Toward the Rational Design of Galactosylated Glycoclusters That Target Pseudomonas aeruginosa Lectin A (LecA): Influence of Linker Arms That Lead to Low-Nanomolar Multivalent Ligands

Article abstract of DOI:10.1002/chem.201602047

Anti-infectious strategies against pathogen infections can be achieved through antiadhesive strategies by using multivalent ligands of bacterial virulence factors. LecA and LecB are lectins of Pseudomonas aeruginosa implicated in biofilm formation. A series of 27 LecA-targeting glycoclusters have been synthesized. Nine aromatic galactose aglycons were investigated with three different linker arms that connect the central mannopyranoside core. A low-nanomolar (K_d=19 nm, microarray) ligand with a tyrosine-based linker arm could be identified in a structure–activity relationship study. Molecular modeling of the glycoclusters bound to the lectin tetramer was also used to rationalize the binding properties observed.

Full text of DOI:10.1002/chem.201602047

DOI: 10.1002/chem.201602047
Full Paper
&
Glycoclusters
Toward the Rational Design of Galactosylated Glycoclusters That
Target Pseudomonas aeruginosa Lectin A (LecA): Influence of
Linker Arms That Lead to Low-Nanomolar Multivalent Ligands
+
[a]
+ [b]
[c]
[a]
[d]
Shuai Wang , Lucie Dupin , Mathieu Noꢀl, Cindy J. Carroux, Louis Renaud,
[b]
[c]
[b]
[c]
Thomas Gꢁhin, Albert Meyer, Eliane Souteyrand, Jean-Jacques Vasseur,
Gꢁrard Vergoten, Yann Chevolot,* FranÅois Morvan,* and Sꢁbastien Vidal*
[e]
[b]
[c]
[a]
Abstract: Anti-infectious strategies against pathogen infec-
tions can be achieved through antiadhesive strategies by
using multivalent ligands of bacterial virulence factors. LecA
and LecB are lectins of Pseudomonas aeruginosa implicated
in biofilm formation. A series of 27 LecA-targeting glycoclus-
ters have been synthesized. Nine aromatic galactose agly-
cons were investigated with three different linker arms that
connect the central mannopyranoside core. A low-nanomo-
lar (K =19 nm, microarray) ligand with a tyrosine-based
d
linker arm could be identified in a structure–activity relation-
ship study. Molecular modeling of the glycoclusters bound
to the lectin tetramer was also used to rationalize the bind-
ing properties observed.
Introduction
at the malleable cell surface and also by the multimeric charac-
ter of most bacterial lectins. The design of synthetic analogues
[2,3]
Bacterial adhesion at the cell surface takes place through mul-
tivalent protein–carbohydrate interactions between the bacte-
rial proteins (e.g., toxins, adhesins, and lectins) and the glyco-
conjugates at the outer cell membrane (e.g., glycolipids and
of oligosaccharides has been largely studied by chemists
and has now provided interesting results in antiadhesive thera-
py against bacterial infections that target, for instance, Escheri-
[4–8]
[9–14]
chia coli
or Pseudomonas aeruginosa.
LecA and LecB are
[
1]
glycoproteins). High avidity is achieved through multivalency
by presenting multiple copies of the oligosaccharidic partner
two soluble tetrameric lectins of Pseudomonas aeruginosa im-
[15]
plicated in biofilm formation. Although LecA binds preferen-
[16]
[17]
tially to galactosides, fucosides are recognized by LecB.
+
The main part of the design of multivalent architectures that
display multiple carbohydrate copies on their periphery is the
choice of the central-core scaffold. The correct balance be-
tween valency and density is required to avoid, on one hand,
low valency and poor affinity, but also, on the other hand,
high valency and steric hindrance toward the protein-binding
partners and hence poor efficacy in this context. The geometry
[
a] Dr. S. Wang, C. J. Carroux, Dr. S. Vidal
Institut de Chimie et Biochimie Molꢀculaires et Supramolꢀculaires
Laboratoire de Chimie Organique 2 - Glycochimie UMR 5246
CNRS - Universitꢀ Claude Bernard Lyon 1
3 Boulevard du 11 Novembre 1918, 69622 Villeurbanne (France)
E-mail: sebastien.vidal@univ-lyon1.fr
4
+
[
b] L. Dupin, Dr. T. Gꢀhin, Dr. E. Souteyrand, Dr. Y. Chevolot
Institut des Nanotechnologies de Lyon (INL) - UMR CNRS 5270
Ecole Centrale de Lyon, Universitꢀ de Lyon
[18]
of the scaffolds also plays a role in the affinity toward lectins.
3
6 Avenue Guy de Collongue, 69134 Ecully cedex (France)
E-mail: yann.chevolot@ec-lyon.fr
Synthetic glycoclusters have been designed based on diverse
[2,19–23]
aromatic-core scaffolds, such as porphyrins,
ful-
and more recently pillarar-
Oligonucleotide chemistry with the powerful phos-
[
c] M. Noꢁl, A. Meyer, Dr. J.-J. Vasseur, Dr. F. Morvan
Institut des Biomolꢀcules Max Mousseron (IBMM) - UMR 5247
CNRS - Universitꢀ Montpellier - ENSCM
Place Eugꢂne Bataillon, CC1704, 34095 Montpellier cedex 5 (France)
E-mail: morvan@univ-montp2.fr
[2,24–28]
[2,9,21,29–32]
lerenes,
calixarenes,
[33–37]
enes.
phoramidite coupling offers a general and accessible platform
for the design of multivalent glycoclusters because valency
can be easily modulated as well as the overall geometry of the
macromolecule with optimal water solubility thanks to the
[
d] Dr. L. Renaud
Institut des Nanotechnologies de Lyon, UMR CNRS 5270
Universitꢀ Claude Bernard Lyon 1, Universitꢀ de Lyon
3 Boulevard du 11 Novembre 1918, 69622 Villeurbanne (France)
[14,38–44]
4
phosphodiester groups.
In this context, a tetravalent
[
e] Dr. G. Vergoten
Unitꢀ de Glycobiologie Structurale et Fonctionnelle (UGSF) - UMR 8576
CNRS - Universitꢀ de Lille 1, Citꢀ Scientifique, Avenue Mendeleiev
mannopyranoside core platform was also introduced and pro-
vided glycoclusters of high affinity toward LecA as antibacterial
[14,43,45,46]
candidates.
Bat C9, 59655 Villeneuve d’Ascq cedex (France)
+
The type of linker used to connect the carbohydrate to the
core also greatly affects the binding properties toward lectins.
