Synthesis of branched-phosphodiester and mannose-centered fucosylated glycoclusters and their binding studies with Burkholderia ambifaria lectin (BambL)

Article abstract of DOI:10.1039/c3ra43807d

Five fucosylated glycoclusters exhibiting 4, 6 or 8 residues were synthesised with two different spatial environments based on mannose-centered and branched-phosphodiester scaffolds. Their synthesis was performed in solution using phosphoramidite chemistry to generate phosphodiester linkages, combined with Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC). The multivalent ligands were evaluated for their ability to bind to Burkholderia ambifaria Lectin (BambL). Binding evaluation was performed through inhibition of hemagglutination (HIA), surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC). All fucosylated glycoclusters exhibited a higher affinity to BambL than methyl α-l-fucoside. A dissociation constant of 43 nM was observed for the fucocluster exhibiting four residues with the branched-phosphodiester spatial environment corresponding to a 22-fold increase in comparison with methyl α-l-fucoside. These multivalent fucoclusters represent the first example of ligands of high affinity to BambL.

Full text of DOI:10.1039/c3ra43807d

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PAPER
Synthesis of branched-phosphodiester and mannose-
centered fucosylated glycoclusters and their binding
studies with Burkholderia ambifaria lectin (BambL)3
Cite this: RSC Advances, 2013, 3,
19515
Caroline Ligeour,a Aymeric Audfray,b Emilie Gillon,b Albert Meyer,a Nicolas Galanos,c
c
a
b
a
´
Sebastien Vidal, Jean-Jacques Vasseur, Anne Imberty* and François Morvan*
Five fucosylated glycoclusters exhibiting 4, 6 or 8 residues were synthesised with two different spatial
environments based on mannose-centered and branched-phosphodiester scaffolds. Their synthesis was
performed in solution using phosphoramidite chemistry to generate phosphodiester linkages, combined
with Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC). The multivalent ligands were evaluated for their
ability to bind to Burkholderia ambifaria Lectin (BambL). Binding evaluation was performed through
inhibition of hemagglutination (HIA), surface plasmon resonance (SPR) and isothermal titration
microcalorimetry (ITC). All fucosylated glycoclusters exhibited a higher affinity to BambL than methyl a-
L-fucoside. A dissociation constant of 43 nM was observed for the fucocluster exhibiting four residues with
the branched-phosphodiester spatial environment corresponding to a 22-fold increase in comparison with
methyl a-L-fucoside. These multivalent fucoclusters represent the first example of ligands of high affinity to
BambL.
Received 4th June 2013,
Accepted 12th August 2013
DOI: 10.1039/c3ra43807d
www.rsc.org/advances
glycoclusters built from different scaffolds have been reported
targeting different plants, bacteria, viruses or human lec-
Introduction
tins.12–18 This strategy led to potent inhibitors of the HIV virus
infection in cellular models.19 Similarly, modification at the
aglycone of mannosides revealed a family of potent inhibitors
of Escherichia coli bacterial infection based on their interac-
tions with the FimH adhesin.20–23 Galactoclusters could also
be used as inhibitors of b-galactosidase.24
Multivalent recognitions between lectins and carbohydrate
ligands occur in biological processes such as cell–cell
communication, inflammation, cancer metastasis and infec-
tion by pathogens (viruses or bacteria).1–3 Several synthetic
multivalent glycoconjugates have been reported4–6 in order to
compete with natural carbohydrate ligands on cell surface and
to control or modulate these interactions leading to potential
therapeutic applications. These resulting neoglycans are
expected to display a much higher binding for the target than
the sum of the constitutive interactions thanks to the so-called
‘‘cluster glycoside effect’’.7,8 The concept of multivalency has
emerged as a promising strategy to design efficient lectin
inhibitors to avoid pathogen infections through anti-adhesive
approaches.9–11 However, the design of good candidates is still
empirical since the precise mode of recognition of multivalent
glycoclusters is not fully understood. Several examples of
Burkholderia ambifaria is an opportunistic pathogen that
has originally been isolated from the environment and from
sputum of cystic fibrosis patients.25 B. ambifaria is a major
component of the rhizosphere of many crop plants including
maize26 and has interesting biocontrol properties due to
pesticide activity.27 However, B. ambifaria belongs to the
Burkholderia cepacia complex (Bcc), a group of genetically
distinct but phenotypically similar Gram-negative bacteria that
cause infection in patients who are immuno-compromised or
suffering from granulomatous disease or cystic fibrosis (CF).28
In this latter case, it is responsible for the ‘‘cepacia syndrome’’
that leads to rapid lung deterioration and death in most
cases.29,30 Most of the Bcc organisms are highly resistant to all
major classes of antibiotics.31 Opportunistic bacteria often use
lectins, i.e. protein receptors with high specificity for glyco-
conjugates, to recognize and adhere to human tissues.10,32,33
More particularly, fucosylated glycoconjugates are present in
higher quantities in CF lungs34 and appear to be a target for
lectins from pathogenic bacteria such as Pseudomonas
aeruginosa35 and Burkholderia cenocepacia.36,37
a
´
´
Institut des Biomolecules Max Mousseron, UMR 5247, CNRS, Universite Montpellier
´
´
1, Universite Montpellier 2, Universite Montpellier 2, CC1704, Place E. Bataillon,
34095, Montpellier Cedex 5, France. E-mail: morvan@univ-montp2.fr;
Fax: (+33)467-042-029
b
´
CERMAV-CNRS, affiliated with Universite Joseph Fourier and ICMG, BP53 38041,
Grenoble, France. E-mail: anne.imberty@cermav.cnrs.fr; Fax: (+33)476-547-203
c
´
´
Institut de Chimie et Biochimie Moleculaires et Supramoleculaires, Laboratoire de
´
Chimie Organique 2-Glycochimie, UMR 5246, CNRS, Universite Claude Bernard Lyon
1, 43 Boulevard du 11 Novembre 1918, F-69622, Villeurbanne, France
Electronic supplementary information (ESI) available: SPR and ITC data, 1H
3
and 13C NMR spectra for all new compounds. See DOI: 10.1039/c3ra43807d
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Fig. 1 Two orthogonal views of BambL trimer complexed with fucose, adapted from the crystal structure of BambL/blood group B complex (PDB code 3ZWE).38 The
three BambL peptide chains are represented by ribbons of different colors (with the accessible surface on right panel) and the six fucose residues are represented by
spheres.
