechanisms
(chelate, aggregative, aggregative chelate, statistical rebind-
ing) that are inherently concentration-dependant. Different
experimental techniques measure different processes.[4,74]
HIA, SPR and ITC methodologies intrinsically generate var-
iations in local analyte concentrations thus influencing equi-
libria in which a particular binding mechanism would be fav-
oured compared to others. This clearly illustrates the re-
quirement of multiple synthetic approaches and experimen-
tal analyses of multivalent interactions for the design of
high-affinity multivalent ligands that could efficiently inhibit
lectin binding.
Experimental Section
General methods: All reagents for synthesis were commercial and used
without further purification. Solvents were distilled over CaH2 (CH2Cl2),
Mg/I2 (MeOH), Na/benzophenone (THF) or purchased dry. All reactions
were performed under an argon atmosphere. Reactions under microwave
activation were performed on a Biotage Initiator system. NMR spectra
were recorded at 293 K, unless otherwise stated, by using a 300 or
400 MHz spectrometer. Shifts are referenced relative to deuterated sol-
vent residual peaks. The following abbreviations are used to explain the
observed multiplicities: s, singlet; d, doublet; t, triplet; q, quadruplet; m,
multiplet and brs, broad singlet. Due to the high conformational flexibili-
ty of the peptoid and calixarene backbones, some signals are missing in
13
the 1H and/or C spectra (e.g. carbonyl, C-triazole, CH2-peptoid and N
À
CH2-triazole). This conformational flexibility could also be observed in
proton spectra as some proton signals are broadened. However, correla-
tion traces were observed in 2D experiments (HSQC and HMBC) and
assignments were deduced from these 2D correlation spots. Melting
points are reported uncorrected. IR spectra were recorded by using an
FTIR spectrometer with ATR attachment. High-resolution (LSIMS)
mass spectra were recorded in the positive mode by using a Thermo Fin-
nigan Mat 95 XL spectrometer. ESI mass spectra were recorded in the
positive mode by using a Thermo Finnigan LCQ spectrometer. High-res-
olution (HR-ESI-QTOF) mass spectra were recorded by using a Bruker
MicrOTOF-Q II XL spectrometer. MALDI-ToF mass spectra were re-
corded in positive-ion reflectron mode by using a Voyager DE-STR spec-
trometer (Applied Biosystem) with CHCA (f-cyano-4-hydroxycinnamic
acid, 10 gLÀ1 in MeOH) and NaI (10 g.LÀ1 in acetone) as the matrix.
TLC was carried out on aluminium sheets coated with silica gel 60 F254
(Merck). TLC plates were inspected by UV light (l=254 nm) and devel-
oped by treatment with a mixture of 10% H2SO4 in EtOH/H2O (95:5 v/
v) followed by heating. Silica gel column chromatography was performed
with silica gel Si 60 (40–63 mm). Optical rotation was measured by using
a Perkin–Elmer polarimeter and values are given in 10À1 degcm2 gÀ1. The
following conditions were used for the synthesis and analyses of 2. IR
spectra were recorded on a Shimadzu FTIR-8400S IR spectrophotometer
and n are expressed in cmÀ1. NMR spectra were recorded on a Bruker
AC 400 spectrometer (1H at 400 MHz, 13C at 100 MHz). HRMS were re-
corded in electrospray ionization mode on a micro q-tof Micromass in-
Conclusion
The design of multivalent glycoconjugates as high-affinity li-
gands of lectins is the source of both fundamental and po-
tential applied research particularly for the development of
new anti-infective agents. The better understanding of the
multivalent lectin–carbohydrate interactions is crucial in this
field and requires the design of several synthetic multivalent
molecular architectures incorporating a large set of core
2154
www.chemeurj.org
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 2146 – 2159

Selectivity among Two Lectins
FULL PAPER
strument (3000 V) with an internal lock mass (H3PO4) and an external
lock mass (Leu-enkephaline). Lyophilized ECA was purchased from
Sigma–Aldrich. Recombinant PA-IL was produced in E. coli and ITC
measurements were performed according to our previously reported pro-
tocols.[24]
CH3CO, CH3); HR-LSIMS-MS: m/z: calcd for C110H165N16O54: 2574.0657
[M+H]+; found: 2574.0697.
(4,8,12,16-Tetra-aza)(4,8,12,16-tetra-N-{1-[(b-d-galactopyranosyloxy)-3,6-
dioxaoct-8-yl]-1,2,3-triazol-4-yl}-4-methyl)(5,9,13,17-tetra-oxo)octadeca-
noic acid (4): Obtained as a freeze-dried white solid (109 mg, 77%) by
following
a variation of method B: Compound 3 (198 mg, 77 mmol,
General procedure for 1,3-dipolar cycloadditions (method A): The
alkyne-functionalized scaffold, copper iodide, iPr2Net and azido-function-
alized galactoside A in degassed DMF were introduced in a Biotage Ini-
tiator 2–5 mL vial. The vial was flushed with argon and protected from
light (aluminium sheet) and the solution was sonicated for 30 s. The vial
was sealed with a septum cap and heated at 1108C under microwave irra-
diation (solvent absorption level: high). After uncapping the vial, the
crude mixture was diluted with EtOAc (200 mL). The organic layer was
washed with HCl (1n, 2ꢅ50 mL), water (4ꢅ50 mL) and brine (50 mL)
unless otherwise stated. The organic layer was dried (Na2SO4), filtered
and evaporated. The crude product was purified by flash silica gel
column chromatography to afford the desired acetylated glycoclusters.
1 equiv) was suspended in THF (5 mL) and aqueous NaOH (4m, 5 mL).