The present study focuses on the design of LecA high-affinity
[
] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201602047.
Chem. Eur. J. 2016, 22, 1 – 11
1
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
[
48]
with the histidine (His) residue His50.
This type of study
[51]
emerged with the design of a C-galactoside with an aromat-
ic moiety that displayed low-micromolar dissociation constants,
Table 1. Aromatic aglycons investigated for improved LecA-binding
properties.
Galactoside
K
d
Number
Authors
[
a]
[b]
[
mm] tested
Chabre
et al.
3
5
7
10
[51]
Figure 1. General structure of mannose-centered glycoclusters.
Cecioni
.8
0
[31]
et al.
glycoclusters based on phosphoramidite coupling with particu-
lar insight into the influence of two linker arms (Linker 1 and
Linker 2; Figure 1) used in the glycocluster architecture. Linker
Kadam
et al.
4.2
11
[13]
1
is directly connected to the mannopyranoside core scaffold,
whereas Linker 2 is connected to the carbohydrate epitope
i.e., galactoside) with a triazole moiety that conjugates both
(
Kadam
4
6
.2
.3
11
2
linkers. The mannose unit used as the tetravalent-core scaffold
was selected as the best candidate among several other sac-
[48]
et al.
[
38,43,45]
charides from earlier studies.
A cyanine fluorescent-dye
Rodrigue
(
Cy3)-labeled 15-mer oligonucleotide sequence was introduced
[49]
et al.
[
47]
for multiplexing on carbohydrate microarrays, thus allowing
[
41]
rapid qualitative and quantitative
binding properties.
screening of the LecA-
[
d
a] The K values were measured by using ITC. [b] Number of additional
aromatic aglycons tested within the study and for which the best ligand
identified is presented herein.
The influence of Linker 1 was studied by using three differ-
ent linkers of increasing lengths: propyl (Pro), diethyleneglycol-
methylene (EG M), and triethyleneglycolmethylene (EG M).
2
3
Access to the carbohydrate epitopes by the lectin moiety is
crucial for optimal binding properties; that is, the shorter
Linker 1 is, the poorer the affinity is to be expected.
as measured by using isothermal titration calorimetry (ITC;
Table 1). Based on the known micromolar affinity of para-nitro-
The nature of the aglycon moiety at the anomeric position
of the galactosides has been carefully studied, and aromatic
derivatives have displayed improved binding properties for the
phenyl b-d-galactoside (K =140 mm), we designed an ana-
d
logue in which the nitro functionality was replaced by an azi-
[31]
doacetamido group as a reactive handle for conjugation to
multivalent core platforms. Similarly, the aromatic aglycon re-
ported by Reymond and co-workers displayed improved bind-
[
3,13,31,43,46,48–51]
lectin.
The present study provides a new set of
nine potential aromatic linkers (Linker 2; Figure 1) with
phenyl, furanyl, thiophenyl, pyridinyl, or tyrosinyl groups. The
influence of the distance to the anomeric carbon atom and
the heteroaromatic structures is discussed.
[13]
ing properties toward LecA.
Further studies by the same
group identified larger aromatic aglycons, such as 2-naphthyl,
[48]
as promising targets, along with the corresponding thiogly-
[49]
After synthetic efforts toward the carbohydrate building
blocks and conjugation to the oligonucleotide scaffolds,
a series of 27 glycoclusters were evaluated by means of DNA-
directed immobilized (DDI) carbohydrate microarrays to assess
their binding properties toward LecA. The results obtained
were rationalized by using molecular-modeling studies for the
best ligands identified through their structure–activity relation-
ships.
coside. Meanwhile, other reports have used multivalent gly-
coclusters with aromatic aglycons in their linker arm. Improved
binding properties toward LecA were demonstrated when con-
jugated to modified oligonucleotides and by using a carbohy-
[43,46]
drate-microarray system for the screening of affinity.
Synthesis of the azido-functionalized carbohydrates
Introduction of a glycol moiety between the aromatic ring and
the anomeric carbon atom (Scheme 1) was achieved by galac-
tosylation of nitrophenoxyethanols 2a–c readily obtained by
alkylation from ethylene carbonate of the corresponding phe-
Results and Discussion
The binding pocket of LecA exhibits a preference for aromatic
[52–54]
aglycons with additional interactions through CH–p stacking
nols 1a–c.
Galactosides 3a–c were converted into the de-
&
&
Chem. Eur. J. 2016, 22, 1 – 11
www.chemeurj.org
2
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
Scheme 1. Synthesis of ethyleneglycol-phenyl-azidoacetamido galactosides GalEGo-PhNAz (5a), GalEGm-PhNAz (5b), and GalEGp-PhNAz (5c; Gal=galactose).
sired azido-functionalized carbohydrates 5a–c through the
anomer 9. Saponification under smooth conditions to the car-
boxylic acid intermediate, followed by condensation with 2-(2-
azidoethoxy)ethanamine and re-acetylation of the crude mix-
ture afforded the furan-based galactoside 10.
A similar synthetic strategy was adopted toward the thio-
phen-based linker arm (Scheme 3). Dicarboxylic acid 11 was
converted into dimethylester 12 and selective mono-saponifi-
cation to acid/ester 13 followed by chemoselective reduction
of the carboxylic acid afforded alcohol 14. Galactosylation was
[
31]
bromo intermediates 4a–c. This three-step process from the
nitro to the azidoacetamido (3a–c!5a–c) moiety was inter-
rupted when the bromo derivatives 4a–c were obtained by
rapid column chromatography on silica gel, thus obtaining op-
timal yields for the final azidation step.
Furan-2,5-dicarboxylic acid (6) was diesterified to dimethyles-
[
55]
ter 7 and selectively monoreduced to the corresponding al-
cohol 8 (Scheme 2). Galactosylation afforded the desired b-
Scheme 2. Synthesis of the furan-based galactosides GalFurEG
benzotriazole, TsOH=toluenesulfonic acid.
2 3
N (10; fur=furanyl). EDCl=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, HOBt=1-hydroxy-
2 3
Scheme 3. Synthesis of the thiophen-based galactosides GalThioEG N (17; Thio=thiophenyl). TBTU=2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate.
Chem. Eur. J. 2016, 22, 1 – 11
www.chemeurj.org
3
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
2 3 2 3
Scheme 4. Synthesis of the phenyl- and pyridine-based galactosides GalPhEG N (19) and GalPyrEG N (21; Pyr=pyridyl).
performed by using a peracetate galactosyl donor to the corre-
sponding galactoside 15. Concomitant saponification of both
acetate protecting groups and the methyl ester followed by
re-acetylation of the crude product provided carboxylic acid
vious results by using more sophisticated galactosyl
[57–59]
donors.