fucose residues were introduced. Two mannose-centered
tetrafucosylated clusters were elaborated using different linker
lengths between the mannose core and the fucose moiety such
as a phosphopropyltriazolyltriethylene glycol (POProTzEG3, 15-
atoms length) and a phosphodiethyleneglycolmethylene-tria-
zolyltriethylene glycol (POEG2MTzEG3, 20-atoms length) in
order to evaluate the influence of the linker length on the
binding properties. A mannose-centered octafucosylated gly-
cocluster was prepared with POProTzEG3 linkers and was used
to evaluate the influence of valency on the binding efficiency.
The second family displays a branched-phosphodiester spatial
arrangement with four or six fucose residues. They were
prepared on a pentaerythritol/1,1,1-(tris-hydroxymethyl)ethane
core with a methylenetriazolyltriethylene glycol (MTzEG3, 12-
atoms length) linker (Fig. 2). The five fucoclusters, exhibiting
4, 6 or 8 residues, were synthesized at hundred-milligram scale
using a combination of phosphoramidite chemistry41 and
Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or "click"
chemistry.42,43 The resulting fucosylated glycoclusters, except
3, exhibit negatively charged phosphodiester linkages that lead
to glycoclusters highly soluble in water and could also bring
additional interaction with the lectin.
We developed such a strategy for the synthesis of glycoclus-
ters 1–5 because the coupling between a hydroxyl and a
phosphoramidite derivative activated with benzylthioltetrazole
is very efficient with quantitative conversion. Thus, the
reaction of phosphoramitides 8a–c on the four hydroxyls of
the mannose core is much more efficient than four alkylations
as performed by Dubber and Lindhorst.44 This strategy
allowed also the straightforward synthesis of mannose-
centered octa-pentynyl using the bis-alkynyl-linker phosphor-
amidite derivative 8c. Furthermore, the introduction of the
alkynyl-linkers proceeded by an one pot procedure and the
work-up was just an aqueous extraction affording the pure
Analysis of the available genomes of B. ambifaria allowed to
identify a new fucose-binding lectin, BambL,38 with no
similarity to lectins from P. aeruginosa and B. cenocepacia but
rather related to lectins from the fungus Aleuria aurantia39 and
from the phytopathogenic bacterium Ralstonia solanacearum.40
BambL shows a six bladed b-propeller architecture resulting
from the association of 3 monomers with two binding sites per
monomer. The overall lectin consequently exhibits 6 binding
sites, all located on the same side of the protein (Fig. 1).
BambL displays a micromolar affinity for fucose and binds
strongly to cell or cell-like surfaces such as human tissues or
mucins. Because of the characteristics (multivalency, topology,
affinity…) of this B. ambifaria lectin, the design of synthetic,
multivalent, high affinity ligands can be of great interest. Such
BambL ligands can be used for the development of anti-
adhesive compounds against B. ambifaria infections and also
for a better understanding of multivalent interactions.
This study reports the design and synthesis of five multi-
valent fucosylated ligands prepared from a mannose-based
core or a branched-phosphodiester scaffold and bearing four,
six or eight epitopes. Their binding properties towards BambL
were evaluated using hemagglutination inhibitory assay (HIA),
surface plasmon resonance (SPR) and isothermal titration
microcalorimetry (ITC).
Results and discussion
Synthesis
Two families of fucosylated glycoclusters were prepared
starting from two different scaffolds. The first family (1 to 3)
was synthesised using a mannose-core on which four or eight
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Fig. 2 Structure of fucoclusters 1 to 5.
corresponding poly-alkynylated scaffolds 9a–c with high yields
(81–98%). Eventually, fucose azide derivatives were conjugated
by CuAAC.
tion. Finally, platforms 9a–c were conjugated with the
azidotriethyleneglycol functionalized fucoside 1050 by CuAAC
using copper nanopowder affording the hydroxylated fucoclus-
ters 1–3 after ammonia treatment and purification by C18
reverse phase chromatography.
The glycomimetics 1–3 were synthesized in three steps
starting from mannose 6 (Scheme 1). Firstly, mannose was
glycosylated by methanol under acidic catalysis using Dowex
50W-X8 resin H+ form affording methyl a-D-mannopyranoside
7.45 Then, each hydroxyl was phosphorylated using the
alkynylated phosphoramidites 8a,46 8b47 or 8c48 leading to
phosphitetriester linkages. The small excess of phosphorami-
dite was hydrolysed by addition of water and phosphitetriester
linkages were then oxidized by means of solid-supported meta-
periodate49 affording the mannose-based platform exhibiting
four or eight pent-4-ynyl residues 9a or 9c but also four
propargyl diethyleneglycyl groups 9b. During the process, the
excess of phosphoramidite (8a–c) was hydrolysed affording a
H-phosphonate diester that was oxidized leading to its
negatively charged phosphodiester derivative. This water
soluble side-product was easily removed by aqueous extrac-
The branched-phosphodiester-shaped fucoclusters 4 and 5
were synthesized, in two steps, using bis-propargyloxymethyl-
2,2-propanol phosphoramidite 1348 which was coupled once to
bis-propargyloxymethyl-2,2-propanol 1148 or twice to bis-
propargyloxymethyl-2,2-propane-1,3-diol 1251 using tetrazole
as activator (Scheme 2). After addition of water to hydrolyse the
excess of 13, solid-supported meta-periodate was added to
oxidize the phosphitetriester linkages into phosphate triesters
affording the tetra-alkynylated 14 and hexa-alkynylated 15
platforms respectively. Each of them was conjugated with
azidotriethyleneglycol functionalized fucoside 10 by CuAAC
using copper nanopowder affording fucoclusters 4 and 5
respectively, after ammonia treatment and purification by C18
reverse phase chromatography
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Scheme 1 Synthesis of mannose-centered tetra- and octa-fucoclusters 1 Man(ProTzEG3–Fuc)4, 2 Man(EG2MTzEG3–Fuc)4 and 3 Man[(ProTzEG3–Fuc)2]4. Pro = propyl,
EG2M = diethyleneglycol methylene, Gray ball represents polystyrene solid support.