After stirring at RT for 16 h, the reaction mixture was neutralized by
using Amberlite IR-120 H+ resin until pH 5 was obtained. After filtra-
tion, washing (MeOH) and concentration, the product was dissolved in
water (5 mL) and then freeze-dried to afford the pure deacetylated glyco-
cluster 4. [a]D =+3.1 (c=0.45 in H2O); 1H NMR (300 MHz, CD3OD):
d=8.19–7.86 (m, 4H; H-triaz), 4.78–4.50 (m, 16H; NCH2C-triaz,
OCH2CH2N-triaz), 4.29 (d, J=6.3 Hz, 4H; H-1), 4.05–3.81 (m, 16H; H-4,
1/2GalOCH2CH2O, OCH2CH2N-triaz), 3.79–3.69 (m, 16H; H-6a, H-6b,
1/2GalOCH2CH2O, 2 ꢅ CH2-peptoid), 3.61 (brs, 28H; 3ꢅCH2-EG3, 2ꢅ
CH2-peptoꢇd), 3.57–3.42 (m, 12H; H-2, H-3, H-5), 3.04–2.47 (m, 8H; 4ꢅ
CH2-peptoꢇd), 2.29–2.14 ppm (m, 3H; CH3); 13C NMR (75 MHz,
CD3OD): d=173.8, 173.5 (2 s, 4C; O=C-peptoid), 144.8 (CIV-triaz), 125.5
(CH-triaz), 104.9 (C-1), 76.6, 74.8, 72.5 (C-2, C-3, C-5), 71.3 (3ꢅCH2-
EG3), 70.3 (1 s; C-4, OCH2CH2N-triaz), 69.6 (GalOCH2-), 62.5 (C-6),
51.4 (OCH2CH2N-triaz), 21.2, 21.7 ppm (CH3, 2conformers); HR-ESI-
QTOF MS (positive mode) m/z: calcd for C74H124N16NaO38: 1867.8155
General procedure for deacetylation of glycoclusters (method B): The
acetylated glycocluster was suspended in distilled MeOH, ultra-pure
water and ultra-pure triethylamine (5:1:1, v/v/v) unless otherwise stated.
The mixture was stirred under argon at room temperature for 3 to 4 days.
Solvents were evaporated, co-evaporated with toluene and the residue
was dissolved in ultra-pure water (5 mL) and then freeze-dried to afford
the pure glycocluster without further purification.
[M+Na]+; found: 1867.8181; calcd for C74H124N16Na2O38
[M+2Na]2+; found: 945.4056.
: 945.4023
tert-Butyl
(4,8,12,16-tetra-aza)(5,9,13,17-tetra-oxo)(4,8,12,16-tetra-N-
(4,8,12,16-Tetra-N-{1-[(2,3,4,6-tetra-O-acetyl-b-d-galactopyranosyloxy)-
3,6-dioxaoct-8-yl]-1,2,3-triazol-4-yl}-4-methyl)(4,8,12,16-tetra-
propargyl)octadecanoate (2): Acetic anhydride (717 mL, 7.63 mmol,
10 equiv) was added to a solution of amine 1 (390 mg, 0.76 mmol) in
CH2Cl2 (25 mL). The mixture was stirred at RT over 5 h and then concen-
trated under reduced pressure. The crude mixture was diluted with
AcOEt (20 mL) and then washed with a saturated solution of NaHCO3
aza)(1,5,9,13-tetra-oxo)cyclohexadecane (6): Obtained as a white foam
(166 mg, 77%) by following method A: Compound 5 (38 mg, 87 mmol,
1 equiv), A (264 mg, 0.522 mmol, 6 equiv), CuI (8 mg, 0.5 equiv) and
iPr2NEt (56 mL, 5 equiv) in DMF (3 mL). Microwave irradiation: 30 min
at 1108C. After concentration of the crude mixture and three co-evapora-
tions with toluene, the residue was purified by Al2O3 flash chromatogra-
phy (EtOAc then EtOAc/MeOH 85:15). Rf =0.15 (EtOAc/MeOH 4:1);
[a]D =À3.5 (c=0.92 in CH2Cl2); 1H NMR (300 MHz, CDCl3): d=7.72–
7.55 (m, 4H; H-triaz), 5.34 (d, J=3.1 Hz, 4H; H-4), 5.19–5.10 (m, 4H;
H-2), 4.99 (dd, J=10.4, 3.1 Hz, 4H; H-3), 4.63–4.41 (m, 20H; H-1,
OCH2CH2N-triaz, NCH2C-triaz), 4.18–4.06 (m, 8H; H-6a, H-6b), 3.98–
3.88 (m, 8H; H-5, 1/2GalOCH2CH2O), 3.87–3.79 (m, 8H; OCH2CH2N-
triaz), 3.78–3.62 (m, 12H; 1/2GalOCH2CH2O, NC(O)CH2CH2N), 3.60–
3.52 (m, 24H; 3ꢅCH2-EG3), 2.88–2.57 (m, 8H; NC(O)CH2CH2N), 2.10,
2.00, 1.94 ppm (3 s, 4ꢅ12H; CH3CO); 13C NMR (100 MHz, CDCl3): d=
172.0, 171.1, 170.5, 170.4, 170.3, 170.2, 170.1, 169.5 (8 s, 8C; C=O), 144.2,
144.1, 143.9, 143.7 (4 s, 4C; CIV-triaz), 124.4, 124.2, 123.6, 122.7 (4 s, 4C;
CH-triaz), 101.4 (C-1), 70.9 (C-3), 70.7 (C-5), 70.6, 70.5, 70.2 (3ꢅCH2-
EG3), 69.5 (OCH2CH2N-triaz), 69.2 (GalOCH2-), 68.9 (C-2), 67.1 (C-4),
61.3 (C-6), 50.2 (OCH2CH2N-triaz), 45.4, 45.3, 44.9, 44.5, 42.5, 41.3, 40.5
(7 s, 8C; NCH2C-triaz, NC(O)CH2CH2N), 32.4, 31.8 (2 s, 4C;
NC(O)CH2CH2N), 28.2 ((CH3)3C-), 20.8, 20.7, 20.7 ppm (3 s, 4ꢅCH3CO;
CH3); HR-MALDI-TOF MS (positive-ion reflectron mode): m/z: calcd
for C104H152N16NaO52: 2479.9636; found: 2479.9638.