Galactoside 24 was obtained in good yield (64%).
Meanwhile, acid 22 was esterified to form allyl ester 25 and
then galactosylated to the intermediate 26 in high yield (90%).
A one-pot three-step process afforded the azido-functionalized
intermediate 27 and removal of the allyl ester moiety led to
the desired galactoside 28.
16. Condensation with 2-(2-azidoethoxy)ethanamine afforded
amide 17 in good yield.
[
56]
The previously reported galactosides 18 and 20 were con-
jugated with 2-(2-azidoethoxy)ethanamine and provided
amides 19 and 21, respectively (Scheme 4). The pyridine deriv-
ative 21 was obtained in lower yields due to difficulty in purify-
ing carboxylic acid 20, the purity of which greatly alters the
outcome of the amide-bond formation.
Conjugation with oligonucleotides
[62]
The azido-functionalized CPG precursor 29 was conjugated
I
with propargyl a-d-mannopyranoside 30 in a Cu-catalyzed
Tyrosine (Tyr) was used as a linker arm through either the
amine or carboxylic acid moiety (Scheme 5). The carboxylic
acid of Fmoc-tyrosine 22 was conjugated with 2-(2-azido-
ethoxy)ethanamine to the corresponding amide 23. Galactosyl-
ation of the phenol moiety of tyrosine has been reported with
azide–alkyne cycloaddition reaction (CuAAC) on a solid support
[63]
by using our standard methodology (Scheme 6). The four
hydroxy groups of the mannose moiety were converted into
[38,46,64]
phosphitetriesters with phosphoramidites 32–34.
Subse-
quent oxidation with iodine/water afforded phosphotriester
linkages. Subsequent release of the dimethoxytrityl (DMTr) at
the primary hydroxy function of the solid support allowed for
elongation of a 15-mer oligonucleotide sequence and Cy3 la-
beling (see the Supporting Information). Treatment with am-
[
57,58]
[59]
galactosyl bromides
or trichloroacetimidates as donors,
[
60,61]
but the readily available galactose peracetate
was used
and provided the best yields. The glycosylation reaction pro-
ceeded under rather simple reaction conditions relative to pre-
2 3
Scheme 5. Synthesis of the tyrosine-based galactosides GalTyrEG N (24) and GalTyrNAz (28). Fmoc=9-fluorenylmethoxycarbonyl.
&
&
Chem. Eur. J. 2016, 22, 1 – 11
www.chemeurj.org
4
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
Scheme 6. Conjugation of azido-functionalized carbohydrate epitopes with the tetrapropargylated oligonucleotide-based scaffolds. Asc=ascorbate, BMT=5-
(benzylmercapto)-1H-tetrazole, CPG=controlled pore glass, DMTr=4,4’-dimethoxytrityl, SPS=solid phase synthesis, A, T, C, G=protected nucleoside phos-
phoramidites of deoxyadenosine (A), deoxythymidine (T), deoxycytidine (C) and deoxyguanosine (G).
monia was used for cleavage from the CPG solid support and
concomitant oligonucleotide deprotection to afford the Cy3-
DNA tagged tetra-propargylated scaffolds 35–37.
linker arms at the phosphotriester site (Linker 1) and no less
than nine structurally different spacer arms between the tri-
azole and galactose moieties (Linker 2; Scheme 6).
A second CuAAC conjugation to introduce the four galacto-
side moieties was performed in solution with the deprotected
carbohydrates 5a–cOH, 10OH, 17OH, 19OH, 21OH, and 28OH or the
protected galactoside 24. Deacetylation was performed by
using the classic treatment of MeOH/Et N/H O. These hydroxy-
Binding studies with microarrays
The 27 glycoclusters prepared were investigated for their bind-
ing properties toward the bacterial lectin LecA from Pseudomo-
nas aeruginosa. First, the influence of Linker 1 was assessed
and the best type of Linker 1 identified. The influence of dif-
ferent examples of Linker 2 on the subfamily of nine glyco-
clusters, which incorporate the optimal Linker 1, was evaluat-
ed in a structure–activity relationships analysis. These investi-
gations were first performed qualitatively by determining the
fluorescence signals of LecA bound to the glycoclusters at the
surface of the microarray and quantitatively by measuring the
dissociation constants (Kd).
3
2
lated carbohydrates were characterized by means of high-reso-
lution mass spectrometric analysis (see Table S1 in the Sup-
porting Information). Conjugations were initially performed in
[45]
water according to our previously established protocol by
using a copper(0) species with microwave assistance for
1.5 hours at 658C (see Method A in the Supporting Informa-
tion). Tris(benzyltriazolylmethyl)amine (TBTA) in methanol was
used as a ligand for a copper(I) species that is known to accel-
[
65]
erate the CuAAC
hour at room temperature (see Method B in the Supporting
Information). Finally, tris(3-hydroxypropyl triazolylmethyl)-
reaction, which was completed within
1
The glycocluster oligonucleotide conjugates were labeled
with Cy3, whereas LecA was tagged with Alexa647. The Cy3
fluorescence signal monitored the relative surface densities of
the different glycoclusters (less than 12% deviation). The
Alexa647 fluorescence signal was used to monitor the binding
of LecA with the glycoclusters (Figure 3).
[
66]
amine (THPTA) was used instead of TBTA, and the reaction
was performed in water with the same efficiency (see Meth-
od C in the Supporting Information). Therefore, glycoclusters
38–40 were obtained with variations in the three different
Chem. Eur. J. 2016, 22, 1 – 11
www.chemeurj.org
5
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
the affinity toward LecA. 2) For the furan-based (d) and TyrNH2
(
h) Linker 1, the opposite was observed. 3) However, no influ-
ence of Linker 1 was observed for the TyrCO H (i) aglycon.
2
4
) Finally, a decrease of affinity from Pro to EG M, then an in-
2
crease from Pro to EG M was observed for the thiophen- (e)
3
and pyridine-based (g) aglycons. These observations are very
difficult to rationalize, but the general trend (i.e., the longer
that Linker 1 is, the better the affinity for the lectin) is quite
logical. This linker is internally positioned in the structure of
the whole glycocluster and might moderately influence the
binding properties toward LecA.
Influence of Linker 2
Figure 2. Structures of mannose-centered galactoclusters with aromatic or
triethyleneglycol aglycons.
