Design and Synthesis of Galactosylated Bifurcated Ligands with Nanomolar Affinity for Lectin LecA from Pseudomonas aeruginosa

Article abstract of DOI:10.1002/cbic.201700154

Lectin A (LecA) from Pseudomonas aeruginosa is an established virulence factor. Glycoclusters that target LecA and are able to compete with human glycoconjugates present on epithelial cells are promising candidates to treat P. aeruginosa infection. A fami

Full text of DOI:10.1002/cbic.201700154

Accepted Article
Title: Design and synthesis of galactosylated bifurcated ligands with
nanomolar affinity for lectin LecA from Pseudomonas aeruginosa
Authors: Anthony Angeli, Muchen Li, Lucie Dupin, Gerard Vergoten,
Mathieu Noel, Mimouna Madaoui, Shuai Wang, Albert Meyer,
Thomas Gehin, Sebastien Vidal, Jean-Jacques Vasseur,
Yann Chevolot, and Francois Morvan
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201700154
Link to VoR: http://dx.doi.org/10.1002/cbic.201700154
A Journal of
www.chembiochem.org

ChemBioChem
10.1002/cbic.201700154
FULL PAPER
Design and synthesis of galactosylated bifurcated ligands with
nanomolar affinity for lectin LecA from Pseudomonas aeruginosa
[
a]
[b]
[b]
[c]
[a]
Anthony Angeli, a Muchen Li, Lucie Dupin, Gérard Vergoten, Mathieu Noël, Mimouna
[
a]
[d]
[a]
[b]
[d]
Madaoui, Shuai Wang, Albert Meyer, Thomas Géhin, Sébastien Vidal, Jean-Jacques
[
a]
[b]
[a]
Vasseur, Yann Chevolot,* François Morvan*
Abstract: Lectin LecA of Pseudomonas aeruginosa is established
as a virulent factor. Glycoclusters targeting LecA able to compete
with human glycoconjugates present on epithelial cells are promising
rectangle (32 and 70 Å away on the width and length,
respectively).[6, 7] Several studies report that galactoclusters can
reach simultaneously two CRDs along the width of one tetramer
leading to chelation.[ LecB (PDB code 2VUD) has more or less
a tetrahedral spatial distribution of the CRDs that lead to a more
difficult chelation.[9] Carbohydrate-lectin interactions usually
occur with low affinities with dissociation constants between mM
to M. To increase the binding, Nature uses multivalency
leading to a cluster effect.[ Along this line, many multivalent
glycoclusters have been synthesized to improve the avidity of
carbohydrate ligands to lectins[11-20] and especially against PA.[8]
However, a high number of carbohydrates is not a guarantee for
a higher affinity due to some steric hindrance. Furthermore, it
has been shown that the topology of the glycocluster is often
more important than the number of carbohydrates to gain a high
affinity for LecA or LecB. Hence glycoclusters targeting LecA or
8]
candidates to treat P. aeruginosa infection.
A family of 32
glycodendrimers of generation 0 and 1 displaying bifurcated bis-
galactosides has been designed to interact with LecA. The influence
of both the central multivalent core and the aglycon of these
glycodendrimers on their affinity toward LecA has been evaluated
using a microarray technology both qualitatively for a rapid screening
of the binding properties but also quantitatively (Kd) leading to high
affinity LecA ligands with Kd values in the low nanomolar range (Kd
10]
=
22 nM for the best one).
Introduction
LecB exhibiting only 3 carbohydrates can reach the nanomolar
range avidity.[21] Furthermore, the simplest the multivalent ligand,
the best for therapeutic applications. There are a few examples
in the literature of bis-galactosides that display high affinity for
LecA. Three bis-galactosides exhibiting a phenyl aglycon display
Pseudomonas aeruginosa (PA) is an opportunistic Gram-
negative bacterium involved in nosocomial diseases. It strongly
affects immunocompromised people and especially cystic
fibrosis patients as it is often related to pulmonary infections.[1]
PA has two soluble lectins LecA (or PA-IL) and LecB (or PA-IIL)
that recognize D-galactose and L-fucose respectively. Both
lectins are involved in biofilm formation.[2-6] LecA (PDB code
K
d
values between 82 and 550 nM corresponding to an increase
[22-24]
of potency per residue between 91 and 159 fold (Figure 1).
The most remarkable bis-galactosides with an increase of
potency per residue between 272 to 3778 fold were those with a
1OKO) has a cobblestone shape with the four carbohydrate
recognition domains (CRDs) located at the corner of the
rigid glucose-triazole backbone reported by Pieters with a K
d
value of 28 nM (Figure 1).[25-28] The docking studies suggested
that such bis-glycosides could chelate simultaneously two
adjacent CRDs.
[
a] Dr A. Angeli, M. Noël, M. Madaoui, A. Meyer, Dr J.-J. Vasseur, Dr F.
Morvan
Institut des Biomolécules Max Mousseron (IBMM) - UMR 5247, CNRS,
Université Montpellier, ENSCM,
Place Eugène Bataillon, CC1704, 34095 Montpellier cedex 5, (France)
E-mail: francois.morvan@umontpellier.fr
[
[
[
b] M. Li, Dr L. Dupin, T. Géhin, Dr Y. Chevolot
Université de Lyon, Institut des Nanotechnologies de Lyon (INL), UMR
CNRS 5270, Site Ecole Centrale de Lyon
36 avenue Guy de Collongue, 69134 Ecully cedex, (France) E-mail: E-
mail: yann.chevolot@ec-lyon.fr
c] Dr G. Vergoten
Unité de Glycobiologie Structurelle et Fonctionnelle (UGSF) - UMR
8576 CNRS - Université de Lille 1,
Cité Scientifique, Avenue Mendeleiev, Bat C9, 59655 Villeneuve
d'Ascq cedex, (France)
d] Dr S. Wang, Dr S. Vidal
Institut de Chimie et Biochimie Moléculaires et Supramoléculaires,
Laboratoire de Chimie Organique 2 – Glycochimie UMR 5246, CNRS -
Université Claude Bernard Lyon 1,
Figure 1. Schematic representation of some bis-galactosides against LecA
43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne, (France)
[25, 26]
[23]
[23]
[24]
with their K
d
values (Compounds G1,
G2, , G3 and G4 ).
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201700154
FULL PAPER
Results and Discussion
We report herein the design, synthesis and binding studies
toward LecA of bis-galactosylated glycodendrimers of
generation 0 and 1 built on two different core scaffolds, one with
a 1,3-propanediol aliphatic core (Figure 2A) and the other with a
1,3-dihydroxybenzene aromatic moiety (Figure 2B). Both
structures have the same distance in terms of number of atoms
between the two functional groups used for conjugation with
galactosides. Since it has been shown that aromatic
galactosides exhibit higher affinity for LecA,[2,
22,
29-32]
we
introduced seven different aromatic -D-galactosides and an
additional triethyleneglycol -D-galactoside for comparison.
These bifurcated bis-galactosides have been designed as a
clamp to chelate two adjacent CRDs of LecA in line with the
finding of Pieters [25, 26] (Figure 2).
Scheme 1. Synthesis of bis-propargyl phosphoramidite 2.
The bis-galactosides were synthesized from eight galactosides
exhibiting different aglycons (Figure 4). Amongst the
galactosides, two were chosen to provide glycoclusters with low
to medium affinities (7a and 7b) based on our previously
reported results,[32] four (7c-f) should display high affinities[30, 32]
and two aglycons are new (7g and 7h) as analogues of aromatic
aglycons 7d and 7e.
CRD
CRD
CRD
CRD
Lectin
Lectin
Figure 2. General structure of bis-galactosides with an aliphatic (A) or
aromatic (B) core. The grey rectangle represents a part of the tetrameric lectin
LecA with two CRDs.
The synthesis of the glycodendrimers was performed by copper
catalyzed azide alkyne cycloaddition (CuAAC)[33, 34] using azido-
functionalized glycosides with different aglycons and bis-
propargylated
tris(hydroxymethyl)ethane
or
3,5-
dihydroxybenzoic acid. In order to rapidly screen the affinity of
these structures toward LecA, they were conjugated to an
oligonucleotide for their subsequent immobilization on a DNA
chip as previously reported.[35,
36]
Both bis-propargylated
scaffolds were prepared as phosphoramidite intermediates 1
and 2 (Figure 3) for their coupling with the oligonucleotide by
phosphoramidite chemistry[37] on solid support. Finally the
glycosides were introduced in solution by CuAAC using the
corresponding azido-functionalized glycosides. The synthesis of
Figure 4. Structures of azido-functionalized -D-galactosides used for the
synthesis of bis-galactosides.