(2ꢅ10 mL),
a solution of KHSO4 (5%; 2ꢅ10 mL) and then brine
(10 mL). The organic layer was dried (MgSO4), filtered and then concen-
trated under reduce pressure. Purification by flash chromatography
(AcOEt/MeOH 95:5) provided compound 2 as a pale-yellow oil (408 mg,
97%). Rf =0.25 (EtOAc); 1H NMR (400 MHz, CDCl3): d=4.11–4.23 (m,
8H), 3.63–3.84 (m, 8H), 2.75–2.90 (m, 6H), 2.52–2.60 (m, 2H), 2.27–2.34
(m, 2H), 2.22 (m, 2H), 2.19 (s, 3H), 2.16, 2.15 1.44 ppm (s, 9H);
13C NMR (100 MHz, CDCl3): d=173.7 (C), 171.4 (4C), 170.9, 170.8,
170.3, 170.1, 170.0, 169.7, 81.4 (C), 81.2, 80.7, 79.1 (4C), 78.6, 78.4, 73.0
(4CH), 72.8, 72.5, 72.0, 71.8, 44.2 (4ꢅCH2), 44.0, 43.7, 43.5, 43.4, 43.3,
43.2, 43.1, 42.8, 42.6, 39.7 (2ꢅCH2), 39.6, 38.8, 38.7, 38.6, 38.3, 38.2, 34.6
(3ꢅCH2), 34.3, 34.1, 33.9, 33.8, 31.9 (3ꢅCH2), 31.8, 31.7, 31.6, 31.5, 31.4,
27.9 (3CH3), 21.2 (CH3), 20.6 ppm; IR (neat): nꢆ=>=1153, 1367, 1417,
1641, 1722, 2120, 2935, 2976, 3246, 3290 cmÀ1; HR-ESI-MS: m/z: calcd
for C30H41N4O6: 553.3026 [M+H]+; found: 553.3034.
tert-Butyl (4,8,12,16-tetra-N-{1-[(2,3,4,6-tetra-O-acetyl-b-d-galactopyra-
nosyloxy)-3,6-dioxaoct-8-yl]-1,2,3-triazol-4-yl}-4-methyl)(4,8,12,16-tetra-
aza)(5,9,13,17-tetra-oxo)octadecanoate (3): Obtained as a colourless gum
(464 mg, 86%) by following method A: 2 (116 mg, 0.21 mmol, 1 equiv),
A (586 mg, 1.16 mmol, 5.5 equiv), CuI (20 mg, 0.11 mmol, 0.5 equiv) and
iPr2NEt (183 mL, 1.05 mmol, 5 equiv) in DMF (3 mL). Microwave irradia-
tion: 15 min at 1108C. After dilution of the crude mixture in EtOAc
(300 mL), the organic layer was washed by NH4Cl (2ꢅ150 mL) and
water (2ꢅ150 mL). The residue was purified by Al2O3 gel flash chroma-
tography (EtOAc then EtOAc/MeOH 4:1). Rf =0.53 (EtOAc/MeOH
95:5); [a]D =À4.7 (c=1.10 in CH2Cl2); 1H NMR (300 MHz, CDCl3): d=
7.85–7.61 (m, 4H; H-triaz), 5.37 (dd, J=3.2, <1 Hz, 4H; H-4), 5.18 (dd,
J=10.4, 8.0 Hz, 4H; H-2), 5.02 (dd, J=10.4, 3.2 Hz, 4H; H-3), 4.66–4.42
(m, 20H; H-1, NCH2C-triaz, OCH2CH2N-triaz), 4.21–4.05 (m, 8H; H-6a,
H-6b), 4.00–3.79 (m, 16H; H-5, 1/2GalOCH2CH2O, 1 ꢅ CH2-EG3), 3.76–
3.66 (m, 8H; 1/2GalOCH2CH2O, 2 ꢅ CH2-peptoid), 3.66–3.52 (m, 28H;
3ꢅCH2-EG3, 2ꢅCH2-peptoid), 2.97–2.42 (m, 8H; 4ꢅCH2-peptoid), 2.13,
2.03, 1.97 (3 s, 4ꢅ12H; CH3CO), 1.84 (s, 3H; CH3), 1.41 ppm (brs, 9H;
tBu); 13C NMR (75 MHz, CDCl3): d=170.5, 170.4, 170.3, 169.6 (4 s; 4ꢅ
CH3CO), 101.5 (C-1), 71.0 (C-3), 70.8 (C-5), 70.8, 70.7, 70.3, 69.5 (4 s;
CH2OCH2CH2OCH2), 69.3 (GalOCH2), 68.9 (C-2), 67.2 (C-4), 61.4 (C-6),
50.4 (OCH2CH2N-triaz), 28.2 (CMe3), 20.9, 20.8, 20.7 ppm (3 s; 4ꢅ
(4,8,12,16-Tetra-aza)(4,8,12,16-tetra-N-{1-[(b-d-galactopyranosyloxy)-3,6-
dioxaoct-8-yl]-1,2,3-triazol-4-yl}-4-methyl)(1,5,9,13-tetra-oxo)cyclohexa-
decane (7): Obtained as a freeze-dried white solid (97 mg, 87%) by fol-
lowing method B: Compound 6 (160 mg, 62 mmol, 1 equiv) was suspend-
ed in methanol (2 mL), water (0.5 mL) and triethylamine (0.5 mL). After
stirring at RT for 4 days and concentration, the mixture was co-evaporat-
ed with toluene three times, dissolved in ultra-pure water (4 mL) and
freeze-dried to afford the pure deacetylated glycocluster. [a]D =+ 0.6
(c=0.95 in H2O); 1H NMR (400 MHz, DMSO+traces CD3OD): d=
8.11–7.83 (m, 4H; H-triaz), 4.54–4.41 (m, 16H; NCH2C-triaz,
OCH2CH2N-triaz), 4.13–4.04 (m, 4H; H-1), 3.86–3.72 (m, 12H; 1/2Ga-
lOCH2CH2O, OCH2CH2N-triaz), 3.62–3.58 (m, 4H; H-4), 3.57–3.40 (m,
36H; 1/2GalOCH2CH2O, H-6a, H-6b, 3ꢅCH2-EG3), 3.35–3.28 (m, 4H;
H-5), 3.28–3.21 ppm (m, 8H; H-2, H-3); 13C NMR (100 MHz, DMSO+
traces CD3OD): d=173.3, 170.4 (2 s, 4C; O=C-peptoid), 143.9 (CIV-triaz),
124.1 (CH-triaz), 103.7 (C-1), 75.4 (C-5), 73.5, 70.7 (2 s, 8C; C-2, C-3),
70.0, 69.9, 69.8 (3ꢅCH2-EG3), 68.9 (OCH2CH2N-triaz), 68.4 (C-4), 68.1
Chem. Eur. J. 2011, 17, 2146 – 2159
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2155