agglutinate red cells by crosslinking glycoconjugates on their
surface, and therefore multivalent glycoclusters can inhibit
this hemagglutination. The potency of the competition is
defined by the minimum inhibitory concentration (MIC)
Interaction of the fucoclusters with BambL
The binding of BambL to the fucosylated glycoclusters was
evaluated according to different assays. BambL is able to
Scheme 2 Synthesis of branched-phosphodiester fucoclusters 4 BPO(MTzEG3–Fuc)4, 5 BPO(MTzEG3–Fuc)6.
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Table 1 HIA and SPR evaluation for binding of aMeFuc and multivalent
glycoclusters to BambL
HIA
MIC (mM)
SPR
IC50 (mM)
Ligand
Valence
ba
ba
aMeFu
1
4
4
8
4
6
62.5
3.9
3.9
0.24
0.24
0.37
1
16
16
256
256
171
16.3
1
1
2
3
4
5
0.191
0.154
0.169
0.462
0.170
85
106
96
35
96
a
b corresponds to the ratio of the aMeFuc value over the value of
the considered fucocluster.
required for inhibiting the agglutination of erythrocytes. This
method is based on visual analysis, and is therefore only semi-
quantitative, but it has the advantage to use the biological
targets for the lectin action. The mannose-centered tetrafuco-
sylated glycoclusters (1 and 2) displayed a similar binding
profiles regardless of the linker length with potencies (b) 16-
fold better than for the monovalent reference methyl a-L-
fucopyranoside aMeFuc (Table 1). aMeFuc was chosen as
control since its aglycon could be considered similar to the
beginning of the triethyleneglycol moiety. The three other
glycoclusters displayed a similar and stronger inhibition of
hemagglutination with potencies between 171 and 256-fold.
SPR experiments were also conducted as inhibition assays,
using increasing concentrations of glycoclusters to inhibit the
binding of circulating BambL to a-fucosides attached on the
chip surface. Sensorgramms showed a very fast association of
the lectin and a very slow dissociation due to multivalent
Fig. 4 Raw ITC data (top) obtained by injections of glycoclusters in a solution of
BambL and the corresponding integrated titration curve (bottom). Left: 150 mM
4 in 13.3 mM BambL. Right: 150 mM 1 in 12.5 mM Bamb.
improvement ratio (b) between 35 and 106-fold. With this
technique, the tetravalent compound 4 seems to be a slightly
weaker inhibitor than the other compounds. This discrepancy
in the binding profile of glycocluster 4 between HIA and SPR
experiments could be attributed to the different experimental
setup for each technique, and ITC data reported below will
select this glycocluster 4 as the best ligand for BambL.
Therefore, the surface-bound fucosides present on the
sensorchip used in the SPR experiments provides a situation
quite different from the ‘‘in solution’’ conditions found in HIA
and ITC experiments where the interacting partners are all
present in solution and none is bound to a surface.
Finally, the binding of BambL to fucoclusters 1–5 was
studied by isothermal titration microcalorimetry (ITC) to
determine the dissociation constant (KD) and stoichiometry
(N) of interaction but also the thermodynamic parameters
such as the enthalpy of binding (DH) (Fig. 4).
interaction of BambL on the surface (Fig. 3A, ESI ). Inhibition
3
curves have been plotted based on the response level at the
end of the equilibrium phase. Inhibition curves obtained with
glycoclusters are different to those with aMeFuc, because of
multivalent interactions of BambL resulting in partial aggrega-
tion and sharper sigmoid shape (Fig. 3B). The resulting IC50
values (Table 1) confirmed that all multivalent glycoclusters
are more potent competitors than aMeFuc with rather similar
Fig. 3 Analysis of BambL/fucosylated glycoclusters interactions by inhibition assay using surface plasmon resonance. (A) Sensorgramms of BambL binding to an
a-fucosylated sensorchip with increasing concentrations of aMeFuc and 3 (from 0.005 mM to 100 mM). (B) Inhibition curve of 3 compared to aMefuc.
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Table 2 Titration microcalorimetry data for the interaction between BambL and fucoclusters 1–5. Standard deviations result from experimental data performed in
duplicates or triplicates
Ligand
Valence
Na
% Fuc bound
KD (nM)
2DH (kJ mol21
)
2TDS (kJ mol21
)
2DG (kJ mol21
)
bb
aMeFucc
1
4
4
8
4
6
6.06
2.34
3.27
1.47
2.37
1.26
100
64
46
51
63
79
960 ¡ 30
111.2 ¡ 0.1
100 ¡ 20
57 ¡ 3
43 ¡ 2
93 ¡ 5
47.8 ¡ 1.8
155 ¡ 1
101.7 ¡ 0.6
240 ¡ 4
146 ¡ 8
273 ¡ 8
13.5
116
61
199
104
233
34.3
40
40
41
42
1
9
10
17
22
10
1
2
3
4
5
40
a
b
Number of glycoclusters per BambL trimeric b-propeller. From Audfray et al.38 c b corresponds to the ratio of the aMeFuc Kd value over
the value of the considered fucocluster.