1
was previously reported[38] while phosphoramidite 2 was
prepared from methyl 3,5-dihydroxybenzoate that was
3
The synthesis of galactoside 7g was performed from 4-
propargylated to give 4, then hydrolyzed with LiOH to reach the
benzoic acid 5.[39] Amidation with 6-amino-hexanol afforded 6
that was finally converted into 2 by reaction of 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite (Scheme 1).
carboxyphenyl 2,3,4,6-tetra-O-acetyl--D-galactopyranoside 8[22]
that was conjugated to 3-azidopropylamine affording the fully
protected derivative 9 (Scheme 2). Acetyl groups were removed
by a solution of methanol, triethylamine, water affording pure 7g
after evaporation. Galactoside 7h was prepared in few steps as
described in Scheme 3. First, 3-azidopropylamine was
conjugated to Fmoc-Lys(Boc)-OH affording 11. The Fmoc group
was removed leading to amine 12 and then condensation with
acid 8 afforded the fully protected derivative 13. Acetyl groups
were removed by a solution of methanol, triethylamine and water
then the Boc group was removed by treatment with a 1N HCl
Figure 3. Structure of bis-propargyl phosphoramidites 1 and 2.
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201700154
FULL PAPER
under microwaves assistance affording the fully deprotected
galactoside 7h.
Scheme 4. Synthesis of propargyl tris(hydroxymethyl)ethane 16
The glycodendrimers were designed using
a
propargyl
diethyleneglycol 18 for the generation series and the
0
propargylated diol 16 for the generation 1. To this end the
tris(hydroxymethyl)ethane 15 was monoalkylated with propargyl
bromide affording the propargylated diol 16 (Scheme 4).
Scheme 2. Synthesis of the phenylcarboxamido-propylazide galactoside 7g.
The synthesis of the oligonucleotide glycodendrimers started
with the immobilization of propargyl diethyleneglycol 18 or
propargyl tris(hydroxymethyl)ethane 16 on the azido-
functionalized solid support 17 by means of CuAAC with CuSO
4
and sodium ascorbate under microwaves assistance[40] leading
to 19 and 20 respectively (Scheme 5). Then, the
phosphoramidites 1 or 2 were coupled on the free hydroxyls and
the oligonucleotides (DNA1-4) were elongated and labeled with a
Cy3 fluorescent dye, using standard phosphoramidite cycle
(
solid phase oligonucleotide synthesis SPOS).[37] The cycle
consists to remove the DMTr group, then couple the nucleoside
phosphoramidite, cap the unreacted species and finally oxidize
the phosphite linkage into phosphate one. A final ammonia
treatment afforded the Cy3-oligonucleotides conjugated with one
(21A/B) or two (22A/B) A or B motifs. Each construction was
tagged with a different oligonucleotide sequence in order to
performed multiplexed analysis of their binding with LecA. [41]
Scheme 3. Synthesis of the phenylcarboxamido-lysine-propylazide
galactoside 7h.
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201700154
FULL PAPER
Scheme 5. Solid phase synthesis of Cy3-oligonucleotide conjugated with generation 0 (21A, 21B) or generation 1 (22A, 22B) dendrimer precursors. SPOS Solid
Phase Oligonucleotides Synthesis: a) 3% Cl
THF, pyridine.
3 2 2 2 3 2 2 2
CCO H in CH Cl ; b) amidite, benzylthiotetrazole, CH CN; c) Ac O, N-methyl-imidazole, THF, pyridine; d) I , H O,
Scheme 6. Synthesis of glycodendrimers conjugated with Cy3-oligonucleotides.
After purification by C18 reverse phase HPLC, the four scaffolds
were conjugated by CuAAC in solution with the eight azido-
functionalized -D-galactosides 7a-h (Figure 4). Thus, 32
oligonucleotide glycodendrimers exhibiting one (23A/Ba-h,
generation 0) or two (24A/Ba-h, generation 1) A or B motifs
were obtained (Scheme 6). Each construction was purified by
HPLC and characterized by MALDI-TOF MS (Table S1).
to determine the binding of LecA to the immobilized
glycodendrimers.
The oligonucleotide glycodendrimers 23A/Ba-h and 24A/Ba-h
labeled with the Cy3 dye were immobilized on the microarray by
DNA Directed Immobilization (DDI).
glycocluster (Man ) as a negative control, a mono-phenyl (Ph-
Gal) galactoside and a tetra-phenyl galactoside [(Ph-Gal)
A
tris-mannosylated
3
4
]
previously described[31] as positive controls were also
immobilized (Figure 5). Their correct immobilization was readily
verified by the Cy3 fluorescence signal which deviated by less
than 11%. It has been demonstrated that immobilization of
3 4
Figure 5. Structure of negative (Man) and positive Ph-Gal and (Ph-Gal)
controls.
1
1
12
glycoconjugates by DDI leads to surface densities of 10 -10
molecules per cm2 with a distance between the immobilized
molecules over 30 nm.[42] Under such conditions, each lectin
molecule interacts with only one glycoconjugate. Therefore, no
surface cluster effect is expected. Alexa 647 labeled LecA was
incubated on the resulting carbohydrate microarray and the
Alexa fluorescence signal was recorded averaged over 32 spots
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201700154
FULL PAPER
showing a high affinity for LecA. However, aglycons biPh
(
23A/Bc) and TyrNH
2
(23A/Be) were slightly poorer (~ 28 500-
29 000 a.u.) than the four other aglycons (> 30 000 a.u.). In
general, a slightly higher fluorescence signal could be observed
for the bis-galactosides built with a B motif.
The increase of the valency, from 2 to 4, is more effective for the
binding to LecA for bis-galactosides with A than B motifs. For A
motif, generation 1 tetravalent glycodendrimers 24 displayed a
higher signal of fluorescence than the generation 0 divalent
glycodendrimers 23. For B motif, the change of fluorescence
intensity was mostly negligible except for low affinity aglycons a
and b, and also to a lower extent for the TyrNH
As a result, glycodendrimers 24Ad-h displayed higher binding to
LecA than the control (Ph-Gal) while all glycodendrimers with a
B motif displayed lower fluorescence signals than the control,
excepted 24Bf (TyrCO
H aglycon).[32] We have recently showed
that this TyrCO aglycon led to mannose-centered
tetragalactocluster of high affinity (K This
19 nM).[32]
2
aglycon (24Be).
4
2
2
H
a
d
=
.
difference of affinity could arise from the general structure and
hence topology of the present glycodendrimers. For motif A, the
distance between two triazole rings of two bis-galactosides is 19
atoms while for motif B it is 35 atoms due to the presence of the
two hexylamide linkers. Hence glycodendrimers 24A could be
envisaged as tetraglycoclusters while glycodendrimers 24B as
two bifurcated bis-galactosides. The binding properties are
mostly influenced by the aglycon while valency appears as a
Figure 6. Screening of the binding properties towards LecA using the
carbohydrate microarray through the Alexa 647 fluorescence signal of labeled
LecA (arbitrary units a.u.). "Upper panel: 23-24Aa-h; Lower panel: 23-24Ba-
h. Divalent compounds: light blue; Tetravalent compounds: blue. Controls:
red". Lectin concentration of 0.12 μM.
secondary parameter. The tetraphenyl galactoside on
a
mannose core (Ph-Gal) displayed a fluorescence signal (36 400
4
a.u.) similar to that of the best digalactoside 23Ah with LysPh
aglycon. This data shows that the topology and the nature of the
aglycon are two important parameters to consider for reaching
high affinity.