S. Vidal et al.
(GalOCH2-), 60.7 (C-6), 49.7 (OCH2CH2N-triaz), 39.5 ppm* (NCH2C-
1719, 1478, 1362, 1188, 1113, 1004, 875 cmÀ1
;
1H NMR (300 MHz,
triaz; signal partially overlapped by residual CHCl3 peak); HR-MALDI-
CDCl3): d=7.15 (s, 12H; H-ar), 3.87–4.15 (m, 24H; ArCH2 OCH2), 1.28
(s, 6H; CꢁCH), 0.81 ppm (s, 54H; CMe3); 13C NMR (75 MHz, CDCl3):
d=153.3, 146.4, 133.4, 126.6 (ArC), 79.7 (CꢁCH), 75.3 (OCH2), 61.1
(CꢁCH), 34.5 (ArCH2), 31.6 ppm (CMe3); HR-ESI-QTOF MS: m/z:
calcd for C84H96NaO6: 1223.7105 [M+Na]+; found: 1223.7045.
TOF MS (positive-ion reflectron mode): m/z: calcd for C72H120N16NaO36
1807.7947 [M+Na]+; found: 1807.8008.
:
5,11,17,23,29,35-Hexa-tert-butyl-38,40,42-tri-({1-[(2,3,4,6-tetra-O-acetyl-b-
d-galactopyranosyloxy)-3,6-dioxaoct-8-yl]-1,2,3-triazol-4-yl}-4-methyle-
neoxy)-37,39,41-trimethoxy-calix[6]arene (11): Obtained as a pale-yellow
foam (79 mg, 65%) by following method A: Compound 10[58] (52 mg,
5,11,17,23,29,35-Hexa-tert-butyl-37,38,39,40,41,42-hexa-({1-[(2,3,4,6-tetra-
O-acetyl-b-d-galactopyranosyloxy)-3,6-dioxaoct-8-yl]-1,2,3-triazol-4-yl}-4-
methyleneoxy)calix[6]arene (14): Obtained as
a pale-yellow foam
46 mmol, 1 equiv),
A (105 mg, 0.210 mmol, 4.5 equiv), CuI (4.4 mg,
(153 mg, 84%) by following method A: Compound 13 (52 mg, 43 mmol,
1 equiv), A (196 mg, 0.390 mmol, 9 equiv), CuI (4 mg, 22 mmol, 0.5 equiv)
and iPr2NEt (40 mL, 0.21 mmol, 5 equiv) in DMF (3 mL). Microwave ir-
radiation: 15 min at 1108C. After workup, the residue was purified by
silica gel flash chromatography (EtOAc then EtOAc/MeOH 9:1). Rf =
0.64 (EtOAc/MeOH 9:1); [a]D =À4.0 (c=0.60 in CH2Cl2); 1H NMR
(400 MHz, CDCl3): d=8.10–6.80 (m; H-ar, H-triaz), 5.37 (d, J=2.5 Hz,
6H; H-4), 5.22–5.13 (m, 6H; H-2), 5.01 (dd, J=10.5, 3.4 Hz, 6H; H-3),
4.62–4.45 (m, 18H; H-1, OCH2CH2N), 4.19–4.07 (m, 12H; H-6a, H-6b),
3.98–3.87 (m, 16H; H-5, 1ꢅCH2-EG3), 3.86–3.76 (m, 6H; 1/2Ga-
lOCH2CH2O), 3.73–3.64 (m, 6H; 1/2GalOCH2CH2O), 3.55 (brs, 36H;
3ꢅCH2-EG3), 2.13, 2.04, 2.03, 1.97 (4 s, 4ꢅ18H; CH3CO), 1.30–0.80 ppm
(m; 6ꢅCMe3); 13C NMR (100 MHz, CDCl3): d=170.5, 170.4, 170.3, 169.6
(4 s; 4ꢅ6C, 4ꢅCH3CO), 101.5 (C-1), 71.1 (C-3), 70.7 (C-5), 70.68, 70.62,
70.3 (3 s; 3ꢅ6C, 3ꢅCH2-EG3), 69.6, 69.2 (2 s; 2ꢅ6C, GalOCH2CH2O, 1 ꢅ
CH2-EG3), 68.9 (C-2), 67.2 (C-4), 61.3 (C-6), 50.2 (OCH2CH2N),
31.6 ppm (brs, 18C; 6ꢅCMe3); HR-ESI-QTOF MS (positive mode): m/z:
calcd for C204H282N18Na2O78: 2138.9219 [M+Na]2+; found: 2138.9302.