ITC measurements performed with the mannose-centered
tetrafucosides 1 and 2 provided similar KD values close to 100
nM that represent a 10-fold improvement compared to
reported data obtained with aMeFuc38 (Table 2). The octava-
lent compound 3 is almost twice as good, as one could expect
with the increase of valency. The highest affinity compound is
the tetravalent branched-phosphodiester-based compound 4
with a KD value of 43 nM, corresponding to a 22-fold increase
in comparison with aMeFuc. The increases of affinity for
multivalent compounds are of the same range as the ones
observed with SPR, although not always in the same order,
again probably due to the different experimental setup.
Concerning the lower affinity of the branched hexafucose 5,
in respect with branched tetracluster 4, it has been reported
several times that the global topology of a glycocluster is often
more important than the number of carbohydrates to gain a
high affinity. The possible explanation is that fucose residues
are too close and lead to a steric hindrance.
towards the bacterial lectin BambL from B. ambifaria. They
displayed a better affinity in comparison with the monovalent
methyl a-L-fucoside with an increase of potency from 9 to 22.
The lowest KD value (43 nM) was found for a tetrameric cluster
with a branched-phosphodiester scaffold illustrating that
spatial arrangement is a very important parameter in the
recognition event since hexa- of octa-valent glycoclusters did
not display much better binding properties towards this lectin.
Even though six binding sites are available for cross-linking
with the fucoside epitopes of the glycoclusters, this chelate
binding mode is most probably not observed in the present
case since moderate potencies were observed. Nevertheless,
the nanomolar affinity measured for these glycoclusters is
highly compatible with applications in anti-adhesive therapy
against bacterial infection.
Analysis of the stoichiometry of binding (N) was performed
by evaluating the ratio of glycocompounds bound per
b-propeller. For the tetravalent compounds, this value varies
approximately from 3 to 2, corresponding to roughly the
binding to the lectin of two to three fucose residues per
tetravalent compounds (46% to 64% of fucose residues
bound). This value is in agreement with the enthalpy of
binding varying from 2150 to 2100 kJ mol21. The compound
4 has higher affinity since 63% of the fucose residues are
bound, resulting in a strong DH, which is associated to
relatively low entropy barrier (DS). The hexavalent 5 and
octavalent 3 compounds have much stronger enthalpy of
binding (with more fucose residues involved) but associated
with higher entropy cost. These glycoclusters involved roughly
5 and 4 fucose residues respectively in the interaction with the
lectin (79% and 51% of fucose bound).
Experimental section
Materials and methods: All reagents were commercial and
used without further purification. Acetonitrile was distilled
over CaH2. Phosphorylations were performed under an argon
atmosphere. NMR spectra were recorded at 293 K using a 400
MHz or 600 MHz spectrometer (Bruker). Shifts are referenced
relative to deuterated solvent residual peaks. MALDI-TOF mass
spectra were recorded on a Voyager mass spectrometer
(Perspective Biosystems, Framingham, MA) equipped with a
nitrogen laser. High-resolution (HR-ESI-QToF) mass spectra
were recorded using a Q-Tof Micromass spectrometer. Thin-
layer chromatography (TLC) was carried out on aluminum
sheets coated with silica gel 60 F254 (Merck). TLC plates were
inspected by UV light (l = 254 nm) and developed by treatment
with a mixture of 10% H2SO4 in EtOH/H2O (1 : 1 v/v) followed
by heating. Reverse phase chromatography was performed
with C18 flash column.
Conclusion
Synthesis
A series of five multivalent L-fucosylated glycoclusters exhibit-
ing four, six or eight residues and with two different
architectures were synthesized, in a few steps, using phos-
phoramidite chemistry and CuAAC ‘‘Click’’ chemistry. The
affinities of the fucoclusters for BambL have been evaluated
via HIA, SPR and ITC studies. These fucosylated glycoclusters
are the first reported and studied for their binding properties
General procedure for phosphorylation. A solution of methyl
a-D-mannopyranoside45 7 (50 mg, 0.26 mmol, 1 eq) in
anhydrous dimethylformamide–acetonitrile (1 : 1.5, v/v) was
stirred for 1.5h with molecular sieves (3 Å). Then, the alkyne
phosphoramidite 8a–c (1.30 mmol, 5 eq) and a solution of
tetrazole (0.4 M in anhydrous CH3CN, 6.4 mL, 2.60 mmol, 10
eq) were added. The mixture was stirred at 30 uC for 2 h and
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the reaction was stopped by addition of water. A26(IO42) resin
(1.0 g, 2.50 mmol, 9.6 eq) was added and the mixture was
stirred for 2 h. After filtration of the resin and addition of
dichloromethane (40 mL), the reaction was washed with an
aqueous saturated solution of NaHCO3 (60 mL) and brine (60
mL). The organic layer was dried (Na2SO4), filtered and
concentrated to afford the desired tetra- or octa-alkynylated
mannose derivatives 9a–c.
1-Methyl-2,3,4,6-tetra-O-pentynylphosphotriester-a-D-man-
nopyranoseide 9a. Obtained as a pale yellow oil (208 mg, 81%).