The fluorescence signal of negative control Man
3
in the
background (60 a.u.) confirmed that no unspecific adsorption of
LecA occurred. For both motifs, excepted in the case of the
methylenepyridyl aglycon (b), all aromatic aglycons lead to an
In order to have a better insight of the influence of each aglycon
and of the topology on the affinity, the values of dissociation
constant (K
high fluorescence signal (>28 000 a.u.) were measured and
compared with the K value of (Ph-Gal) (Table 1).
d
) for the 12 glycodendrimers (23A/Bc-h) exhibiting
increased affinity compared to the triethyleneglycol (EG
3
) linker
(
a). This increased binding has been reported several times in
the literature [13, 22, 29, 30, 32] and is the result of a face-to-edge or
d
4
edge-to-edge  stacking between the aromatic ring and the
His50 of the lectin. The fluorescence signal observed after
d
Table 1. K values of the best bis-galactosides.
binding of LecA to bis-galactosides with a EG
3
aglycon were low
Glycodendrimer
K
d
(nM)
Rank
10
5eq
11
4
5eq
2
(
23Aa 2569 a.u., 23Ba 1672 a.u.), more surprisingly the
23Ac
23Ad
62 ± 2
38 ± 1.5
68 ± 1
35 ± 1
38 ± 2.5
32 ± 6
fluorescent signals for bis-galactosides with a methylenepyridyl
aglycon (23Ab and 23Bb) were lower (<1000 a.u.). Increasing
the valency from two (23Ab and 23Bb) to four (24Ab and 24Bb),
improved significantly the binding by a factor of nearly 4 and 20,
respectively. Interestingly, the lower the binding of the divalent
clusters was, the higher was the gain due to increased valency
from 2 to 4. The same trend was observed for glycocluster with
23Ae
Valency 2
23Af
23Ag
23Ah
2
3Bc
3Bd
3Be
23Bf
75 ± 3.5
56 ± 1.5
75 ± 11.5
40 ± 4.5
33 ± 1.5
22 ± 2
12eq
2
9
12eq
8
3
1
2
Valency 2
Valency 4
3
EG aglycon (23Aa vs 24Aa and 23Ba vs 24Ba). These mono-
23Bg
and bis-digalactosides exhibited lower fluorescence signal than
the mono-phenyl galactoside (Ph-Gal) (25 000 a.u.). This result
confirmed the high affinity for LecA of glycodendrimers built with
aromatic galactoside. All the other bis-galactosides with A and B
motifs exhibited a high fluorescence signal above 28 000 a.u.
23Bh
39[43]
(Ph-Gal)
4
7
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201700154
FULL PAPER
d
Dissociation constants (K ) were measured using our previously
reported protocol.[44] It is based on incubation of DDI immobilized
glycoclusters with increasing concentrations of lectin. The
resulting isotherms are linearized and the K
from the y-intercepts.[45] All measured K
between 22 and and 75 nM. This data confirmed the high affinity
of these glycodendrimers for LecA. K values are in agreement
with the fluorescence signals confirming that biPh (c) and
TyrNH (e) aglycons displayed lower affinities for LecA than the
d
values calculated
d
values are ranked
d
2
others (rank 10-12).
The bis-galactosides (23A/Bh) with the Lys-Ph aglycon
d
displayed the lowest K values regardless from the motif (A 32
nM or B 22 nM, rank 1-2). Glycoclusters built with Lys-Ph-
galactoside have been reported previously.[22] In such a case,
the increased binding was attributed to the interaction of the
ammonium function from the lysine lateral chain with the Asp47
residue of LecA. This improved binding was not observed for
Figure 7. Bis-galactoside 23Ah interacting with LecA. Red circles show the
ammonium of the lysine chains. Sticks represent 23Ah and ball and sticks in
yellow represent some amino acids of monomers A and B of LecA.
For compound 23Bh, one ammonium group of a lysine chain
exhibits two strong ionic interactions with Asp47 and Glu49 of
monomer B while the ammonium group of the other lysine chain
interacts through a hydrogen bond donor to Gln40 with the
monomer A (Figure 8). The phenyl ring in the monomer B has
two - interactions with His50 and Tyr36 while the other phenyl
ring has a - interaction with Pro38 (monomer A). This double
strong ionic interaction could explain why this bis-galactoside
2 d
bis-galactosides 23Ag and 23Bg with TyrNH aglycons (K = 68
and 75 nM, respectively, rank 11-12). Therefore, for these
glycoclusters, the amine function of the tyrosine did not seem to
engage any additional interaction with the lectin. The amine is
probably located too far from the Asp47 residue. However, the
bis-galactosides 23A/Bf with TyrCO
affinity with K values (23Af 35 nM and 23Bf 40 nM; rank 4 and
) that are nearly twice lower than the bis-galactosides TyrNH
Finally, bis-galactosides with phenyl aglycons (d and g) have
similar K values (23Ad,g and 23Bg 33-38 nM, rank 3-5),
2
H aglycon displayed a high
d
8
2
.
d
displayed the lowest E and K values.
d
excepted for 23Bd (56 nM, rank 9) suggesting that inverting the
amide function with respect to the aromatic ring and addition of
two methylenes have a limited effect on the binding to LecA.
The two glycoclusters 23A/Bh exhibiting the best affinities were
further investigated by using molecular simulations and docking
techniques. An empirical calculation of the potential energy of
interaction E of both bis-galactosides 23A/Bh gave -216 and -
d
260 kcal/mol respectively that is consistent with the K values.
For all galactoside, we observed the characteristic coordination
of the C3 and C4 hydroxyls to the lectin-bound calcium ion and
C6 hydroxyls of the galactose form a hydrogen bond with each
His50 residue of adjacent LecA monomer.[5, 22, 46, 47] For 23Ah,
one ammonium group of a lysine chain interacts through a
hydrogen bond to Gln40 (monomer A) while the ammonium
group of the other lysine chain gives a strong ionic interaction
with Glu49 (monomer B). Both phenyl rings of the bis-
galactoside form a - interaction with both Pro38s (Figure 7).
Figure 8. Bis-galactoside 23Bh interacting with LecA. Red circles show the
ammonium of the lysine chains. Sticks represent 23Bh and ball and sticks in
yellow represent some amino acids of monomers A and B of LecA.
Conclusions
In the present contribution, 32 glycodendrimers exhibiting eight
different aglycons were synthesized on two different central
motifs (A corresponding to an aliphatic linker and B to an
aromatic linker) and tagged with
a
Cy3-fluorescent
oligonucleotide sequence. Thanks to their immobilization on a
DNA microarray, their binding to LecA from Pseudomonas
aeruginosa has been rapidly, qualitatively and quantitatively
evaluated. The results showed that the effect of the central core
on the affinity of the glycoclusters was limited. In contrast, the
nature of the aglycon led to strong difference of affinity. The
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201700154
FULL PAPER
galactoclusters with triethyleneglycol or methylenepyridyl
aglycons bound poorly to LecA. In contrast, those with phenyl,
biphenyl, naphthyl and phenyl-lysine aglycons showed high
30
min,
benzotriazol-1-yloxytris(dimethylamino)-phosphonium
hexafluorophosphate (2.07 g, 4.2 mmol) was added and the mixture was
stirred during 90 min. The solvent was removed in vacuo, and the crude
product was dissolved in EtOAc. The organic layer was washed with satd
d
affinity with K values between 22 to 75 nM. While the increase
NaHCO
Na SO
flash chromatography (20% EtOAc in cyclohexane) to afford the 6 (1.13 g,
97%). H NMR (400 MHz, CDCl ) δ 7.07 (d, J = 2.3 Hz, 2H, Ar), 6.79 (t, J
= 2.3 Hz, 1H, Ar), 4.77 (d, J = 2.4 Hz, 4H, CH
2H, CH OH), 3.36 (t, J = 7.2 Hz, 2H, NHCH ), 2.97 (t, J = 2.4 Hz, 2H,
CCH), 1.68-1.52 (m, 4H, NHCH CH and CH CH OH), 1.47-1.39 (m, 4H,
EtCH CH ) δ 169.5, 160.2, 137.9,
EtOH). 13C NMR (100 MHz, CDCl
08.0, 106.2, 79.4, 77.1, 62.9, 57.0, 41.0, 33.6, 30.4, 27.9, 26.6. HR-ESI-
3
(2 x 20 mL) and brine (2 x 20mL). The organic layer was dried
), and concentrated in vacuo. The crude product was purified on
of valency from two to four has a noticeable beneficial effect on
the binding of glycoclusters with A motif, it was mostly negligible
for those with B motif. Our results are in good agreement with
the literature and suggest that the primary parameter for
enhanced binding is the structure of the aglycon. In particular, as
already reported by several groups including ourselves, the
aromatic aglycon is a major contributor to the binding through
(
2
4
1
3
2
CC), 3.55 (t, J = 6.6 Hz,
2
2
2
2
2
2
2
2
3
1
 and s-p interactions between the phenyl of the aglycon and
11 4
QToF MS (positive mode): m/z calcd. for C13H O
[M+H]+ 330.1705,
the amino acids of lectin (His50, Tyr36and Pro38). In the present
work, we showed that the introduction of an additional lysine
found 330.1705.