23 mmol, 0.5 equiv) and iPr2NEt (40 mL, 0.23 mmol, 5 equiv) in DMF
(3 mL). Microwave irradiation: 15 min at 1108C. After workup, the resi-
due was purified by silica gel flash chromatography (EtOAc then EtOAc/
MeOH 9:1). Rf =0.73 (EtOAc/MeOH 9:1); [a]D =À4.6 (c=0.57 in
CH2Cl2); 1H NMR (300 MHz, CDCl3): d=7.90 (s, 3H; H-triaz), 7.26 (s,
6H; H-ar), 6.64 (s, 6H; H-ar), 5.35 (d, J=3.3 Hz, 3H; H-4), 5.17 (dd, J=
10.4, 7.9 Hz, 3H; H-2), 5.08 (s, 6H; OCH2C-triaz), 4.99 (dd, J=10.4,
3.3 Hz, 3H; H-3), 4.61–4.47 (m, 15H; H-1, Ar-CH2-Ar, OCH2CH2N-
triaz), 4.18–4.02 (m, 6H; H-6a, H-6b), 3.90 (m, 12H; 1/2GalOCH2CH2O,
H-5, OCH2CH2N-triaz), 3.72–3.63 (m, 3H; 1/2GalOCH2CH2O), 3.60–
3.46 (m, 18H; 3ꢅCH2-EG3), 3.34 (d, J=15.1 Hz, 6H; Ar-CH2-Ar), 2.15
(s, 9H; OCH3), 2.12, 2.01, 2.00, 1.95 (4 s, 4ꢅ9H; 4ꢅCH3CO), 1.36 (s,
27H; 3ꢅCMe3), 0.78 ppm (s, 27H; 3ꢅCMe3); 13C NMR (75 MHz,
CDCl3): d=170.5, 170.3, 170.2, 169.5 (4 s; CH3CO), 154.4 (CIV-ar), 151.6
(CIV-ar), 146.2 (CIV-C(CH3)3), 145.0 (CIV-C(CH3)3), 144.7 (CIV-triaz), 133.7
ACHTUNGTRENNUNG ACHTUNGTRENNUGN
(CIV-OMe), 133.0 (CIV-OMe), 128.2 (CH-ar), 124.1 (CH-triaz), 123.7
(CH-ar), 101.4 (C-1), 71.0 (C-3), 70.70 (C-5), 70.68 (2ꢅCH2-EG3), 70.3
(1ꢅCH2-EG3), 69.6 (GalOCH2-), 69.2 (OCH2CH2N-triaz), 68.9 (C-2),
67.2 (C-4), 66.5 (OCH2C-triaz), 61.3 (C-6), 60.2 (OCH3), 50.3
(OCH2CH2N-triaz), 34.3 (CMe3), 34.0 (CMe3), 31.7 (CMe3), 31.2 (CMe3),
28.8 (Ar-CH2-Ar), 20.9, 20.8, 20.7 ppm (3 s, 12C; 12ꢅCH3CO); HR-
MALDI-TOF MS (positive-ion reflectron mode): m/z: calcd for
5,11,17,23,29,35-Hexa-tert-butyl-37,38,39,40,41,42-hexa({1-[(b-d-galacto-
pyranosyloxy)-3,6-dioxaoct-8-yl]-1,2,3-triazol-4-yl}-4-methyleneoxy)ca-
lix[6]arene (15): Obtained as a freeze-dried pale-yellow solid (129 mg,
79%) by following method B: Compound 14 (215 mg, 51 mmol) was sus-
pended in methanol (15 mL), water (3 mL) and triethylamine (3 mL).
After stirring at RT for 4 days and concentration, the mixture was dis-
solved in ultra-pure water (5 mL) and then freeze-dried to afford the
pure deacetylated glycocluster. [a]D =+14.6 (c=0.35 in H2O); 1H NMR
(400 MHz, DMSO+eD2O): d=8.40–7.10 (m; H-ar, H-triaz), 5.00–4.70
(m, 12H; OCH2C-triaz), 4.60–4.40 (m, 12H; OCH2CH2N-triaz), 4.07 (m,
6H; H-1), 3.90–3.70 (m, 18H; OCH2CH2N, 1/2GalOCH2CH2O), 3.60–
3.40 (m, 70H; H-4, H-6a, H-6b, 1/2GalOCH2CH2O, 3 ꢅ CH2-EG3), 3.30–
3.20 (m, 18H; H-2, H-3, H-5), 2.60–2.40 (m; Ar-CH2-Ar), 1.20–0.60 ppm
(m; 6ꢅCMe3); 13C NMR (100 MHz, DMSO+eD2O): d=103.7 (C-1),
75.3, 73.5, 70.6 (C-2, C-3, C-5), 70.0, 69.9, 69.8 (3 s; 3ꢅ6C, 3ꢅCH2-EG3),
69.1 (OCH2CH2N), 68.2 (C-4), 68.0 (GalOCH2CH2O), 60.6 (C-6), 49.6
C
138H189N9NaO42: 2667.2825 [M+Na]+; found: 2667.2908.
5,11,17,23,29,35-Hexa-tert-butyl-38,40,42-tri({1-[(b-d-galactopyranosy-
loxy)-3,6-dioxaoct-8-yl]-1,2,3-triazol-4-yl}-4-methyleneoxy)-37,39,41-tri-
methoxy-calix[6]arene (12): Obtained as
a freeze-dried white solid
(55 mg, 86%) by following method B: Compound 11 (79 mg, 30 mmol)
was suspended in methanol (15 mL), water (3 mL) and triethylamine
(3 mL). After stirring at RT for 3 days and concentration, the mixture
was dissolved in ultra-pure water (4 mL) and then freeze-dried to afford
the pure deacetylated glycocluster. [a]D =À2.8 (c=0.32 in DMSO);
1H NMR (300 MHz, CD3OD): d=8.29 (s, 3H; H-triaz), 7.32 (s, 6H; H-
ar), 6.72 (s, 6H; H-ar), 5.11 (s, 6H; OCH2C-triaz), 4.70–4.61 (m, 6H;
OCH2CH2N-triaz), 4.53 (d, J=15.7 Hz, 6H; Ar-CH2-Ar), 4.23 (d, J=
7.4 Hz, 3H; H-1), 4.01–3.87 (m, 6H; OCH2CH2N-triaz), 3.81 (d, J=
2.4 Hz, 3H; H-4), 3.78–3.62 (m, 9H; H-6a, H-6b, GalOCH2-), 3.62–3.38
(m, 27H; H-2, H-3, H-5, 3ꢅCH2-EG3), 3.28–3.22 (m, 6H; Ar-CH2-Ar),
2.19 (s, 9H; OCH3), 1.40 (s, 27H; 3ꢅCMe3), 0.80 ppm (s, 27H; 3ꢅ
CMe3); 13C NMR (75 MHz, CD3OD): d=155.5 (CIV-ar), 152.7 (CIV-ar),
147.2 (CIV-ar), 147.2 (CIV-ar), 145.4 (CIV-triaz), 135.1 (CIV-OMe), 134.4
(CIV-OMe), 129.4 (CH-ar), 126.9 (CH-triaz), 124.9 (CH-ar), 105.1 (C-1),
76.6, 74.9, 72.5 (C-2, C-3, C-5), 71.5, 71.4 (2 s; 6C, 2ꢅCH2-EG3), 70.5
(OCH2CH2N-triaz), 70.3 (C-4), 69.6 (1ꢅCH2-EG3), 68.8 (GalOCH2-),
66.9 (OCH2C-triaz), 62.5 (C-6), 61.3 (OCH3), 51.5 (OCH2CH2N-triaz),
35.2 (CMe3), 35.1 (CMe3), 32.1 (CMe3), 31.9 (CMe3), 30.7 ppm (Ar-CH2-
Ar); HR-ESI-QTOF MS (positive mode): m/z: calcd for
C114H165N9NaO30: 2163.1560 [M+Na]+; found: 2163.1568.