1H NMR (300 MHz, D2O) d 4.98 (d, J = 2.10 Hz, 1H, H-1), 4.87–
4.57 (m, 3H, H-2, H-5, H-6), 4.37–4.12 (m, 16H, OCH2CH2CN,
POCH2CH2), 3.94–3.89 (m, 1H, H-6), 3.45 (s, 4H, OCH3, H-3),
3.40 (m, 1H, H-4), 2.88–2.78 (m, 8H, CH2CN), 2.39–2.34 (m, 4H,
CH2CH2CCH), 2.08–1.90 (m, 8H, POCH2CH2), 1.73–1.64 (m,
4H, CH2CCH). 31P NMR (162 MHz, CDCl3) d 21.65–23.01 (m,
1P). 13C NMR (100 MHz, CDCl3) d 115.5 (CN), 98.3 (C-1), 81.5
(OCH2CCH), 68.5 (CH2CCH, C-2, C-5, C-6), 65.6 (C-3, C-4), 60.9
(2s, POCH2), 55.7 (OCH3), 27.7 (POCH2CH2), 18.7 (CH2CN),
13.1 (CH2CH2CCH). MS MALDI-TOF+ m/z calcd for
C39H55N4O18P4 [M + H]+: 991.76 found 991.86. HR-ESI-QToF
MS (positive mode): m/z calcd for C39H55N4O18P4 [M + H]+
991.2465 found 991.2462.
1-Methyl-2,3,4,6-tetra-O-propargyldiethyleneglycylphosphotriester-
a-D-mannopyranoseide 9b. Obtained as a colorless oil (279 mg, 87%).
1H NMR (400 MHz, CDCl3) d 4.93 (d, J = 2.42 Hz, 1H, H-1), 4.84–4.79
(m, 1H, H-6), 4.73–4.59 (m, 2H, H-2, H-5), 4.37–4.18 (m, 16H,
POCH2CH2CN, POCH2CH2), 4.17–4.12 (m, 8H, OCH2CCH), 3.86–3.80
(m, 1H, H-6), 3.68 (m, 8H, POCH2CH2), 3.63 (s, 17H, OCH2CH2O,
H-3), 3.61–3.57 (m, 1H, H-4), 3.38 (s, 3H, OCH3), 2.82–2.74 (m, 8H,
CH2CN), 2.46 (m, 4H, OCH2CCH). 31P NMR (162 MHz, CDCl3) d
21.67–23.11 (m, 1P). 13C NMR (100 MHz, CDCl3) d 117.1 (CN), 98.3
(C-1), 79.6 (OCH2CCH), 74.9 (CH2CCH, C-2, C-5, C-6), 70.2–69.7 (2m,
POCH2CH2, C-3, C-4), 69.1 (OCH2CH2O), 67.8–62.3 (5 m, POCH2),
58.3 (OCH2CCH), 55.7 (OCH3), 19.5 (CH2CN). MALDI-TOF+ m/z calcd
for C47H71N4O26P4 [M + H]+: 1231.96 found 1231.19. HR-ESI-QToF
MS (positive mode): m/z calcd for C47H71N4O26P4 [M + H]+ 1231.3297
found 1231.3307.
The nanopowder copper was eliminated by centrifugation.
Then the reaction was diluted in CH2Cl2 (15 mL), and washed
with brine (3 6 15 mL). The organic layer was dried (Na2SO4),
filtered and concentrated to dryness. The resulting product
was dissolved in acetone (5 mL) and concentrated ammonia
solution (30%) was added (20 mL). The mixture was stirred 1 h
at room temperature. After evaporation, the crude product was
dissolved in milliQ water, and the solution was passed through
a column filled with DOWEX-50W X8 resin, Na+ form. After
concentration, the residue was purified by C18 flash column
chromatography (40 g) (water/CH3CN/triethylammonium acet-
ate buffer 0.1 M pH 7.7, 97/0/3 to 47/50/3) to afford the desired
glycoconjugates (1–5).
Glycocluster 1. Obtained as a pale yellow oil (61 mg, 60%)
according to general procedure for CuAAC reactions: 9a (49
mg, 0.049 mmol) with azido-derivative 10 (105 mg, 0.235
1
mmol, 4.8 eq). H NMR (400 MHz, D2O) d 7.96–7.91 (m, 4H,
H-triaz), 4.95 (d, J = 2.8 Hz, 1H, H-1 man), 4.92 (d, J = 2.8 Hz,
4H, H-1 fuc), 4.63–4.61 (m, 8H, CH2N-triaz), 4.48–4.40 (m, 3H,
H-2 man, H-3 man, H-5 man), 4.10 (q, J = 4.0 Hz, J = 8.4 Hz, 4H,
H-5 fuc), 4.00 (m, 9H, OCH2CH2N-triaz, H-6 man), 3.91 (m, 5H,
H-6 man, H-3 fuc), 3.85–3.76 (m, 20H, CH2O-fuc, POCH2CH2,
H-2 fuc), 3.68 (m, 28H, OCH2CH2, H-4 fuc), 3.55 (t, J = 3.6 Hz, J
= 6.4 Hz, 1H, H-4 man), 3.45 (s, 3H, OCH3), 2.88–2.78 (m, 8H,
CH2CH2C-triaz), 1.98 (s, 8H, CH2CH2C-triaz), 1.24 (d, J = 4.4
Hz, 12H, H-6 fuc). 13C NMR (100 MHz, D2O) 123.9 (CH-triaz),
98.8 (C-1 fuc, C-1 man), 72.0 (C-4 fuc), 70.9, 70.7 (C-6 man, C-3
fuc), 69.9–69.6 (C-2 fuc, C-4 man, OCH2CH2O), 69.0 (C-5 fuc),
68.3 (OCH2CH2N-triaz), 67.0 (POCH2CH2), 66.8 (CH2O-fuc),
65.5, 65.2, 65.0, (C-2 man, C-3 man, C-5 man), 50.3 (OCH3),
50.1 (CH2N-triaz), 29.9 (CH2CH2CH2), 29.7 (CH2C-triaz), 15.5
(C-6 fuc). MS MALDI-TOF2 m/z calcd for C75H133N12O46P4 [M
2 H]2: 2062.81 found 2062.90. HR-ESI-QToF MS (positive
mode): m/z calcd for C75H137N12O46P4 [M + 3H]3+ 688.5900
found 688.5888.