Linker 2 was introduced to probe the influence of 1) the regio-
isomery of the aromatic ring a–c, 2) the distance of this ring
from the anomeric carbon atom of galactose (i.e., a–c versus j),
and 3) the nature of the aromatic aglycon.
Aromatic aglycons drastically improve the binding of glyco-
[
3,13,31,43,46,48–51]
clusters to LecA.
Three previously reported aro-
Among the 31 glycoclusters evaluated, the one moiety with
a triethyleneglycol aglycon species (Linker 2) 38k displayed
[
46]
matic glycoclusters 38j, 39j, and 40j
and glycocluster
8k, which does not possess an aromatic aglycon, but a tri-
[
43]
3
[43]
the lowest affinity toward LecA. This result confirmed the re-
ethyleneglycol linker, were also included in the study for com-
parison (Figure 2). Two negative controls were included in the
quirement of an aromatic aglycon to increase the affinity for
LecA.
[
47]
microarray measurements. TriMan (see the Supporting Infor-
mation) displayed three mannose residues that should not in-
teract with LecA. A second negative control (ssDNA) corre-
sponded to a single-stranded DNA (Figure 3). The Alexa647
fluorescence signal was less than 56 AU for these negative
controls, thus showing that no nonspecific binding occurred.
Glycoclusters that incorporate the para-substituted EGpara-
Ph (c), TyrCO H (i), and para-Ph (j) moieties displayed, at a simi-
2
lar level, the best affinity for LecA. The next glycoclusters with
a good affinity incorporated TyrNH2 (h) and EGmeta-Ph (b).
Then, on the order of decreasing affinity are the thiophen-
based (e), meta-Bn (f), furan-based (d), pyridine-based (g), and
finally EGortho-Ph (a) components. From the tested molecules,
it appears that the influence of Linker 2 was generally greater
than the influence of Linker 1.
Influence of Linker 1
The influence of Linker 1 was rather dependent on the nature
of the aglycon with four different trends seen: 1) For the
phenyl-based aglycons a–c, f, and j, the longer Linker 1 (from
Glycoclusters 38a–c, in which a flexible glycol (EG) chain
was introduced between the anomeric center of the galactose
residue and the aromatic ring, were compared. The para-sub-
stituted glycocluster 38c provided the best binding properties
propyl to triethyleneglycolmethylene, EG M) was, the better
3
Figure 3. Alexa647–LecA fluorescence signal at l=635 nm as a function of the glycoclusters. The average value was calculated from 32 points per glycoclus-
ter.
&
&
Chem. Eur. J. 2016, 22, 1 – 11
www.chemeurj.org
6
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
toward LecA, better than the meta-regioisomer 38b, whereas
the ortho-derivative 38a was mostly detrimental to the bind-
The influence of the aromatic moiety was also confirmed.
Glycoclusters 40 with a furanyl (d) or pyridinyl (g) ring exhibit-
ing properties. The same influence was observed for the EG M
ed the weakest binding with K values of around 200 nm,
2
d
3
9 and EG M 40 species in the Linker 1 series. The ortho-sub-
whereas those glycoclusters with thiophenyl (e) or meta-benzyl
3
stitution pattern (a) probably generates significant steric hin-
drance because the amino acids of LecA are close to the car-
bohydrate-recognition domain. This effect is probably less im-
portant for the meta-regioisomer (b), whereas while the para-
substituted derivative (c) is the least congested, thus providing
the best affinity observed.
(f) groups exhibited better affinities with K values of around
80 nm, but were still about seven- and threefold less potent,
d
respectively, than glycocluster 40j with a phenyl linker (K =
d
28 nm).
As the stabilisation involves a face-to-edge p–p interaction
between the ring and His50 of LecA, this interaction might be
improved in the case of the thiophenyl (e) moiety based on
the higher aromaticity due to the lower electronegativity of
the sulfur atom relative to the oxygen atom in the furanyl ring
(d). Furthermore, the addition of a methylene moiety on going
from the para-Ph (j) to the meta-Bn (f) groups was detrimental,
probably because the oxygen atom provides an enrichment in
electron density on the aromatic ring of 40j, which is benefi-
cial for a better face-to-edge p–p interaction with the His50
residue.
Finally, a comparison of Linker 2 in the series of d–g pro-
vides insight to the influence of the aromatic ring. In this case,
the order of influence within the glycoclusters was observed
to be thiophen (e)>meta-Bn (f)>furan (d)>pyridine (g). The
pyridine-based (g) Linker 2 was superior among these four
congeners only in the case of glycocluster 40, and then it was
only superior to the furan-based (d) Linker 2.
Measurements of dissociation constants (K ) on microarrays
d
[13]
Reymond and co-workers reported that an amine group
To gain a better understanding of the influence of Linker 2,
(as a lysine residue of the core moiety of the peptide-based
glycocluster) close to the aromatic moiety can interact with
the aspartic acid (Asp) residue Asp47 of LecA, thus leading to
stabilization of the interaction through coulombic forces. In
dissociation constants (K ) were measured by means of micro-
d
array to provide quantitative insight into the glycocluster-bind-
ing properties toward LecA (Table 2). The K values were deter-
d
our case, the free amine group of the TyrNH -based glycoclus-
2
ter 40h did not improve the binding relative to glycocluster
Table 2. Dissociation constants (K
ray.
d
) measured by carbohydrate microar-
4
0j (K =47 and 28 nm, respectively). In contrast, the introduc-
d
tion of a carboxylic acid group into the TyrCO H-based glyco-
2
2
Glycocluster
Linker 2
K
d
[nm]
Rank
R
cluster 40i led to the best binding with a K value of 19 nm,
d
thus being the best LecA ligand identified to date by using mi-
croarrays.
4
4
4
4
4
4
4
4
4
4
0a
0b
0c
0d
0e
0 f
0g
0h
0i
EGortho-Ph
EGmeta-Ph
EGpara-Ph
furanyl
thiophenyl
meta-Bn
304
52
39
202
77
81
10
5
3
8
6
6
8
4
1
2
0.963
0.993
0.996
0.991
0.997
0.985
0.986
0.995
0.994
[
[
[
[
a]
a]
a]
a]
Molecular simulations
pyridyl
197
47
TyrNH
TyrCO
para-Ph
2
Investigations were performed by using molecular-simulation
studies to rationalize the binding properties observed on the
microarrays and their implications in the binding of the galac-
tosides and their aglycons in the binding site of LecA. The stoi-
chiometry typically observed in such mannose-centered glyco-
2
H
19
28
[
b]
[c]
0j
–
[a] Could not be distinguished because the root-square deviation (RSD)
of the K
than 8%. [b] The K
d
[
measurements determined by means of microarrays was less
67]
[46]
d
value previously reported. [c] Does not apply.