(OCH2CH2N), 46.0 (Ar-CH2-Ar), 31.6 (brs; 18C, 6ꢅC
MALDI-TOF MS (positive-ion reflectron mode): m/z: calcd for
156H235N18O54: 3224.6191 [M+H]+; found: 3224.6185.
ACHTUGNERTN(NUNG CH3)3); HR-
C
5,10,15,20-Tetra(4-propargyloxy-phenyl) porphyrin (17):[66,67] Propionic
acid (100 mL) was added to a 500 mL round-bottomed flask flushed with
argon. The mixture was heated at 1208C upon vigorous stirring and a
mixture of para-propargyl-benzaldehyde (16)[68] (3.6 g, 22.5 mmol,
1 equiv) and pyrrole (1.6 mL, 22.5 mL, 1 equiv in 5 mL of propionic acid)
was added dropwise. After 1 h, the mixture was cooled to RT (2 h). The
crude product was precipitated by cooling the mixture with an ice-bath
and adding methanol (250 mL). Filtration afforded a purple gum that
was dissolved in dichloromethane. After evaporation and re-dissolution
in a minimal quantity of chloroform, dropwise incorporation of methanol
yielded the pure porphyrin 17 (1.09 g, 23%) as a deep-purple shiny solid.
Rf =0.49 (PE/CH2Cl2 1:1); 1H NMR (300 MHz, CDCl3): d=8.87 (s, 8H;
H-porph), 8.14 (d, J=8.4 Hz, 8H; H-ar), 7.36 (d, J=8.4 Hz, 8H; H-ar),
4.98 (d, J=1.9 Hz, 8H; OCH2CꢁCH), 2.70 (t, J=1.9 Hz, 4H; CꢁCH),
À2.76 ppm (s, 2H; NH).
5,11,17,23,29,35-Hexa-tert-butyl-37,38,39,40,41,42-hexapropargyloxy-cal-
ix[6]arene (13): Calix[6]arene 8[56] (1.77 g, 1.90 mmol) was added to a so-
lution of NaH (60 wt.% in oil, 1.307 g, 32.7 mmol, 17 equiv) in anhydrous
THF (50 mL). The reaction mixture was refluxed for 30 min and then
propargyl bromide (3.49 mL, 30.1 mmol, 16 equiv) was added dropwise.
After 15 h of refluxing, the reaction mixture was concentrated to 1/3 of
the initial volume and HCl (1m, 50 mL) was carefully added at 08C. The
organic layer was then extracted with chloroform (3ꢅ30 mL), washed
with saturated NH4Cl (2ꢅ30 mL), dried (Na2SO4), filtered and concen-
trated under reduce pressure. The crude residue was triturated in diethyl-
ether and then filtered, giving pure compound 13 (401 mg, 18%) as a
slightly pale-yellow solid. M.p. 2208C dec; IR (KBr): n˜ =3288, 2953,
5,10,15,20-Tetra(4-propargyloxy-phenyl) porphyrin·Zn (18):[66,67] Porphy-
rin (17) (500 mg, 0.60 mmol, 1 equiv) and ZnCl2 (410 mg, 3 mmol,
5 equiv) were introduced into a Biotage Initiator 2–5 mL vial. The vial
was flushed with argon and protected from light (aluminium sheet). An-
hydrous and degassed DMF (4.5 mL) and then Et3N (585 mL, 4.2 mmol,
7 equiv) were added. The vial was sealed with a septum cap and heated
at 1208C for 15 min under microwave irradiation (solvent absorption
2156
www.chemeurj.org
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 2146 – 2159

Selectivity among Two Lectins
FULL PAPER
level: high). After uncapping the vial, the crude mixture was diluted with
EtOAc (250 mL). The organic layer was washed with water (3ꢅ100 mL)
and brine (100 mL). The organic layer was dried (Na2SO4), filtered and
evaporated. The crude product was crystallized (CHCl3/MeOH) to afford
the pure zinc–porphyrin 18 as shiny purple crystals (434 mg, 87%). Rf =
0.20 (PE/CH2Cl2 1:1); 1H NMR (300 MHz, CDCl3): d=8.97 (s, 8H; H-
porph), 8.14 (d, J=8.4 Hz, 8H; H-ar), 7.34 (d, J=8.4 Hz, 8H; H-ar), 4.97
(d, J=1.9 Hz, 8H; OCH2CꢁCH), 2.69 ppm (t, J=1.9 Hz, 4H, CꢁCH).
ried out by the addition of 12.5 mL lectin solution (at the required con-
centration) to 25 mL of sequential dilutions of glycoclusters, monomer
molecules and controls. These solutions were then incubated at 258C for
2 h and then 12.5 mL of 4% erythrocyte solution was added followed by
an additional incubation at 258C for 30 min. The minimum inhibitory
concentration for each molecule was determined by simple eye detection.