Glycocluster 2. Obtained as a pale yellow oil (51 mg, 54%)
according to general procedure for CuAAC reactions: 9b (50
mg, 0.041 mmol) with azido-derivative 10 (73 mg, 0.164 mmol,
1
1-Methyl-2,3,4,6-tetra-O-bis-pentynylphosphotriester-a-D-
mannopyranoseide 9c. Obtained as a pale yellow oil (263 mg,
4 eq). H NMR (400 MHz, D2O) d 8.21 (s, 4H, H-triaz), 5.01 (s,
1H, H-1 man), 4.95 (d, J = 2.8 Hz, 4H, H-1 fuc), 4.71 (t, J = 3.2
Hz, J = 6.0 Hz, 8H, CH2N-triaz), 4.46–4.35 (m, 3H, H-2 man, H-3
man, H-5 man), 4.17 (d, J = 4.4 Hz, 4H, H-5 fuc), 4.14 (q, J = 4.4
Hz, J = 8.8 Hz, 8H, OCH2C-triaz), 4.06 (t, 9H, OCH2CH2N-triaz,
H-6 man), 3.96 (d, J = 2.4 Hz, 1H, H-6 man), 3.94 (dd, 4H, H-3
fuc), 3.87 (m, 8H, POCH2CH2), 3.84 (d, J = 2.8 Hz, 4H, H-2 fuc),
3.82 (m, 30H, CH2O-fuc, OCH2CH2O), 3.73 (m, 26H,
OCH2CH2O), 3.68 (d, J = 3.2 Hz, 4H, H-4 fuc), 3.57 (t, J = 3.6
Hz, J = 6.4 Hz, 1H, H-4 man), 3.47 (s, 3H, OCH3), 1.27 (d, J = 4.4
Hz, 12H, H-6 fuc). 13C NMR (100 MHz, D2O) 98.8 (C-1 fuc, C-1
man), 72.0 (C-4 fuc), 69.7 (C-2 fuc, C-3 fuc, C-2 man, C-3 man,
C-4 man), 69.4 (CH2O-fuc), 69.2 (OCH2CH2N-triaz), 68.9
(OCH2C-triaz), 68.3 (C-5 man), 67.0 (C-6 man), 66.8 (C-5 fuc),
63.4 (POCH2CH2), 55.3 (OCH3), 50.2 (CH2N-triaz), 15.5 (C-6
fuc). MS MALDI-TOF2 m/z calcd for C83H149N12O54P4 [M 2
H]2: 2303.02 found 2303.29. HR-ESI-QToF MS (positive mode):
m/z calcd for C83H153N12O54P4 [M + 3H]3+: 768.6182 found
768.6204.
1
98%). H NMR (600 MHz, CDCl3) d 4.89 (d, J = 1.57 Hz, 1H,
H-1), 4.78–4.76 (m, 1H, H-2), 4.65–4.62 (m, 1H, H-5), 4.46–4.42
(m, 1H, H-6), 4.24–4.13 (m, 16H, POCH2CH2), 3.89–3.86 (m,
1H, H-6), 3.38–3.35 (m, 4H, OCH3, H-3), 3.32 (m, 1H, H-4), 2.30
(m, 16H, CH2CCH), 1.97 (m, 8H, CH2CCH), 1.88 (m, 16H,
POCH2CH2). 13C NMR (100 MHz, CDCl3) d 98.5 (C-1), 82.6
(OCH2CCH), 69.6–69.4 (4 s, CH2CCH, C-2, C-5, C-6), 64.2, 64.1
(2 s, C-3, C-4), 55.6 (OCH3), 47.2 (POCH2), 29.2–29.0 (m,
POCH2CH2), 14.8 (CH2CH2CCH). MS MALDI-TOF (positive
mode) m/z calcd for C47H67O18P4 [M + H]+: 1043.92 found
1043.80. HR-ESI-QToF MS (positive mode): m/z calcd for
C47H67O18P4 [M + H]+ 1043.3278 found 1043.3319.
General procedure for CuAAC conjugation and deacetyla-
tion. The alkyne-functionalized compounds (9a–c, 14, 15) 1.0
eq and azido-functionalized fucoside derivative (10) (4 to 9.6
eq) were dissolved in dioxane (1.4 mL) with triethylammonium
acetate buffer (175 mL, 0.1 M, pH 7.7) with nanopowder copper
(y2 mg). The resulting mixture was stirred overnight at 70 uC.
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Glycocluster 3. Obtained as a pale yellow oil (167 mg, 98%)
RSC Advances
layer was dried (Na2SO4), filtered and concentrated to afford
1
the desired hexa-alkynylated derivative 15 (203 mg, 97%). H
according to general procedure for CuAAC reactions: 9c (49
mg, 0.047 mmol) with azido-derivative 10 (200 mg, 0.447
NMR (400 MHz, CDCl3) d 4.29–4.25 (m, 4H, POCH2CH2), 4.13–
4.09 (m, 16H, OCH2CCH, POCH2Cq), 3.98 (d, J = 4.28 Hz, 4H,
OCH2Cq), 3.54 (s, 4H, POCH2Cq), 3.40–3.36 (m, 8H, OCH2Cq),
2.79–2.76 (m, 4H, CH2CN), 2.47–2.45 (m, 6H, OCH2CCH), 0.99
(s, 6H, CH3). 13C NMR (100 MHz, CDCl3) d 116.6 (CN), 79.9
(OCH2CCH), 75.2 (OCH2CCH), 74.8 (OCH2Cq), 67.3, 66.2
(POCH2Cq), 62.1 (POCH2CH2), 58.8 (OCH2CCH), 40.6 (Cq-tris),
37.1 (Cq), 19.8 (CH2CN), 17.0 (CH3). MS MALDI-TOF2 m/z
calcd for C39H53N2O14P2 [M + H]+: 835.79 found 835.33. HR-
ESI-QToF MS (positive mode): m/z calcd for C39H53N2O14P2 [M
+ H]+ 835.2972 found 835.2978.