(
ammonium chain) allows additional ionic and hydrogen bound
interactions with the amino acids of LecA leading to bis-
galactosides 23A/Bh with values of 32 and 22 nM
6
-(3,5-bis(Prop-2-ynoxy)benzamido)hexyl)-(2-cyanoethyl)-N-
diisopropyl phosphoramidite 2: N-(6-Hydroxyhexyl)-3,5-bis(prop-2-
ynoxy)benzamide 6 (543 mg, 1.65 mmol) was added to a solution of
K
d
respectively. Indeed, as investigated by molecular simulations
and docking studies, additional electrostatic interactions further
reinforced the affinity through strong ionic interactions between
Asp47 and Glu49 and the ammonium of the lysine lateral chain
and additional hydrogen bonds with Gln40.
The synthesis of both bis-galactosides 23A/Bh without the DNA
tag is in progress for subsequent evaluation of their anti-
infectious activity against Pseudomonas aeruginosa.
DIEA (645 µL, 4.0 mmol), CH
mixture was stirred for 30 min on 4Å molecular sieves under argon, then
-cyanoethyl N,N-diisopropylchlorophosphoramidite (239 mg, 2.0 mmol)
was added and the mixture was stirred for 1 h. After addition of water (1
mL) and 15 min stirring, cyclohexane (20 mL) was added and the
solvents weres removed in vacuo. The crude residue was purified on
2 2
Cl (5 mL) and acetonitrile (5 mL). The
2
silica gel chromatography (NEt
leading to 2 (823 mg, 94%). H NMR (400 MHz, CDCl
3
10% and EtOAc 40% in cyclohexane)
) δ 7.06 (t, J = 5.0
1
3
Hz, 1H, NH), 7.01 (d, J = 2.3 Hz, 2H, Ar), 6.71 (t, J = 2.3 Hz, 1H, Ar), 4.76
(
(
d, J = 2.4 Hz, 4H, CH
q, J = 7.0 Hz, 2H, NCH
CN), 1.62-1.53 (m, 4H, NHCH
.35 (m, 4H, EtCH CH EtOH), 1.16 (m, 12H, 4xCH
) δ 166.9, 159.5, 138.4, 118.3, 107.5, 105.7, 79.3, 77.1, 64.2 (d),
2
CC), 3.81-3.53 (m, 6H, 2xCH
), 2.84 (t, J = 2.4 Hz, 2H, CCH), 2.63 (t, J = 6.0
CH and CH CH OH), 1.43-
). 13C NMR (100 MHz,
2 3
OP, NCHMe ), 3.31
2
Hz, 2H, CH
1
CDCl
2
2
2
2
2
Experimental Section
2
2
3
3
General methods
5
9.2 (d), 56.8, 43.7 (d), 40.5, 31.8 (d), 30.1, 27.3, 26.4, 24.9 (d), 21.1 (d).
3
1P NMR (162 MHz, CDCl
3
) δ 147.0 (s, 1P). HR-ESI-QToF MS (positive
All reagents for synthesis were commercial and used without further
purification. Solvents were commercial and used without further
purification. All moisture sensitive reactions were performed under an
argon atmosphere. NMR solvents were purchased from Euriso-Top.
NMR spectra were recorded at 293 K using a 200, 300, 400 or a 600
MHz spectrometer (Bruker). Shifts (δ) are referenced relative to
deuterated solvent residual peaks and expressed in parts per million
+
mode): m/z calcd. for C28
H
41 3 5
N O P [M+H] 530.2784, found 530.2784.
N-(3-Azidopropyl)-4-carboxamidophenyl
galactopyranoside 9: To a solution of 4-carboxyphenyl 2,3,4,6-tetra-O-
acetyl--D-galactopyranoside
dichloromethane (2 mL), 3-azidopropylamine (84 mg, 0.72 mmol) and
DIEA (157 L, 0.96 mmol) were added with a few beads of molecular
sieves (4 Å). After 1 h stirring under argon, BOP (283 mg, 0.62 mmol)
was added and the solution was stirred for 1 h at rt. After evaporation,
the mixture was purified on silica gel (15 g) using cyclohexane/EtOAc
2,3,4,6-tetra-O-acetyl--D-
8[22]
(224 mg, 0.48 mmol) in
(ppm). Coupling constants are expressed in hertz (Hz). The following
abbreviations are used to explain the observed multiplicities: s (singlet), d
(
(
doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), sbr
broad singlet). High-resolution (HR-ESI-QToF) mass spectra were
(
(
7:3 to 3:7 v/v) affording
cyclohexane/EtOAc, 2:8, v/v). []
CN) δ 7.82-7.64 (m, 2H, Ar), 7.12-6.99 (m, 2H, Ar), 5.40 (dd, J
3.5, 1.1 Hz, 1H, H4), 5.32-5.27 (m, 2H, H1, H2), 5.24-5.12 (m, 1H, H3),
.27-4.19 (m, 1H, H5), 4.15 and 4.09 (2dd, J = 11.3 and 5.4 Hz, 2H, H6),
.41 and 3.39 (2t, J = 6.6 Hz, 4H, CH CH CH ), 2.11 (s, 1H, COCH ),
.99 (s, 3H, COCH ), 1.98 (s, 3H, COCH ), 1.93 (s, 3H, COCH ), 1.80 (p,
). 13C NMR (100 MHz, CD
CH CN) δ 170.9, 170.8,
9
(153.3 mg, 58%).
= 0.0 (c = 1.0, MeOH). H NMR (400
R
f
=
0.43
recorded using a Q-Tof Micromass spectrometer. MALDI-TOF mass
spectra were recorded on a Voyager mass spectrometer (Perspective
Biosystems, Framingham, MA) equipped with a nitrogen laser. Thin layer
chromatography (TLC) was carried out on aluminum sheets coated with
1
D
MHz, CD
3
=
4
3
1
2
2
2
N
3
3
254 nm) and developed by treatment with a mixture of 10% H
2 4
SO in
3
3
3
EtOH/H O (1:1 v/v), vanillin, KMnO or phosphomolybdic acid followed by
2
4
J = 6.6 Hz, 2H, CH
2
N
2 3
3
heating. Silica gel column chromatography was performed with silica gel
Si 60 (40–63 mm). Reverse phase chromatography was performed with
a C18 reversed phase flash column.
1
6
70. 5, 170.1, 166.9, 159.6, 130.1, 129.5, 116.7, 98.9, 71.8, 71.1, 69.1,
7.9, 62.0, 49.5, 37.4, 29.1, 20.5, 20.48, 20.4, 20.39. HR-ESI-QToF MS
11 [M + H]+ 551.1989, found
(positive mode): m/z calcd. for C24H N O
31 4
551.1993.
Compounds 5,[39] 7a,[48] 7b,[32] 7c,[30] 7d,[29] 7e,[32] 7f,[32] 7i,[49] 8,[22] were
prepared according to the literature.
N-(3-azidopropyl)-4-carboxamidophenyl -D-galactopyranoside 7g:
Compound 9 was dissolved in a solution of MeOH/H O/Et N (4:1:1 v/v/v)
2
3
N-(6-Hydroxyhexyl)-3,5-bis(prop-2-ynoxy)benzamide 6: 3,5-bis-(prop-
2
and stirred 3 h at rt. After evaporation, galactoside 7g was obtained. HR-
-ynoxy)benzoic acid 5[39] (810 mg, 3.5 mmol) and 6-amino hexanol (450
[M+H]+
ESI-QToF MS (positive mode): m/z calcd. for
83.1567, found 383.1565.