Surface plasmon resonance studies (SPR): SPR inhibition experiments
were performed on a Biacore 3000 instrument at 258C. Measurements
were carried out on 2 channels with 2 immobilised sugars: a-l-fucose
(channel 1), a-d-galactose (channel 2). Immobilization of sugars was per-
formed at 258C by using running buffer (HBS) at 5 mLminÀ1. Immobili-
zation on each channel (CM5 Chip) was performed independently as fol-
lows. First, channel was activated by injecting a fresh mixture of EDC/
NHS (35 mL, 420 s). Then, a solution of strepatavidin (100 mg/mL in Na
acetate pH 5 buffer) was injected (50 mL, 600 s). Remaining reactive spe-
cies were quenched by injecting ethanolamine (1m, 35 mL, 420 s). Finally
a solution of the desired biotinylated–polyacrylamide–sugar (Lectinity,
200 mg/mL) was coated onto the surface (50 mL, 600 s) through streptavi-
din–biotin interaction. This procedure led to 804 RU (fucoside) and 796
RU (galactoside) of immobilized sugars on channel 1 and 2, respectively.
Inhibition experiments were performed with the galactosylated channel 2
and plots represent substracted data (channel 2–channel 1).
5,10,15,20-Tetra{1-[(2,3,4,6-tetra-O-acetyl-b-d-galactopyranosyloxy)-3,6-
dioxaoct-8-yl]-1,2,3-triazol-4-yl}-4-methyleneoxy-phenyl
porphyrin·Zn
(19): Obtained as a purple gum (104 mg, 64%) by following method A:
Compound 18 (50 mg, 56 mmol, 1 equiv), A (169 mg, 0.34 mmol, 6 equiv),
CuI (5.3 mg, 28 mmol, 0.5 equiv) and iPr2NEt (49 mL, 0.28 mmol, 5 equiv)
in DMF (2.5 mL). After workup (without the washing with acidic solu-
tion), the residue was purified by silica gel flash chromatography (EtOAc
then EtOAc/MeOH 95:5). Rf =0.30 (EtOAc/MeOH 9:1); 1H NMR
(300 MHz, CDCl3): d=8.92 (s, 8H; H-porph), 8.11 (d, J=8.5 Hz, 8H; H-
ar), 7.72 (s, 4H; H-triaz), 7.25* (d, J=8.5 Hz, 8H; H-ar; signal partially
overlapped by residual CHCl3 peak), 5.34 (dd, J=3.3, <1 Hz, 4H; H-4),
5.18 (dd, J=10.5, 7.9 Hz, 4H; H-2), 4.97 (dd, J=10.5, 3.3 Hz, 4H; H-3),
4.86 (brs, 8H; PhOCH2C-triaz), 4.50 (d, J=7.9 Hz, 4H; H-1), 4.41 (t, J=
4.9 Hz, 8H; OCH2CH2N-triaz), 4.15–4.02 (m, 8H; H-6a, H-6b), 3.98–3.89
(m, 4H; 1/2GalOCH2CH2O), 3.88–3.83 (m, 4H; H-5), 3.79 (t, J=4.9 Hz,
8H; OCH2CH2N-triaz), 3.71–3.65 (m, 4H; 1/2GalOCH2CH2O), 3.64–3.51
(m, 24H; GalOCH2CH2OCH2CH2O), 2.10, 2.00, 1.95, 1.94 ppm (4 s, 4ꢅ
12H; CH3CO); 13C NMR (75 MHz, CDCl3): d=170.5, 170.3, 170.2, 169.6
(4 s; CH3CO), 157.9 (CIV-ar), 150.5 (CIV-porph), 143.7 (CIV-triaz), 136.4
(CIV-ar), 135.8 (CH-ar), 131.8 (CH-porph), 123.8 (CH-triaz), 120.4 (Ph-
CIV-porph), 112.9 (CH-ar), 101.4 (C-1), 71.0 (C-3), 70.8 (C-5), 70.8, 70.74,
70.68 (3 s; 12C; GalOCH2CH2OCH2CH2O), 69.4 (OCH2CH2N-triaz),
69.3 (GalOCH2-), 68.9 (C-2), 67.1 (C-4), 62.0 (PhOCH2C-triaz), 61.3 (C-
6), 50.4 (OCH2CH2N-triaz), 20.9, 20.8, 20.74, 20.70 (4 s; CH3CO);
MALDI-TOF MS (positive-ion reflectron mode): m/z: calcd for
Conditions for ECA: The running buffer for ECA experiments is
HEPES (10 mm), CaCl2 (2 mm), MnCl2 (2 mm), Tween P20 0.005%,
pH 7.4. Inhibition studies consisted in the injection (50 mL, 10 mLminÀ1
,
dissociation: 60 s) of incubated (>1 h, RT) mixtures of ECA (0.5 mm)
and various concentrations of inhibitor (2-fold cascade dilutions). For
each inhibition assay, ECA (0.5 mm) without inhibitor was injected to ob-
serve the full adhesion of the lectin onto the sugar-coated surface (0%
inhibition). The CM5 chip was fully regenerated by successive injections
of d-galactose (10 mL, 100 mm in running buffer) and NaCl (2ꢅ10 mL, 1m
in running buffer).
136H160N16O52Zn: 2912.97 [M]+; found 2912.92.