[14]
clusters with LecA was measured as n=0.5. The calculations
were performed by considering that two branches of the same
glycocluster would interact simultaneously with two binding
sites of the same LecA tetramer.
mined only for the EG M (40) Linker 1 series, which had been
3
identified as the optimal linker for this position in the macro-
Glycoclusters 40c, 40h, and 40i (Figures 4–6) were investi-
gated by using molecular simulations and docking techniques.
Glycocluster 40c was selected to evaluate the influence of the
ethyleneglycol moiety introduced at the anomeric center upon
binding to LecA. Glycoclusters 40h and 40i provided the best
molecule. The quantitative K values (Table 2) were in agree-
d
ment with the qualitative data obtained by direct fluorescence
(Figure 3).
The preference in the substitution pattern at the phenyl ring
was confirmed to be para (c)>meta (b)@ortho (a). Indeed, the
Kd values decreased by nearly sixfold between the ortho and
meta isomers and by nearly eightfold between the ortho and
para isomers.
binding properties from the K measurements and were there-
d
fore further investigated. Because the high affinities observed
point toward chelate-binding modes as previously observed
[21,31,50]
for this lectin,
an empirical calculation of the potential
A comparison between 40c and 40j showed that the intro-
duction of an ethyleneglycol group between the galactose and
phenyl rings had only a limited and small negative effect on
energy of interaction DE was carried out for the six different
possible combinations for the simultaneous binding of the two
galactose epitopes displayed at the tip of two branches at-
tached at the core mannopyranoside ring (Table 3).
the affinity (K =39 and 28 nm, respectively).
d
Chem. Eur. J. 2016, 22, 1 – 11
www.chemeurj.org
7
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
Furthermore, the His58 residue from the B monomer is in hy-
drogen-bond contact with the O2 branch. Finally, branch O6
also interacts with both monomers with hydrogen bonds
formed toward the His50A, Glu49A, and Gln40B residues
(
Glu=glutamic acid, Gln=glutamine).
Glycocluster 40h with a tyrosine–amine aglycon provided
another good DE-to-K correlation when considering branches
d
ꢀ
1
from positions 3 and 4 (K =47 nm, DE=ꢀ232 kcalmol ). The
d
galactoside epitopes displayed on branches at positions 3 and
4
interact in the LecA binding site through the usual interac-
tions, and no additional intermolecular bonding was observed
Figure 5). The remaining stabilizing interactions could be ac-
(
counted for by the interaction of branches 2 and 6 with other
parts of the glycocluster itself (if considering that these
branches would not interact with another LecA tetramer in so-
lution). The O3 galactoside interacts with the lectin monomer
B through hydrogen bonds with the His50 residue, which
again does not interact with the aromatic moiety but the 6-OH
group of the galactose moiety (Figure 5c). Nevertheless, a van
der Waals contact between the aromatic aglycon and the g-
proton of the proline residue Pro38 could be identified with
a distance from 2.70 to 3.85 ꢃ (Figure 5c). The amine group
was not engaged in any electrostatic interaction with the pro-
tein.
Figure 4. Glycocluster 40c interacting with LecA. a) Top view, b) side view,
c) binding-site close-up view of the O3 galactose residue. Calcium cations
are represented as spheres. Gal2, Gal3, Gal4, and Gal6 are the galactose epi-
topes displayed at the end of each linker arm of the mannopyranoside hy-
droxy groups. Dashed lines represent the hydrogen-bonding contacts.
Glycocluster 40i with a tyrosinyl carboxylate group led to
the most stable complex, in agreement with the lowest
K value measured (19 nm). A good DE-to-K agreement was
A good DE-to-K correlation was observed for glycocluster
d
4
0c with branches at the O3 and O4 atoms of the mannopyra-
d
d
ꢀ
1
noside ring (K =39 nm, DE=ꢀ239 kcalmol ). The galactoside
found with branches issued from positions 2 and 4 of the man-
d
ꢀ
1
epitope at position 3 interacts with the lectin monomer B
through hydrogen bonds with the Tyr36, His50, and Asn107
residues (Figure 4c; Asn=asparagine). The His50 residue usual-
nopyranoside ring (K =19 nm, DE=ꢀ308 kcalmol ). The gal-
d
actose epitope at the O2 branch interacts with monomer B of
the tetrameric lectin (Figure 6). The carboxylate group in the
tyrosine-based linker is essentially exposed to the solvent, but
also creates additional interactions with the Pro38 and Glu39
amino acids. The O4 branch interacts with monomer A of the
same tetrameric lectin. The carboxylate group present in the
linker is again engaged in a hydrogen bond with the His50 res-
[13,48]
ly involved in CH–p stacking with other aromatic aglycons
now interacts with the OH-6 group of the galactose residue
and the carbonyl bond of the amide group present on the
aglycon. The same carbonyl bond also interacts with the Tyr36
residue. The O4 branch is only connected to monomer A
through hydrogen bonds with the Thr39 and His50 residues.
idue (Figure 6c). The TyrCO H aglycon therefore causes the de-
2
Figure 5. Glycocluster 40h interacting with LecA. a) Top view, b) side view, c) binding-site close-up view of the O3 galactose residue. Calcium cations are rep-
resented as spheres. Gal2, Gal3, Gal4, and Gal6 are the galactose epitopes displayed at the end of each linker arm of the mannopyranoside hydroxy groups.
Dashed lines represent hydrogen-bonding contacts and also distances for the van der Waals interactions with Pro38.
&
&
Chem. Eur. J. 2016, 22, 1 – 11
www.chemeurj.org
8
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
Figure 6. Glycocluster 40i interacting with LecA. a) Top view, b) side view, c) binding-site close-up view of the O4 galactose residue. Calcium cations are repre-
sented as spheres. Gal2, Gal3, Gal4, and Gal6 are the galactose epitopes displayed at the end of each linker arm of the mannopyranoside hydroxy groups.
Dashed lines represent hydrogen-bonding contacts.
“
lead” for the design of potential anti-infectious agents against
Table 3. Empirical potential energy of interaction (DE) calculated from
each possible two-set of branches from the mannopyranoside scaffold
used for docking.
Pseudomonas aeruginosa lung infection.