16 23 4 7
C H N O
mg, 3.85 mmol) were dissolved in anhydrous CH
2 2
Cl (10 mL) with dry
3
DIEA (1.14 mL, 7.0 mmol) with 4Å molecular sieves under argon. After
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201700154
FULL PAPER
FmocLys(Boc) (3-azidopropyl)amide 11: To
a
solution of Fmoc-
3
CD OD) δ ppm: 7.91-7.77 (d, J = 8.8 Hz, 2H, Ar), 7.23-7.07 (d, J = 8.8 Hz,
Lys(Boc)-OH (1.0 g, 2.13 mmol) in CH
5.5 mmol) followed by 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide
EDC (1.2 g, 6.3 mmol) and HOBt (850 mg, 6.29 mmol) were added. The
mixture was stirred at room temperature for 2 days. CH Cl was added
and the organic layer was washed with a 1M HCl (10 mL), a white
precipitate was formed and removed by filtration. The organic layer was
2
Cl
2
(20 mL), 3-azidopropylamine
2H, Ar), 4.95 (d, J = 7.7 Hz, 1H, H1), 4.46 (dd, J = 8.8, 5.7 Hz, 1H, CH
Lys), 3.92 (d, J = 3.4 Hz, 1H, H4), 3.85-3.69 (m, 4H, H2, H5, H6), 3.60
(
(dd, J = 9.7, 3.4 Hz, 1H, H3), 3.36 (t, J = 6.7 Hz, 2H, CH
(m, 2H, CH EtN ), 3.04 (t, J = 6.7 Hz, 2H, CH ), 1.90-1.74 (m, 4H, CH
CH CH ), 1.38-1.56 (m, 4H, CH , CH ), 1.41 (s, 9H, 3 x CH
NMR (100 MHz, CD OD) δ ppm: 174.8, 169.8, 162.0, 130.3, 128.8, 117.3,
2 3
N ), 3.30-3.27
2
2
2
3
2
2
,
C
13
2
N
2 3
2
2
3
).
3
washed with water, dried (Na
residue was purified by silica gel flash chromatography (CH
5:5) to afford compound 11 (753 mg, 58%) as an amorphous white solid.
= 0.33 (CH Cl ) δ ppm: 7.73
/MeOH 95:5). 1H NMR (400 MHz, CDCl
d, J = 7.5 Hz, 2H, CHAr), 7.55 (d, J = 7.4 Hz, 2H, CHAr), 7.37 (t, J = 7.5
Hz, 2H, CHAr), 7.27 (t, J = 7.4 Hz, 2H, CHAr), 6.83 (bs, 1H, NH amide),
2
SO
4
) and evaporated under vacuum. The
102.4, 77.1, 74.8, 72.2, 70.2, 62.4, 55.6, 50.1, 41.0, 37.8, 32.8, 30.6,
2
Cl /MeOH
2
29.7, 28.8, 24.4. HR-ESI-QToF MS (positive mode): m/z calcd. for
+
9
R
(
C
27
H
43
N
6
O
10 [M+H] 611.3041, found 611.3045.
f
2
2
3
4
7
-(β-D-Galactopyranosyloxy)-benzamide Lys (3-azidopropyl) amide
h: Compound 14 (636 mg, 1 mmol) was solubilized in HCl 1N (10 mL).
5
4
4
CH
(
.86 (bd, J = 5.6 Hz, 1H, NH urethane), 4.78 (s, 1H, NH urethane), 4.46-
The mixture was stirred at 70°C for 5 min under microwave irradiation.
Polyvinyl pyridine (PVP 2% cross-linked, 60 mesh, 8 meq/g) was added
to neutralize the reaction. The mixture was filtered and the PVP washed
.24 (m, 2H, CH
H, CH CH CH
CH
100 MHz, CDCl
2
Fmoc), 4.16 (m, 2H, CHLys/CHFmoc), 3.37 – 3.19 (m,
), 3.08 (m, 2H, CH ), 1.77-1.68 (m, 4H, CH
, CH
) δ ppm: 172.2, 156.6, 156.3, 127. 8, 127.1, 125.2,
20.0, 79.2, 67.1, 54.9, 49.1, 47.1, 39.9, 37.0, 32.1, 29.6, 28.7, 28.5,
2
2
2
N
3
2
2
,
13
2
2
N
3
), 1.55-1.27 (m, 4H, CH
2
2 3
, 1.42 (s, 9H, 3CH ). C NMR
with MeOH. The solvent was evaporated under vacuum to afford
3
compound 8 (518 mg, 97%) as an amorphous white solid. [α]
20
= -2.0 (c
D
1
2
1
=
7
1
2
1.0 H O). H NMR (400 MHz, D
2
O) δ ppm: 7.89-7.79 (m, 2H, Ar), 7.28-
.18 (m, 2H, Ar), 5.17 (d, J = 7.5 Hz, 1H, H1), 4.42 (dd, J = 8.7, 6.0 Hz,
H, CH Lys), 4.02 (dd, J = 3.2, 0.8 Hz, 1H, H4), 3.97-3.74 (m, 5H, H2,
2.6. HR-ESI-QToF MS (positive mode): m/z calcd. for C29
H N O
39 6 5
+
[M+H] 551.2982, found 551.2985.
H3, H5, H6), 3.37 (t, J = 6.6 Hz, 2H, CH
NHCH EtN ), 3.01 (t, J = 7.7 Hz, 2H, CH ), 1.98-1.65 (m, 6H, CH
CH CH , CH ). C NMR (100 MHz, D O) δ ppm:
174.2, 170.2, 159.7, 129.4, 127.3, 116.2, 115.4, 100.1, 75.5, 72.5, 70.4,
68.4, 60.7, 54.6, 48.7, 39.2, 36.7, 30.4, 27.6, 26.3, 22.3. HR-ESI-QToF
[M+H]+ 511.2516, found
35 6 8
MS (positive mode): m/z calcd. for C23H N O
2 3
N ), 3.35-3.28 (m, 2H,
Lys(Boc) (3-azidopropyl) amide 12: A solution of FmocLys(Boc) (3-
azidopropyl) amide 11 (639 mg, 1.16 mmol) in piperidine/THF (4:6, v/v)
was stirred at rt for 4 h. The solvent was coevaporated with toluene (7
2
3
2
2
,
13
2
2
N
3
2
), 1.50 (m, 2H, CH
2
2
times) and CHCl
3
(3 times) to obtain a yellow oil purified by silica gel
Cl /MeOH 9:1) to afford Lys(Boc) (3-
flash chromatography (CH
2
2
azidopropyl) amide (358 mg, 92%) as
a
clear oil.
) δ ppm: 7.51 (d, J = 4.3
CH CH , CH
), 1.90-1.64 (m, 5H, CH, CH , CH CH ),
). C NMR (100 MHz, CDCl ) δ
ppm: 175.1, 156.1, 79.2, 55.0, 49.3, 40.1, 36.6, 34.5, 29.9, 28.9, 28.4,
R
f
=
0.43
511.2522.
1
2
(CH Cl
2
/MeOH 9:1). H NMR (400 MHz, CDCl
3
Hz, 1H, NH), 4.59 (s, 1H, NH), 3.41-3.22 (m, 5H, CH
lys), 3.18-3.02 (m, 2H, CH
.59-1.28 (m, 12H, CH-7’-CH, 3 x CH
2
2
2 3
N
2-Methyl-2-[(prop-2-ynoxy)methyl]propane-1,3-diol
16.
Sodium
2
2
2
2 3
N
hydride (400 mg, 10 mmol) was added to
a
solution of 1,1,1-
13
1
3
3
tris(hydroxymethyl)ethane (1.2 g, 10 mmol) in dry THF (30 mL) under
argon atmosphere. Then, a solution of propargyl bromide (0.52 mL, 5
mmol) in dry THF (10 mL) was added dropwise at 0°C. The mixture was
stirred overnight and water was added to quench the reaction. After 15
min, the crude mixture was coevaporated with toluene, and directly
purified by silica flash chromatography (EtOAc 50% to 100% in
2
2.9. HR-ESI-QToF MS (positive mode): m/z calcd. for C14
H N O
29 6 3
+
[M+H] 329.2301, found 329.2300.