Glycocluster 4. Obtained as a pale yellow oil (113 mg, 99%)
according to general procedure for CuAAC reaction: 14 (33 mg,
0.065 mmol) with azido-derivative 10 (139 mg, 0.312 mmol, 4.8
eq). 1H NMR (600 MHz, D2O) d 8.06 (s, 4H, H-triaz), 4.89 (d, J =
3.9 Hz, 4H, H-1 fuc), 4.64 (t, J = 5.0 Hz, 8H, CH2N-triaz), 4.62 (s,
8H, OCH2-triaz), 4.07 (q, J = 6.6 Hz, 4H, H-5 fuc), 3.99 (t, J = 5.0
Hz, 8H, OCH2CH2N-triaz), 3.87 (dd, J = 10.4 Hz, J = 3.4 Hz, 4H,
H-3 fuc), 3.82–3.77 (m, 12H, CH2O-fuc, H-2 fuc), 3.75 (s, 4H,
POCH2Cq), 3.67–3.64 (m, 28H, OCH2CH2O, H-4 fuc), 3.41 (d, J =
3.4 Hz, 8H, OCH2-Cq), 1.21 (d, J = 6.6 Hz, 12H, H-6 fuc), 0.86 (s,
6H, CH3). 31P NMR (243 MHz, D2O) d 0.66 (s, 1P). 13C NMR
(150 MHz, D2O) d 144.4 (Cq-triaz), 125.5 (CH-triaz), 98.8 (C-1),
72.6 (OCH2Cq), 69.9–69.7 (4s, C-2, C-3, C-4, OCH2CH2O), 69.0
(C-5), 68.3 (OCH2CH2N-triaz), 67.0 (POCH2Cq), 66.8 (CH2O-fuc),
63.8 (OCH2-triaz), 50.2 (CH2N-triaz), 40.6 (Cq-tris), 16.5 (CH3),
15.5 (C-6). MS MALDI-TOF2 m/z calcd for C70H122N12O36P [M
2 H]2: 1738.75 found 1738.09. HR-ESI-QToF MS (positive
mode): m/z calcd for C70H125N12O36P [M + 2H]++ 870.4029
found 870.4041.
1
mmol, 9.6 eq). H NMR (600 MHz, D2O) d 7.93–7.88 (m, 8H,
H-triaz), 4.98 (d, J = 3.9 Hz, 1H, H-1 man), 4.93 (dd, J = 7.5 Hz, J
= 3.8 Hz, 8H, H-1 fuc), 4.65–4.61 (m, 16H, CH2N-triaz), 4.29–
4.22 (m, 3H, H-2 man, H-3 man, H-5 man), 4.13–4.08 (m, 8H,
H-5 fuc), 4.03–3.90 (m, 26H, OCH2CH2N-triaz, H-6 man, H-3
fuc), 3.88–3.79 (m, 40H, CH2O-fuc, POCH2CH2, H-2 fuc), 3.76–
3.67 (m, 56H, OCH2CH2O, H-4 fuc), 3.58 (t, J = 4.91 Hz, 1H, H-4
man), 3.51 (s, 3H, OCH3), 2.87–2.82 (m, 16H, CH2CH2C-triaz),
1.98 (s, 16H, CH2CH2C-triaz), 1.25 (t, J = 7.12 Hz, 24H, H-6 fuc).
13C NMR (150 MHz, D2O) 148.9 (Cq-triaz), 125.0 (CH-triaz),
100.00 (C-1 fuc, C-1 man), 73.2 (C-4 fuc), 71.1, 71.0, 70.9, 70.6,
70.2 (C-2 fuc, C-4 fuc, C-4 man, OCH2CH2O, C-6 man, C-3 fuc),
69.5 (C-5 fuc), 68.2–67.9, 66.2 (C-2 man, C-3 man, C-5 man,
CH2O-fuc, POCH2CH2, OCH2CH2N-triaz), 51.5 (OCH3), 51.3
(CH2N-triaz), 30.9 (CH2CH2CH2), 30.8 (CH2C-triaz), 16.7 (C-6
fuc). MS MALDI-TOF2 m/z calcd for C143H251N24O74P4 [M +
H]+: 3614.54 found 3614.39.
Tetraalkynylated branched-phosphodiester-like compound
14. A solution of bis-propargyloxymethyl-2,2-propanol 11 (127
mg, 0.65 mmol, 1 eq) in anhydrous acetonitrile (6.3 mL) was
stirred for 1 h with molecular sieves (3 Å). Then, bis-
propargyloxymethyl-2,2-propanol phosphoramidite 13 (309
mg, 0.78 mmol, 1.2 eq) and tetrazole (114 mg, 1.63 mmol,
2.5 eq) were added. The mixture was stirred at 30 uC overnight
2
and the reaction was stopped by addition of water. A26(IO4
)
resin (2.5 g, 6.25 mmol, 9.6 eq) was added and the mixture was
stirred overnight. After filtration of the resin and addition of
dichloromethane (100 mL), the reaction was washed with an
aqueous saturated solution of NaHCO3 (150 mL) and brine
(150 mL). The organic layer was dried (Na2SO4), filtered and
concentrated to afford the desired tetra-alkynylated derivative
14 (85 mg, 26%). 1H NMR (400 MHz, CDCl3) d 4.26 (dt, J = 8.45
Hz, J = 6.42 Hz, 2H, POCH2CH2), 4.14 (dd, 8H, J = 2.36 Hz, J =
1.78 Hz, OCH2CCH), 3.56 (s, 4H, POCH2Cq), 3.50 (d, J = 2.60
Hz, 8H, OCH2Cq), 2.78 (td, J = 6.41 Hz, J = 0.59 Hz, 2H, CH2CN),
2.43 (t, J = 2.37 Hz, 4H, OCH2CCH), 0.90 (s, 6H, CH3). 13C NMR
(100 MHz, CDCl3) d 113.1 (CN), 79.7 (OCH2CCH), 74.5
(OCH2CCH), 74.0 (OCH2Cq), 68.3 (POCH2Cq), 61.7
(POCH2CH2), 58.7 (OCH2CCH), 40.5 (Cq-tris), 19.6 (CH2CN),
17.4 (CH3). MS MALDI-TOF2 m/z calcd for C25H35NO8P [M +
H]+: 508.52 found 508.25. HR-ESI-QToF MS (positive mode): m/
z calcd for C25H35NO8P [M + H]+ 508.2100 found 508.2078.