[
a]
[b]
Glycocluster
Branches
2–6 3–4
Mean
[kcalmol ]
Acknowledgements
ꢀ
1
2–3
2–4
3–6
4–6
4
4
4
0c
0h
0i
ꢀ187 ꢀ215 ꢀ226 ꢀ239 ꢀ197 ꢀ170 ꢀ206
ꢀ199 ꢀ220 ꢀ236 ꢀ232 ꢀ259 ꢀ192 ꢀ223
ꢀ314 ꢀ308 ꢀ289 ꢀ174 ꢀ148 ꢀ303 ꢀ256
This project was supported by the Agence Nationale de la Re-
cherche (Glycomime project ANR-12-BSV5-0020). The authors
thank the Universitꢁ Claude Bernard Lyon 1, Ecole Centrale de
Lyon, Universitꢁ Lille 1, Universitꢁ de Montpellier and the CNRS
for financial support. F.M. is from Inserm. Dr. R. Simon and C.
Duchamp are gratefully acknowledged for the mass-spectro-
metric analyses.
[
a] Branches used for the dual docking from the mannopyranoside scaf-
fold. The numbers used refer to the positions on the pyranose ring.
b] Average DE values for all the possible combinations of the branches.
[
viation of the His50 residue from the previously reported T-
shaped CH–p interaction observed in the crystal structure
toward an ionic interaction of the carboxylate group with the
imidazole side chain. This ionic interaction is more energetical-
ly efficient than the CH–p contact, thus explaining the im-
proved affinity measured.
Keywords: glycoclusters
oligonucleotides · Pseudomonas aeruginosa
·
microarrays
·
multivalency
·
[
1] C. A. Bewley, Protein–Carbohydrate Interactions in Infectious Diseases,
RSC, Cambridge, 2006.
[
[
[
[
2] Y. M. Chabre, R. Roy, Chem. Soc. Rev. 2013, 42, 4657–4708.
3] S. Cecioni, A. Imberty, S. Vidal, Chem. Rev. 2015, 115, 525–561.
4] D. Abgottspon, B. Ernst, Chimia 2012, 66, 166–169.
5] C. K. Cusumano, J. S. Pinkner, Z. Han, S. E. Greene, B. A. Ford, J. R. Crow-
ley, J. P. Henderson, J. W. Janetka, S. J. Hultgren, Sci. Transl. Med. 2011, 3,
Conclusions
LecA-targeting glycoclusters have been extensively studied,
and the design of potent ligands of this lectin is of prime inter-
est in antiadhesive therapies against infection by Pseudomonas
aeruginosa. The binding properties of these ligands have been
improved by using aromatic aglycons (typically O-phenyl galac-
tosides). A family of 27 glycoclusters was synthesized with
a combination of two linker arms that connect the central
mannopyranoside core to the galactoside moeities. Aromatic
aglycons, such as phenyl, furanyl, thiophenyl, pyridyl, or tyro-
109ra115.
[
6] Z. Han, J. S. Pinkner, B. Ford, E. Chorell, J. M. Crowley, C. K. Cusumano, S.
Campbell, J. P. Henderson, S. J. Hultgren, J. W. Janetka, J. Med. Chem.
2
012, 55, 3945–3959.
[
7] M. Totsika, M. Kostakioti, T. J. Hannan, M. Upton, S. A. Beatson, J. W. Ja-
netka, S. J. Hultgren, M. A. Schembri, J. Infect. Dis. 2013, 208, 921–928.
8] S. Vanwetswinkel, A. N. Volkov, Y. G. J. Sterckx, A. Garcia-Pino, L. Buts,
W. F. Vranken, J. Bouckaert, R. Roy, L. Wyns, N. A. J. van Nuland, J. Med.
Chem. 2014, 57, 1416–1427.
[
[
9] A. M. Boukerb, A. Rousset, N. Galanos, J.-B. Mꢁar, M. Thꢁpaut, T. Grand-
jean, E. Gillon, S. Cecioni, C. Abderrahmen, K. Faure, D. Redelberger, E.
Kipnis, R. Dessein, S. Havet, B. Darblade, S. E. Matthews, S. de Bentz-
mann, B. Guꢁry, B. Cournoyer, A. Imberty, S. Vidal, J. Med. Chem. 2014,
sinyl groups, were investigated and a low-nanomolar (K =
d
19 nm) ligand with a tyrosine-based linker arm was identified
in a structure–activity relationship study. The role of the lectin
amino acid side chains in the binding of the three best glyco-
clusters was studied by using molecular modeling to rational-
ize the binding properties observed. The glycocluster that in-
corporated the tyrosine-based linker arm appears to be a new
5
7, 10275–10289.
[
[
10] C. Chemani, A. Imberty, S. de Bentzman, P. Pierre, M. Wimmerovꢄ, B. P.
Guery, K. Faure, Infect. Immun. 2009, 77, 2065–2075.
11] H.-P. Hauber, M. Schulz, A. Pforte, D. Mack, P. Zabel, U. Schumacher, Int.
J. Med. Sci. 2008, 5, 371–376.
Chem. Eur. J. 2016, 22, 1 – 11
www.chemeurj.org
9
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
[
[
12] E. M. V. Johansson, S. A. Crusz, E. Kolomiets, L. Buts, R. U. Kadam, M.
Cacciarini, K.-M. Bartels, S. P. Diggle, M. Cꢄmara, P. Williams, R. Loris, C.
Nativi, F. Rosenau, K.-E. Jaeger, T. Darbre, J.-L. Reymond, Chem. Biol.
[40] F. Morvan, S. Vidal, E. Souteyrand, Y. Chevolot, J.-J. Vasseur, RSC Adv.
2012, 2, 12043–12068.
[41] A. Goudot, G. Pourceau, A. Meyer, T. Gehin, S. Vidal, J.-J. Vasseur, F.
Morvan, E. Souteyrand, Y. Chevolot, Biosens. Bioelectron. 2013, 40, 153–
160.
[42] C. Ligeour, A. Audfray, E. Gillon, A. Meyer, N. Galanos, S. Vidal, J.-J. Vas-
seur, A. Imberty, F. Morvan, RSC Adv. 2013, 3, 19515–19524.
[43] B. Gerland, A. Goudot, C. Ligeour, G. Pourceau, A. Meyer, S. Vidal, T.
Gehin, O. Vidal, E. Souteyrand, J.-J. Vasseur, Y. Chevolot, F. Morvan, Bio-
conjugate Chem. 2014, 25, 379–392.