4
-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyloxy)-benzamide
Lys(Boc) (3-azidopropyl) amide 13: Lys(Boc) (3-azidopropyl)amide 4
337 mg, mmol) and 4-(2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosyloxy)-benzoic acid 5 (472 mg, 1 mmol) were solubilised
in CH Cl (4.5 mL). The solution was stirred with molecular sieve 4Ǻ for
0 min. BOP (505.6 mg, 1 mmol) and DIEA (340 µL, 2 mmol) were
1
cyclohexane) leading to compound 16 (480 mg, 61%). H NMR (400 Mz,
(
1
CDCl
3
) 4.16 (d, J = 2.4 Hz, 2H, CH
2
C≡CH), 3.62-3.54 (m, 6H, CH
2
), 2.45
(t, J = 2.4 Hz, 1H, ≡CH), 0.85 (s, 3H, CH
literature data.[39]
3
). In agreement with the
2
2
3
added and the mixture was stirred at rt for 1 h. The solvent was
evaporated under vacuum and the residue was purified by silica gel flash
chromatography (cyclohexane/EtOAc) to afford compound 13 (820 mg,
Immobilization of alkynes 16 and 18 on azido-functionalized solid
support 17 by CuAAC: An aqueous solution of propargyl diethylene
glycol 18 or compound 16 (0.2 M, 50 µL, 10 mol), freshly prepared
f 2 2
= 0.41 (CH Cl H
/MeOH 95:5). 1
9
9%) as an amorphous white solid. R
NMR (400 MHz, CDCl ) δ ppm: 7.79 (d, J = 8.7 Hz, 2H, Ar), 7.06-6.99 (d,
J = 8.7 Hz 2H, Ar), 6.90 (bs, 1H, NHCO), 5.55-5.43 (m, 2H, H2, H4),
.22-5.04 (m, 2H, H1, H3), 4.59 (m, 1H, CH Lys), 4.23-4.09 (m, 3H, H5,
H6), 3.39-3.29 (m, 4H, CH CH CH ), 3.11 (m, 2H, CH ), 2.18 (s, 3H,
CO), 2.06 (s, 3H, CH CO), 2.06 (s, 3H, CH CO), 2.01 (s, 3H,
CO), 1.99-1.88 (m, 1H, CH), 1.84-1.74 (m, 3H, CH CH , CH),
), 1.47-1.35 (m, 11H, CH , 3xCH
). 1 C NMR
) δ ppm: 171.0, 169.3, 169.2, 169.1, 158.5, 128.0, 127.3,
aqueous solutions of CuSO (0.04 M, 25 µL, 1 mol) and sodium
4
3
ascorbate (0.1 M, 50 µL, 5 mol), TEAAc buffer (2 M, 25 µL), tris(3-
hydroxypropyltriazolylmethyl)amine (THPTA, 0.1 M, 20 µL, 2 mol) and
MeOH (80 µL) were added to 1 µmol of azide solid support 17. The
resulting mixture was heated at 65°C under microwave for 30 min at 250
5
2
2
2
N
3
2
CH
CH
3
3
3
3
rpm. The solution was removed and CPG beads were washed with H
2
O
2
2 3
N
3
(
5 mL), MeOH (5 mL) and CH Cl (5 mL) then dried affording 19 and 20.
2
2
1.59-1.48 (m, 2H, CH
2
2
3
(100 MHz, CDCl
3
Glyco-oligonucleotide synthesis: The oligonucleotide synthesis on
solid support was performed on a DNA synthesizer Applied Biosystems
1
16.0, 98.0, 76.2, 70.2, 70.0, 67.5, 65.8, 60.4, 52.5, 48.1, 39.0, 36.1,
1.0 , 28.5, 27.6, 27.4 , 21.7, 19.7, 19.6, 19.5. HR-ESI-QToF MS
3
(381A or 394 DNA synthesizer). Reactions under microwave activation
50 6
positive mode): m/z calcd. for C35H N O
14Na [M+Na]+ 801.3283, found
(
were performed on an Anton Paar Monowave 300 system. Solutions of
Cap A, Cap B and iodide were purchased from Link Technologies as well
801.3286.
3
as the commercial solid supports. Solutions of TCA and CH CN for DNA
4
-(β-D-Galactopyranosyloxy)-benzamide Lys(Boc) (3-azidopropyl)
synthesis were purchased from Biosolve. Cy3-amidite was purchased
from GE Healthcare. All glycooligonucleotides were purified and
analyzed by C18 reversed-phase HPLC (Macherey-Nagel, Nucleodur
amide 14: Compound 6 was solubilized in MeOH/H
v/v/v) and the mixture was stirred at rt for 2.5 h. The solvent was
2
O/NEt (8 mL, 5:1:2,
3
evaporated to afford compound 14 (695 mg, quantitative) as an
4
.6×75 mm, 3 mM) on a Dionex Ultimate 3000 system with a Reodyn
1
amorphous white solid. R
f
2
= 0.13 (CH Cl
2
/MeOH 9:1). H NMR (400 MHz,
injector and a detector UV DAD 3000. Oligonucleotides were dosed by
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201700154
FULL PAPER
UV-Vis spectrophotometry at 550 nm on a Varian Cary 300 Bio UV-Vis
spectrophotometer.
lyophilized. The Table S1 reports the method used according to each
glycocluster.
General procedure for introduction of phosphoramidites 1 and 2 on
Microarray studies
solid-supported scaffolds 19 and 20: Solid-supported scaffolds 19 and
2
0 (1 µmol) were treated, on a DNA synthesizer, with phosphoramidites 1
Microarray fabrication: Borosilicate glass slides (Glass D, Schott)
bearing 40 microwells fabricated by xurography [42] were chemically
and 2 using only coupling and oxidation steps. For the coupling step,
benzylmercaptotetrazole was used as an activator (0.3 M in anhydrous
CH CN) and phosphoramidites 1 and 2 (0.2 M in anhydrous CH CN)
3 3
were introduced once on 10 and twice on 11 with a 30 s coupling time.
Oxidation was performed with 0.1 M commercial solution of iodide for 15
s.
modified with tert-butyl (dimethylamino)silylundecanoate (TDSUM) to
introduce carboxylic acids on the surface. After deprotection (glacial
formic acid) and activation (DIC, NHS in THF, 0.1 M), amino-modified
oligonucleotides (25 μM in PBS 10X, pH 8.5) were printed with a
sciFLEXARRAYER s3 system piezoelectric leading to 32 repetitions of
each sequence per well. The slides were washed at 70°C in SDS 0.1%
for 30 min. To avoid non-specific adsorption, blocking was performed
with a 4% solution of BSA in PBS 1X (pH 7.4) for 2 h and washed (3×3
minutes in PBS 1X-Tween20 0.5%, pH 7.4 then 3×3 min in PBS 1X then
rinsed with water).
General procedure for elongation of DNA sequences and labelling
with Cy3: The DNA sequences were synthesized on the solid-supported
scaffolds (LCAA-CPG, 500 Å) at the 1 µmol scale on a DNA synthesizer
(
ABI 394) by phosphoramidite chemistry. For the coupling step,
benzylmercaptotetrazole was used as an activator (0.3 M in anhydrous
CH CN), and commercially available nucleoside phosphoramidites (0.1 M
in anhydrous CH CN) were introduced with a 20 s coupling time and Cy3
amidite (0.06 M in anhydrous CH CN) with a 180 s coupling time. The
capping step was performed with acetic anhydride using commercial
solution (Cap A, Ac O/Pyridine/THF, 1:1:8, v/v/v; Cap B, 10% N-
methylimidazole in THF) for 15 s. Each oxidation was performed with
commercial solution of iodide (0.1 M in THF/pyridine/H O, 78:20:2, v/v/v)
for 15 s. Detritylation was performed with 3% TCA in CH Cl for 65 s.
3
Each galactocluster was labelled with Cy3 and displayed a specific DNA
sequence complementary to one of the sequence covalently immobilized
in the microwell. Consequently, the galactoclusters were specifically
addressed on the array by DNA/DNA duplex formation. Galactoclusters
3
3
2
(in PBS 1X, pH 7.4) were mixed (final concentration 1 μM each) and 2 μL
of the resulting mixture were poured into each microwell. Incubation was
performed in a humidity saturated chamber at 37°C for 3 h. Next, the
slides were washed successively with SSC 2X, SDS 0.1% at 51°C for 1
min, SSC 2X for 5 min at rt and rinsed with water. In order to assess the
relative surface density of the clusters, slides were scanned at 532 nm
(excitation of Cy3) by using the Axon microarray scanner. Image analysis
was performed using the Genepix 4100 A software package. For each
galactoclusters, the Cy3 mean fluorescence signal was averaged.