5,10,15,20-Tetra{1-[(b-d-galactopyranosyloxy)-3,6-dioxaoct-8-yl]-1,2,3-tri-
Conditions for PA-IL: The running buffer for PA-IL experiments is
HEPES (10 mm), NaCl (150 mm), CaCl2 (10 mm), Tween P20 0.005%,
C
pH 7.4. Inhibition studies consisted in the injection (150 mL, 10 mLminÀ1
,
azol-4-yl}-4-methyleneoxy-phenyl porphyrin·Zn (20): Obtained as
freeze-dried purple foam (66 mg, 99%) by following method B: Com-
pound 19 (86 mg, 29 Mmol) was suspended in methanol (5 mL), dichloro-
methane (1 mL), water (1 mL) and triethylamine (1 mL). After stirring
at RT for 4 days and concentration, the mixture was dissolved in ultra-
pure water (5 mL) and then freeze-dried to afford the pure deacetylated
glycocluster. 1H NMR (400 MHz, DMSO+eD2O): d=8.81 (s, 8H; H-
porph), 8.39 (s, 4H; H-triaz), 8.09 (d, J=8.5 Hz, 8H; H-ar), 7.47 (d, J=
8.5 Hz, 8H; H-ar), 5.44 (brs, 8H; PhOCH2C-triaz), 4.64 (t, J=5.1 Hz,
8H; OCH2CH2N-triaz), 4.12 (d, J=7.2 Hz, 4H; H-1), 3.92 (t, J=5.1 Hz,
8H; OCH2CH2N-triaz), 3.89–3.80 (m, 4H; H-6a), 3.64–3.46 (m, 40H; H-
5, H-6b, GalOCH2CH2OCH2CH2O), 3.38–3.23 ppm (m, 12H; H-2, H-3,
H-4); 13C NMR (100 MHz, DMSO+eD2O): d=157.8 (CIV-ar), 149.7 (CIV-
porph), 142.8 (CIV-triaz), 135.4 (CIV-ar), 135.3 (CH-ar), 131.7 (CH-porph),
125.4 (CH-triaz), 120.0 (Ph-CIV-porph), 113.0 (CH-ar), 103.66 (C-1), 75.2,
73.4, 70.5 (3 s, C-2, C-3, C-4), 69.9, 69.8, 69.7 (3 s, 12C; Ga-
lOCH2CH2OCH2CH2O), 68.9 (OCH2CH2N-triaz), 68.1 (C-5), 67.9 (C-6),
61.5 (PhOCH2C-triaz), 60.5 (GalOCH2-), 49.7 ppm (OCH2CH2N-triaz);
MALDI-TOF MS (positive-ion reflectron mode): calcd for
a
dissociation: 120 s) of incubated (>1 h, RT) mixtures of PA-IL (5 mm)
and various concentrations of inhibitor (2-fold cascade dilutions). For
each inhibition assay, PA-IL (5 mm) without inhibitor was injected to ob-
serve the full adhesion of the lectin onto the sugar-coated surface (0%
inhibition). The CM5 chip was fully regenerated by successive injections
of d-galactose (2ꢅ30 mL, 100 mm in running buffer).
For both experimental settings, binding was measured as RU over time
after blank subtraction, and data were then evaluated by using the BIAe-
valuation Software, Version 4.1. For IC50 evaluation, the response (Req-
fitted) was considered as the amount of lectin bound to the sugar surface
at equilibrium in the presence of a defined concentration of inhibitor. In-
hibition curves were obtained by plotting the percentage of inhibition
against the inhibitor concentration (on a logarithmic scale) by using
Origin 7.0 software (OriginLab Corp.) and IC50 values were extracted
from a sigmoidal fit of the inhibition curve.
Isothermal titration microcalorimetry (ITC): Recombinant lyophilized
PA-IL was dissolved in buffer (0.1m Tris-HCl, 6 mm CaCl2, pH 7.5) and
degassed (see the Supporting Information for concentration details). Pro-
tein concentration was checked by measurement of optical density by
using a theoretical molar extinction coefficient of 28000. Carbohydrate li-
gands were dissolved directly into the same buffer, degassed, and placed
in the injection syringe. ITC was performed with a VP-ITC MicroCa-
lorimeter from MicroCal Incorporated. PA-IL was placed into the
1.4478 mL sample cell, at 258C. Titration was performed with 10 mL in-
jections of carbohydrate ligands every 300 s. Data were fitted with Micro-
Cal Origin 7 software, according to standard procedures. Fitted data
yielded the stoichiometry (n), the association constant (Ka) and the en-
thalpy of binding (DH). Other thermodynamic parameters (i.e. changes
in free energy, DG, and entropy, DS) were calculated from the equation
DG=DHÀTDS=ÀRTlnKa in which T is the absolute temperature and
C
104H128N16O36Zn: 2240.80 [M]+; found: 2240.78.
Hemagglutination inhibition assays (HIA): Hemagglutination inhibition
assays were performed in U-shaped 96-well microtitre plates. Rabbit er-
ythrocytes were bought from Biomerieux and used without further wash-
ing. The erythrocytes were diluted to a 4% solution in NaCl (150 mm).
Lectin solutions of 2 mg/mL were prepared in Tris/HCl (20 mm), NaCl
(100 mm) and CaCl2 (100 mm). The hemagglutination unit (HU) was first
obtained by the addition of 25 mL of the 4% erythrocyte solution to
25 mL aliquots of sequential (two-fold) lectin dilutions. The mixture was
incubated at 258C for 60 min. The HU was measured as the minimum
lectin concentration required to prevent hemagglutination. For the fol-
lowing lectin-inhibition assays, lectin concentrations of four times that of
the hemagglutination unit were used. For PA-IL, this concentration was
found to be 6 and 20 mg/mL for ECA. Subsequent assays were then car-
R=8.314 JmolÀ1 KÀ1
formed for each ligand tested.
. Two or three independent titrations were per-
Chem. Eur. J. 2011, 17, 2146 – 2159
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2157

S. Vidal et al.
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Acknowledgements
The authors thank the Universitꢁ Claude Bernard Lyon 1, Universitꢁ
Blaise Pascal-Clermont-Ferrand II and the CNRS for financial support.
S.C. thanks the Rꢁgion Rhꢈne-Alpes (Cluster de Recherche Chimie) and
the CNRS (Programme Interdisciplinaire: Chimie pour le Dꢁveloppe-
ment Durable) for additional funding. Financial help is acknowledged
from Vaincre la Mucovisdicose and GDR Pseudomonas. A.I. acknowl-
edges funding from ANR Glycoasterix. M.W. acknowledges the Czech
Ministry of Education (MSM0021622413, ME08008). Part of the research
has received funding from the European Communityꢀs Seventh Frame-
work Programme under grant agreement no. 205872. Dr D. Bouchu, C.
Duchamp and N. Henriques are gratefully acknowledged for mass spec-
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Received: September 13, 2010
Published online: January 5, 2011
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2159