[44] C. Ligeour, L. Dupin, A. Angeli, G. Vergoten, S. Vidal, A. Meyer, E. Sou-
teyrand, J.-J. Vasseur, Y. Chevolot, F. Morvan, Org. Biomol. Chem. 2015,
13, 11244–11254.
2008, 15, 1249–1257.
13] R. U. Kadam, M. Bergmann, M. Hurley, D. Garg, M. Cacciarini, M. A. Swi-
derska, C. Nativi, M. Sattler, A. R. Smyth, P. Williams, M. Cꢄmara, A. Stock-
er, T. Darbre, J.-L. Reymond, Angew. Chem. Int. Ed. 2011, 50, 10631–
10635; Angew. Chem. 2011, 123, 10819–10823.
[
[
14] C. Ligeour, O. Vidal, L. Dupin, F. Casoni, E. Gillon, A. Meyer, S. Vidal, G.
Vergoten, J.-M. Lacroix, E. Souteyrand, A. Imberty, J.-J. Vasseur, Y. Chevo-
lot, F. Morvan, Org. Biomol. Chem. 2015, 13, 8433–8444.
15] A. Imberty, M. Wimmerovꢄ, C. Sabin, E. P. Mitchell, Structures and Roles
of Pseudomonas Aeruginosa Lectins, in Protein–Carbohydrate Interactions
in Infectious Diseases (Ed.: C. A. Bewley), RSC Biomolecular Sciences,
Cambridge, 2006, Chapter 3, pp. 30–48.
[45] G. Pourceau, A. Meyer, Y. Chevolot, E. Souteyrand, J. J. Vasseur, F.
Morvan, Bioconjugate Chem. 2010, 21, 1520–1529.
[
16] G. Cioci, E. P. Mitchell, C. Gautier, M. Wimmerova, D. Sudakevitz, S.
Pꢁrez, N. Gilboa-Garber, A. Imberty, FEBS Lett. 2003, 555, 297–301.
17] E. Mitchell, C. Houles, D. Sudakevitz, M. Wimmerova, C. Gautier, S. Pꢁrez,
A. M. Wu, N. Gilboa-Garber, A. Imberty, Nat. Struct. Biol. 2002, 9, 918–
[46] F. Casoni, L. Dupin, G. Vergoten, A. Meyer, C. Ligeour, T. Gehin, O. Vidal,
E. Souteyrand, J.-J. Vasseur, Y. Chevolot, F. Morvan, Org. Biomol. Chem.
2014, 12, 9166–9179.
[
[47] Y. Chevolot, C. Bouillon, S. Vidal, F. Morvan, A. Meyer, J.-P. Cloarec, A.
Jochum, J.-P. Praly, J.-J. Vasseur, E. Souteyrand, Angew. Chem. Int. Ed.
2007, 46, 2398–2402; Angew. Chem. 2007, 119, 2450–2454.
[48] R. U. Kadam, D. Garg, J. Schwartz, R. Visini, M. Sattler, A. Stocker, T.
Darbre, J. L. Reymond, ACS Chem. Biol. 2013, 8, 1925–1930.
[49] J. Rodrigue, G. Ganne, B. Blanchard, C. Saucier, D. Giguꢆre, T. C. Shiao, A.
Varrot, A. Imberty, R. Roy, Org. Biomol. Chem. 2013, 11, 6906–6918.
[50] M. L. Gening, D. V. Titov, S. Cecioni, A. Audfray, A. G. Gerbst, Y. E. Tsvet-
kov, V. B. Krylov, A. Imberty, N. E. Nifantiev, S. Vidal, Chem. Eur. J. 2013,
19, 9272–9285.
[51] Y. M. Chabre, D. Giguꢆre, B. Blanchard, J. Rodrigue, S. Rocheleau, M.
Neault, S. Rauthu, A. Papadopoulos, A. A. Arnold, A. Imberty, R. Roy,
Chem. Eur. J. 2011, 17, 6545–6562.
[52] L. Fumagalli, C. Bolchi, S. Colleoni, M. Gobbi, B. Moroni, M. Pallavicini, A.
Pedretti, L. Villa, G. Vistoli, E. Valoti, Bioorg. Med. Chem. 2005, 13, 2547–
2559.
[53] P. K. Hashim, R. Thomas, N. Tamaoki, Chem. Eur. J. 2011, 17, 7304–7312.
[54] W. H. Steel, F. Damkaci, R. Nolan, R. A. Walker, J. Am. Chem. Soc. 2002,
124, 4824–4831.
[55] A. Boyer, M. Lautens, Angew. Chem. Int. Ed. 2011, 50, 7346–7349;
Angew. Chem. 2011, 123, 7484–7487.
[56] S. Wang, J. A. Cuesta-Seijo, D. Lafont, M. M. Palcic, S. Vidal, Chem. Eur. J.
2013, 19, 15346–15357.
[57] F. Arena, J. B. Singh, E. Gianolio, R. Stefania, S. Aime, Bioconjugate Chem.
2011, 22, 2625–2635.
9
21.
18] D. Deniaud, K. Julienne, S. G. Gouin, Org. Biomol. Chem. 2011, 9, 966–
79.
[
[
9
19] S. Cecioni, S. Faure, U. Darbost, I. Bonnamour, H. Parrot-Lopez, O. Roy,
C. Taillefumier, M. Wimmerovꢄ, J.-P. Praly, A. Imberty, S. Vidal, Chem. Eur.
J. 2011, 17, 2146–2159.
20] Y. Chen, H. Vedala, G. P. Kotchey, A. Audfray, S. Cecioni, A. Imberty, S.
Vidal, A. Star, ACS Nano 2012, 6, 760–770.
[
[
21] D. Sicard, S. Cecioni, M. Iazykov, Y. Chevolot, S. E. Matthews, J.-P. Praly, E.
Souteyrand, A. Imberty, S. Vidal, M. Phaner-Goutorbe, Chem. Commun.
2
011, 47, 9483–9485.
22] H. Vedala, Y. Chen, S. Cecioni, A. Imberty, S. Vidal, A. Star, Nano Lett.
011, 11, 170–175.
23] D. Sicard, Y. Chevolot, E. Souteyrand, A. Imberty, S. Vidal, M. Phaner-
Goutorbe, J. Mol. Recognit. 2013, 26, 694–699.
[
[
[
[
2
24] S. Cecioni, V. Oerthel, J. Iehl, M. Holler, D. Goyard, J.-P. Praly, A. Imberty,
J.-F. Nierengarten, S. Vidal, Chem. Eur. J. 2011, 17, 3252–3261.