2
2
2
General procedure for déprotection: The CPG beads bearing modified
oligonucleotides were transferred to a 4 mL screw top vial and treated
with 2 mL of concentrated aqueous ammonia for 15 h at 25°C. The
supernatants were withdrawn and evaporated affording crude 21A-B and
2
2A-B.
General procedures for CuAAC in solution:
Method A: To solution of 5’-fluorescent-3’-alkyne platform
Probing Galactocluster-LecA interaction on microarray: The
interaction of LecA (purchased from Elicityl, Crolles, France) with the
galactoclusters was interrogated with Alexa647-LecA (Invitrogen
microscale Alexa labeling kit). 2 μL of Alexa647-LecA (0.12 μM, 7.5 μM
CaCl2, 2% BSA in PBS1X) were added in each well. Incubation was
performed at 37°C for 3 h in a water saturated chamber. The slides were
washed successively with PBS1X (pH 7.4)-Tween20 0.02% for 5 min,
rinsed with water and scanned at 635 nm (excitation).
a
oligonucleotides 21A-B and 22A-B (1 mM in water) were added
glycoside derivatives (20 mM, 12 eq.), THPTA (10 mM, 3 eq.), and 1 mg
0
Cu . The resulting mixture was sonicated, stirred for 15 min at 65°C then
15 min at room temperature. The crude solutions were centrifuged and
the supernatant were directly purified by C18 reversed phase HPLC. The
products were coevaporated with water and lyophilized.
Dissociation constant determination on microarray:[42] LecA
concentration was varied from 0.01 nM to 2 μM. Alexa647 fluorescence
signal was recorded and from the resulting isotherm, the Kd values were
determined using the following equation:
Method B: To
a
solution of 5’-fluorescent-3’-alkyne platform
oligonucleotides 21A-B and 22A-B (1 mM in water) were added
glycoside derivatives (20 mM, 12 eq.), THPTA (10 mM, 3 eq.), TEAAc (2
0
M, 10 µL), water (20 µL) and 1 mg Cu . The resulting mixture was
[LecA]/FI = 1/FImax*[LecA] + Kd/FImax
sonicated, stirred for 15 min at 65°C then 15 min at room temperature.
The crude solutions were centrifuged and the supernatant were directly
purified by C18 reversed phase HPLC. The products were coevaporated
with water and lyophilized.
Where FI represents the fluorescence signal recorded at 635 nm for the
given concentration of LecA and FImax is the maximum fluorescence
signal obtained at 635 nm.
Method C: To
a
solution of 5’-fluorescent-3’-alkyne platform
In silico molecular docking: The three dimensional structure of LecA
was retrieved from the Protein Data Bank (www.rcsb.org) under the PDB
code 2VXJ. For both glycoclusters (23Ah and 23Bh) the building
procedure of the lectin-ligand complex is depicted as follows: The
galactoses endings are brought close to the crystallographic galactose
ligands. The terminal sugars of the two glycocluster branches are then
removed and the chemical bonds with galactose moieties of LecA are
built. LecA and its two galactose groups are considered as aggregates
and the complex is optimized. A classical Monte Carlo conformational
searching procedure has been performed as described in the BOSS
software.[50]
oligonucleotides 21A-B and 22A-B (1 mM in water) were added
glycoside derivatives (20 mM, 12 eq.), THPTA (10 mM, 3 eq.), TEAAc (2
M, 8 µL), MeOH (70 µL), water (30 µL) and 1 mg Cu . The resulting
mixture was sonicated then stirred for 3h under microwave at 65°C. The
crude solutions were centrifuged and the supernatant were directly
applied on a Nap10. After lyophilization the residue was treated overnight
with concentrated ammonia. After evaporation, the solution was
extracted with ethyl acetate. Then the compound was purified by C18
reversed phase HPLC. The products were coevaporated with water and
0
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201700154
FULL PAPER
For the final retained structure, the constraints are removed and the
whole complex is relaxed. In the minimization procedures, the
spectroscopic empirical energy function SPASIBA and the corresponding
parameters are used.[51-53] In the same way, an empirical potential energy
of interaction E for the lectin-ligand complexes using the simple
expression Einteraction = Ecomplex - Eprotein - Eligand was evaluated using the
same force field.
[19] M. Gingras, Y. M. Chabre, M. Roy and R. Roy, Chem. Soc. Rev. 2013,
42, 4823-4841.
[20] C. Muller, G. Despras and T. K. Lindhorst, Chem. Soc. Rev. 2016, 45,
3275-3302.
[21] C. Ligeour, L. Dupin, A. Angeli, G. Vergoten, S. Vidal, A. Meyer, E.
Souteyrand, J. J. Vasseur, Y. Chevolot and F. Morvan, Org. Biomol.
Chem. 2015, 13, 11244-11254.
[
22] R. U. Kadam, M. Bergmann, M. Hurley, D. Garg, M. Cacciarini, M. A.
Swiderska, C. Nativi, M. Sattler, A. R. Smyth, P. Williams, M. Camara,
A. Stocker, T. Darbre and J. L. Reymond, Angew. Chem. Int. Ed. 2011,
Acknowledgements
50, 10631-10635.
[
[
23] M. L. Gening, D. V. Titov, S. Cecioni, A. Audfray, A. G. Gerbst, Y. E.
Tsvetkov, V. B. Krylov, A. Imberty, N. E. Nifantiev and S. Vidal, Chem.
Eur. J. 2013, 19, 9272-9285.
This project was supported by the Agence Nationale de la
Recherche (Glycomime project ANR-12-BSV5-0020). The
authors thank the University Claude Bernard Lyon 1, Ecole
Centrale de Lyon, University of Lille 1, University of Montpellier
and the CNRS for financial support. A.A. thanks the University of
Montpellier for a research studentship. F.M. is from Inserm.
24] A. Novoa, T. Eierhoff, J. Topin, A. Varrot, S. Barluenga, A. Imberty, W.
Romer and N. Winssinger, Angew. Chem. Int. Ed. 2014, 53, 8885-8889.
25] F. Pertici and R. J. Pieters, Chem. Commun. 2012, 48, 4008-4010.
26] F. Pertici, N. J. de Mol, J. Kemmink and R. J. Pieters, Chem. Eur. J.
[
[
2013, 19, 16923-16927.
[
27] A. V. Pukin, A. J. Brouwer, L. Koomen, H. C. Q. van Ufford, J. Kemmink,
Keywords: glycoclusters • microarrays • multivalency •
oligonucleotides • Pseudomonas aeruginosa • LecA
N. J. de Mol and R. J. Pieters, Org. Biomol. Chem. 2015, 13, 10923-
10928.
[
[
[
28] O. Fu, A. V. Pukin, H. C. Q. van Ufford, J. Kemmink, N. J. de Mol and R.
J. Pieters, Chemistryopen 2015, 4, 463-470.
29] S. Cecioni, J. P. Praly, S. E. Matthews, M. Wimmerova, A. Imberty and
S. Vidal, Chem. Eur. J. 2012, 18, 6250-6263.
[
[
[
1]
2]
3]
V. E. Wagner and B. H. Iglewski, Clin. Rev. Allergy Immunol. 2008, 35,
24-134.
1
30] F. Casoni, L. Dupin, G. Vergoten, A. Meyer, C. Ligeour, T. Gehin, O.
Vidal, E. Souteyrand, J. J. Vasseur, Y. Chevolot and F. Morvan, Org.
Biomol. Chem. 2014, 12, 9166-9179.
N. Garber, U. Guempel, A. Belz, N. Gilboagarber and R. J. Doyle,
Biochim. Biophys. Acta 1992, 1116, 331-333.
E. Mitchell, C. Houles, D. Sudakevitz, M. Wimmerova, C. Gautier, S.
Perez, A. M. Wu, N. Gilboa-Garber and A. Imberty, Nat. Struct. Biol.
[
[
31] B. Gerland, A. Goudot, C. Ligeour, G. Pourceau, A. Meyer, S. Vidal, T.