Hexaalkynylated branched-phosphodiester-like compound
15. A solution of bis-propargyloxymethyl-2,2-propane-1,3-diol
12 (54 mg, 0.25 mmol, 1 eq) in anhydrous acetonitrile (2.0 mL)
and a solution of tetrazole (0.4 M in anhydrous CH3CN, 3.1
mL, 1.25 mmol, 5 eq) was stirred for 1.5 h with molecular
sieves (3 Å). Then, bis-propargyloxymethyl-2,2-propanol phos-
phoramidite 13 (252 mg, 0.64 mmol, 2.5 eq) were added. The
mixture was stirred at 30 uC for 4 h and the reaction was
stopped by addition of water. A26(IO42) resin (500 mg, 1.25
mmol, 5 eq) was added and the mixture was stirred for 1 h.
After filtration of the resin and addition of dichloromethane
(30 mL), the reaction was washed with an aqueous saturated
solution of NaHCO3 (30 mL) and brine (30 mL). The organic
Glycocluster 5. Obtained as a pale yellow oil (120 mg, 98%)
according to general procedure for CuAAC reactions: 15 (36
mg, 0.043 mmol) with azido-derivative 10 (127 mg, 0.284
mmol, 6.6 eq). 1H NMR (300 MHz, D2O) d 8.04 (s, 6H, H-triaz),
4.87 (d, J = 3.6 Hz, 6H, H-1 fuc), 4.60–4.56 (m, 24H, CH2N-triaz,
OCH2-triaz), 4.04 (q, J = 6.9 Hz, 6H, H-5 fuc), 3.94 (m, 12H,
OCH2CH2N-triaz), 3.86 (dd, J = 8.4 Hz, J = 3.3 Hz, 6H, H-3 fuc),
3.81–3.71 (m, 26H, CH2O-fuc, H-2 fuc, POCH2Cq), 3.63 (m,
42H, OCH2CH2O, H-4 fuc), 3.52–3.49 (m, 12H, OCH2-Cq), 1.20
(dd, J = 10.2 Hz, J = 6.6 Hz, 18H, H-6 fuc), 0.84 (s, 6H, CH3). 31
P
NMR (243 MHz, D2O) d 0.48 (s, 1P). 13C NMR (150 MHz, D2O) d
145.7 (Cq-triaz), 126.5 (CH-triaz), 99.9 (C-1), 73.1 (OCH2-Cq),
71.1–70.8 (4 s, C-2, C-3, C-4, OCH2CH2O), 69.4 (C-5), 68.2
(OCH2CH2N-triaz), 67.9 (2 s, POCH2Cq, CH2O-fuc), 65.1 (OCH2-
triaz), 51.4 (CH2N-triaz), 48.0 (Cq-tris), 16.7 (CH3), 16.6 (C-6).
MS MALDI-TOF2 m/z calcd for C105H183N18O56P2 [M 2 H]2:
2655.61 found 2655.67. HR-ESI-QToF MS (positive mode): m/z
calcd for
886.0662.
C105H187N18O56P2 [M +
3H]3+ 886.0605 found
Binding studies
Hemagglutination inhibition assays (HIA). Hemagglutination
inhibition assays (HIA) were performed in U-shaped 96-well
microtitre plates. Rabbit erythrocytes were purchased from
Biomerieux and used without further washing. Erythrocytes
were diluted to a 8% solution in NaCl (100 mM). BambL
´
solutions of 3 mM were prepared in TRIS-HCl 20 mM (TRIS =
19522 | RSC Adv., 2013, 3, 19515–19524
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tris(hydroxymethyl)aminomethane), NaCl 100 mM, and CaCl2
100 mM. The hemagglutination unit (HU) was first obtained
by addition of the 4% erythrocyte solution (50 mL) to aliquots
(50 mL) of sequential (twice) lectin dilutions. The mixture was
incubated at 25 uC for 30 min. The HU was measured as the
minimum lectin concentration required to observe hemagglu-
tination. For the following lectin-inhibition assays, lectin
concentrations of 4 HU were used. For BambL, this concentra-
tion was found to be 3 mM. Subsequent inhibition assays were
then carried out by the addition of lectin solution (25 mL, at the
required concentration) to sequential dilutions (50 mL) of
glycoclusters, monomer molecules, and controls. These solu-
tions were incubated at 37 uC for 30 min, then 8% erythrocyte
solution (25 mL) was added, followed by an additional
incubation at 37 uC for 1 h. The minimum inhibitory
concentration for each molecule was determined for each
duplicate
temperature and R = 8.314 J mol21
titrations were performed for each ligand tested.
K
21. Two independent
Acknowledgements
This work was financially supported by ANR-12-BSV5-0020.
´
C.L. thanks the Universite Montpellier 2 for the award of a
research studentship. N.G. was awarded a PhD studentship
´
ˆ
from Region Rhone-Alpes Cluster Chimie. A.A. is supported by
ANR-11-BSV5-002. The COST actions CM1102 and BM1003 and
the Labex ARCANE (ANR-11-LABX-003) are thanked for
financial support. F.M. is member of Inserm.
References
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RSC Advances
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19524 | RSC Adv., 2013, 3, 19515–19524
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