25] J.-F. Nierengarten, J. Iehl, V. Oerthel, M. Holler, B. M. Illescas, A. Munoz,
N. Martin, J. Rojo, M. Sanchez-Navarro, S. Cecioni, S. Vidal, K. Buffet, M.
Durka, S. P. Vincent, Chem. Commun. 2010, 46, 3860–3862.
26] M. Durka, K. Buffet, J. Iehl, M. Holler, J.-F. Nierengarten, J. Taganna, J.
Bouckaert, S. P. Vincent, Chem. Commun. 2011, 47, 1321–1323.
27] I. Nierengarten, J.-F. Nierengarten, Chem. Asian J. 2014, 9, 1436–1444.
28] A. MuÇoz, D. Sigwalt, B. M. Illescas, J. Luczkowiak, L. Rodrꢅguez-Pꢁrez, I.
Nierengarten, M. Holler, J.-S. Remy, K. Buffet, S. P. Vincent, J. Rojo, R. Del-
gado, J.-F. Nierengarten, N. Martꢅn, Nat. Chem. 2016, 8, 50–57.
29] S. Cecioni, O.-A. Argintaru, T. Docsa, P. Gergely, J.-P. Praly, S. Vidal, New J.
Chem. 2009, 33, 148–156.
[
[
[
[58] A. Vargas-Berenguel, M. Meldal, H. Paulsen, K. J. Jensen, K. Bock, J.
Chem. Soc. Perkin Trans. 1 1994, 3287–3294.
[59] N. E. Fahmi, L. Dedkova, B. Wang, S. Golovine, S. M. Hecht, J. Am. Chem.
Soc. 2007, 129, 3586–3597.
[60] H. Lin, D. A. Thayer, C.-H. Wong, C. T. Walsh, Chem. Biol. 2004, 11, 1635–
1642.
[61] N. Yao, G. Fung, H. Malekan, L. Ye, M. J. Kurth, K. S. Lam, Carbohydr. Res.
2010, 345, 2277–2281.
[
[
[
30] S. Cecioni, S. E. Matthews, H. Blanchard, J.-P. Praly, A. Imberty, S. Vidal,
Carbohydr. Res. 2012, 356, 132–141.
31] S. Cecioni, J.-P. Praly, S. E. Matthews, M. Wimmerovꢄ, A. Imberty, S. Vidal,
Chem. Eur. J. 2012, 18, 6250–6263.
[
[
32] A. Dondoni, A. Marra, Chem. Rev. 2010, 110, 4949–4977.
33] S. P. Vincent, K. Buffet, I. Nierengarten, A. Imberty, J.-F. Nierengarten,
Chem. Eur. J. 2016, 22, 88–92.
[62] G. Pourceau, A. Meyer, J.-J. Vasseur, F. Morvan, J. Org. Chem. 2009, 74,
6837–6842.
[63] C. Bouillon, A. Meyer, S. Vidal, A. Jochum, Y. Chevolot, J. P. Cloarec, J. P.
Praly, J. J. Vasseur, F. Morvan, J. Org. Chem. 2006, 71, 4700–4702.
[64] A. Meyer, G. Pourceau, J. J. Vasseur, F. Morvan, J. Org. Chem. 2010, 75,
6689–6692.
[
[
[
34] G. Yu, Y. Ma, C. Han, Y. Yao, G. Tang, Z. Mao, C. Gao, F. Huang, J. Am.
Chem. Soc. 2013, 135, 10310–10313.
35] I. Nierengarten, K. Buffet, M. Holler, S. P. Vincent, J.-F. Nierengarten, Tet-
rahedron Lett. 2013, 54, 2398–2402.
36] K. Buffet, I. Nierengarten, N. Galanos, E. Gillon, M. Holler, A. Imberty, S. E.
Matthews, S. Vidal, S. P. Vincent, J.-F. Nierengarten, Chem. Eur. J. 2016,
[65] T. R. Chan, R. Hilgraf, K. B. Sharpless, V. V. Fokin, Org. Lett. 2004, 6, 2853–
2855.
[66] V. Hong, S. I. Presolski, C. Ma, M. G. Finn, Angew. Chem. Int. Ed. 2009, 48,
9879–9883; Angew. Chem. 2009, 121, 10063–10067.
[67] L. Dupin, F. Zuttion, T. Gehin, A. Meyer, M. Phaner-Goutorbe, J. J. Vas-
seur, E. Souteyrand, F. Morvan, Y. Chevolot, ChemBioChem 2015, 16,
2329–2336.
22, 2955–2963.
[
[
37] N. Galanos, E. Gillon, A. Imberty, S. E. Matthews, S. Vidal, Org. Biomol.
Chem. 2016, 14, 3476–3481.
38] B. Gerland, A. Goudot, G. Pourceau, A. Meyer, V. Dugas, S. Cecioni, S.
Vidal, E. Souteyrand, J.-J. Vasseur, Y. Chevolot, F. Morvan, Bioconjugate
Chem. 2012, 23, 1534–1547.
[39] B. Gerland, A. Goudot, G. Pourceau, A. Meyer, S. Vidal, E. Souteyrand, J.-
Received: May 2, 2016
J. Vasseur, Y. Chevolot, F. Morvan, J. Org. Chem. 2012, 77, 7620–7626.
Published online on && &&, 0000
&
&
Chem. Eur. J. 2016, 22, 1 – 11
www.chemeurj.org
10
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
FULL PAPER
Sweet tyrosine: A series of 27 glyco-
clusters that target lectin A (LecA) have
been synthesized. Aromatic aglycons,
such as phenyl, furanyl, thiophenyl, pyr-
idyl, or tyrosinyl, were investigated and
&
Glycoclusters
S. Wang, L. Dupin, M. Noꢁl, C. J. Carroux,
L. Renaud, T. Gꢀhin, A. Meyer,
E. Souteyrand, J.-J. Vasseur, G. Vergoten,
Y. Chevolot,* F. Morvan,* S. Vidal*
a low-nanomolar (K =19 nm) LecA
d
ligand with a tyrosine-based linker arm
was further investigated by molecular
modelling (see figure; His=histidine).
&
& – &&
Toward the Rational Design of
Galactosylated Glycoclusters That
Target Pseudomonas aeruginosa Lectin
A (LecA): Influence of Linker Arms
That Lead to Low-Nanomolar
Multivalent Ligands
Chem. Eur. J. 2016, 22, 1 – 11
www.chemeurj.org
11
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