Gehin, O. Vidal, E. Souteyrand, J. J. Vasseur, Y. Chevolot and F.
Morvan, Bioconjugate Chem. 2014, 25, 379-392.
2
002, 9, 918-921.
R. Loris, D. Tielker, K. E. Jaeger and L. Wyns, J. Mol. Biol. 2003, 331,
61-870.
[
[
4]
5]
32] S. Wang, L. Dupin, M. Noel, C. J. Carroux, L. Renaud, T. Gehin, A.
Meyer, E. Souteyrand, J. J. Vasseur, G. Vergoten, Y. Chevolot, F.
Morvan and S. Vidal, Chem. Eur. J. 2016, 22, 11785-11794.
33] V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless, Angew.
Chem. Int. Ed. 2002, 41, 2596-2599.
8
G. Cioci, E. P. Mitchell, C. Gautier, M. Wimmerova, D. Sudakevitz, S.
Perez, N. Gilboa-Garber and A. Imberty, FEBS Lett. 2003, 555, 297-
[
[
[
301.
[
[
6]
7]
A. Imberty, M. Wimmerova, E. P. Mitchell and N. Gilboa-Garber, Microb.
Infect. 2004, 6, 221-228.
34] C. W. Tornoe, C. Christensen and M. Meldal, J. Org. Chem. 2002, 67,
3057-3064.
D. Sicard, S. Cecioni, M. Iazykov, Y. Chevolot, S. E. Matthews, J. P.
Praly, E. Souteyrand, A. Imberty, S. Vidal and M. Phaner-Goutorbe,
Chem. Commun. 2011, 47, 9483-9485.
35] Y. Chevolot, C. Bouillon, S. Vidal, F. Morvan, A. Meyer, J. P. Cloarec, A.
Jochum, J. P. Praly, J. J. Vasseur and E. Souteyrand, Angew. Chem.
Int. Ed. 2007, 46, 2398-2402.
[
[
8]
9]
S. Cecioni, A. Imberty and S. Vidal, Chem. Rev. 2015, 115, 525-561.
K. Buffet, E. Gillon, M. Holler, J. F. Nierengarten, A. Imberty and S. P.
Vincent, Org. Biomol. Chem. 2015, 13, 6482-6492.
[
[
[
36] F. Morvan, S. Vidal, E. Souteyrand, Y. Chevolot and J. J. Vasseur, Rsc
Adv. 2012, 2, 12043-12068.
37] S. L. Beaucage and M. H. Caruthers, Tetrahedron Lett. 1981, 22, 1859-
[
[
10] J. J. Lundquist and E. J. Toone, Chem. Rev. 2002, 102, 555-578.
11] J. L. Jimenez Blanco, C. Ortiz Mellet and J. M. Garcia Fernandez,
Chem. Soc. Rev. 2013, 42, 4518-4531.
1
862.
38] C. Ligeour, A. Meyer, J. J. Vasseur and F. Morvan, Eur. J. Org. Chem.
012, 1851-1856.
2
[
12] M. C. Galan, P. Dumy and O. Renaudet, Chem. Soc. Rev. 2013, 42,
[
[
39] S. Eppel and M. Portnoy, Tetrahedron Lett. 2013, 54, 5056-5060.
40] C. Bouillon, A. Meyer, S. Vidal, A. Jochum, Y. Chevolot, J. P. Cloarec, J.
P. Praly, J. J. Vasseur and F. Morvan, J. Org. Chem. 2006, 71, 4700-
4599-4612.
[
[
13] Y. M. Chabre and R. Roy, Chem. Soc. Rev. 2013, 42, 4657-4708.
14] A. Martinez, C. Ortiz Mellet and J. M. Garcia Fernandez, Chem. Soc.
Rev. 2013, 42, 4746-4773.
4702.
[
[
[
[
[
41] Y. Chevolot, J. Zhang, A. Meyer, A. Goudot, S. Rouanet, S. Vidal, G.
Pourceau, J. P. Cloarec, J. P. Praly, E. Souteyrand, J. J. Vasseur and F.
Morvan, Chem. Commun. 2011, 47, 8826-8828.
[
[
15] J.-L. Reymond, M. Bergmann and T. Darbre, Chem. Soc. Rev. 2013,
42, 4814-4822.
16] A. Bernardi, J. Jimenez-Barbero, A. Casnati, C. De Castro, T. Darbre, F.
Fieschi, J. Finne, H. Funken, K.-E. Jaeger, M. Lahmann, T. K. Lindhorst,
M. Marradi, P. Messner, A. Molinaro, P. V. Murphy, C. Nativi, S.
Oscarson, S. Penades, F. Peri, R. J. Pieters, O. Renaudet, J.-L.
Reymond, B. Richichi, J. Rojo, F. Sansone, C. Schaeffer, W. B.
Turnbull, T. Velasco-Torrijos, S. Vidal, S. Vincent, T. Wennekes, H.
Zuilhof and A. Imberty, Chem. Soc. Rev. 2013, 42, 4709-4727.
42] L. Dupin, F. Zuttion, T. Gehin, A. Meyer, M. Phaner-Goutorbe, J. J.
Vasseur, E. Souteyrand, F. Morvan and Y. Chevolot, ChemBioChem
2015, 16, 2329-2336.
43] 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.
Chevolot and F. Morvan, Org. Biomol. Chem. 2015, 13, 8433-8444.
44] A. Goudot, G. Pourceau, A. Meyer, T. Gehin, S. Vidal, J. J. Vasseur, F.
Morvan, E. Souteyrand and Y. Chevolot, Biosens. Bioelectron. 2013, 40,
[
[
17] V. Wittmann and R. J. Pieters, Chem. Soc. Rev. 2013, 42, 4492-4503.
18] N. Spinelli, E. Defrancq and F. Morvan, Chem. Soc. Rev. 2013, 42,
153-160.
4557-4573.
45] S. Park and I. Shin, Org. Lett. 2007, 9, 1675-1678.
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201700154
FULL PAPER
[
46] R. U. Kadam, M. Bergmann, D. Garg, G. Gabrieli, A. Stocker, T. Darbre
and J. L. Reymond, Chem. Eur. J. 2013, 19, 17054-17063.
[50] W. L. Jorgensen and J. Tirado-Rives, J. Comput. Chem. 2005, 26,
1689-1700.
[
[
47] B. Blanchard, A. Imberty and A. Varrot, Proteins 2014, 82, 1060-1065.
48] Z. Szurmai, L. Szabo and A. Liptak, Acta Chim. Hung. 1989, 126, 259-
[51] P. Derreumaux and G. Vergoten, J. Chem. Phys. 1995, 102, 8586-8605.
[52] P. Lagant, D. Nolde, R. Stote, G. Vergoten and M. Karplus, J. Phys.
Chem. A 2004, 108, 4019-4029.
269.
[
49] F. Morvan, A. Meyer, A. Jochum, C. Sabin, Y. Chevolot, A. Imberty, J.
[53] G. Vergoten, I. Mazur, P. Lagant, J. C. Michalski and J. P. Zanetta,
Biochimie 2003, 85, 65-73.
P. Praly, J. J. Vasseur, E. Souteyrand and S. Vidal, Bioconjugate Chem.
2007, 18, 1637-1643.
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201700154
FULL PAPER
Entry for the Table of Contents (Please choose one layout)
Layout 1:
FULL PAPER
A series of 32 glycodendrimers of
generation 0 and 1 targeting Lectin A
A. Angeli, M. Li, L. Dupin, G. Vergoten,
M. Noël, M. Madaoui, S. Wang, A.
Meyer, T. Géhin, S. Vidal, J.J. Vasseur,
Y. Chevolot,* .F. Morvan*
(
LecA) has been synthesized. Bis-
galactosides exhibiting phenyl and
lysine aglycons are able to tightly bind
LecA with a low nanomolar K
2 nM)
d
.( 22 to
Page No. – Page No.
3
Design and synthesis of
galactosylated bifurcated ligands
with nanomolar affinity for lectin
LecA from Pseudomonas aeruginosa
Layout 2:
FULL PAPER
Author(s), Corresponding Author(s)*
((Insert TOC Graphic here; max. width: 11.5 cm; max. height: 2.5 cm))
Page No. – Page No.
Title
Text for Table of Contents
This article is protected by copyright. All rights reserved.