CuAAC synthesis of resorcin[4]arene-based glycoclusters as multivalent ligands of lectins

Article abstract of DOI:10.1039/c1ob05676j

Synthetic multivalent glycoclusters show promise as anti-adhesives for the treatment of bacterial infections. Here we report the synthesis of a family of tetravalent galactose and lactose functionalised macrocycles based on the resorcin[4]arene core. The development of diastereoselective synthetic routes for the formation of lower-rim propargylated resorcin[4]arenes and their functionalistion via Cu-catalyzed azide-alkyne click chemistry is described. ELLA binding studies confirm that galactose sugar clusters are effective ligands for the PA-IL bacterial lectin of Pseudomonas aeruginosa while poor binding for the lactose-based monovalent probe and no binding could be measured for the multivalent glycoclusters was observed for the human galectin-1.

Full text of DOI:10.1039/c1ob05676j

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PAPER
CuAAC synthesis of resorcin[4]arene-based glycoclusters as multivalent
ligands of lectins†
Zahid H. Soomro,a,b Samy Cecioni,c,d Helen Blanchard,e Jean-Pierre Praly,c Anne Imberty,d Se´bastien Vidal*c
and Susan E. Matthews*a
Received 29th April 2011, Accepted 1st July 2011
DOI: 10.1039/c1ob05676j
Synthetic multivalent glycoclusters show promise as anti-adhesives for the treatment of bacterial
infections. Here we report the synthesis of a family of tetravalent galactose and lactose functionalised
macrocycles based on the resorcin[4]arene core. The development of diastereoselective synthetic routes
for the formation of lower-rim propargylated resorcin[4]arenes and their functionalistion via
Cu-catalyzed azide–alkyne click chemistry is described. ELLA binding studies confirm that galactose
sugar clusters are effective ligands for the PA-IL bacterial lectin of Pseudomonas aeruginosa while poor
binding for the lactose-based monovalent probe and no binding could be measured for the multivalent
glycoclusters was observed for the human galectin-1.
We have previously examined the role of multivalent ligand
topology in determining binding affinity at the PA-IL lectin
Introduction
using a series of tetrafunctionalised calixarenes.5d We have also
studied the influence of other core scaffolds such as porphyrins,5g,5h
b-peptoids,5h calix[6]arenes5h or fullerenes.5e,5f Resorcin[4]arenes
offer the opportunity to investigate topology further through the
preparation of macrocycles bearing sugars at the lower-rim in the
two readily available rccc boat and rctt chair diastereoisomers.
Resorcin[4]arenes, octafunctionalised with sugar moieties at the
upper-rim have previously been prepared through amine induced
ring opening of a lactone.8 Such derivatives have been investigated
for a number of biological applications including DNA delivery9
and protein binding studies. Treatment with maltonolactone
resulted in glucose terminated derivatives which could bind ef-
fectively to concanavalin A, whereas reaction with lactonolactone
gave specificity for binding to peanut lectin, a galactose specific
protein.10 When sialic acid residues were introduced at the upper-
rim, binding to Japanese horseshoe crab lectin was observed
in addition to inhibition of virus mediated haemagglutination
in the presence of influenza A.11 This ability to interfere with
cellular interactions has been further explored by the synthesis of
resorcin[4]arenes functionalised with polysaccharide chondroitin
sulphate which were able to inhibit adhesion-triggered cell growth
in baby hamster kidney cells.12
Functionalisation of resorcin[4]arenes at the lower rim with
sugars has received considerably less attention, with a single
publication outlining the stepwise synthesis of carbohydrate
containing macrocycles in which the product adopted the rctc
diamond configuration and the sugar is in its open chain form.13
In this study, we report the synthesis of tetravalent glycoclusters
based on the resorcin[4]arene core in two specific topologies, the
rccc boat and the rctt chair stereoisomers (Fig. 1) and their bio-
logical evaluation against two lectins, the galactose-specific PA-IL
The first step of bacterial infection is the colonization of tissues
through adhesion of bacteria to host cells. This process usually
relies on the recognition of carbohydrate epitopes present at the
surface of cells by receptors (lectins) displayed on the surface of
bacteria.1 Pseudomonas aeruginosa is an opportunistic bacterium
responsible for more than 20% of hospital acquired infections and
is also largely involved in pulmonary infections for cystic fibrosis
and intensive care unit patients.2 This bacterium expresses two
lectins which are involved in the binding event responsible for
infection: the galactose-specific lectin LecA from Pseudomonas
aeruginosa (PA-IL) and the fucose-specific lectin LecB from
Pseudomonas aeruginosa (PA-IIL).3 Even though the affinity of the
lectins for their carbohydrate is relatively weak, the lung bacterial
infection could be diminished by using galactose and fucose as
lectin competitors.4 The design of high affinity ligands of PA-IL5
and/or PA-IIL6 is therefore a promising anti-adhesive strategy7
for fighting bacterial infection.
aSchool of Pharmacy, University of East Anglia, Norwich, UK, NR4 7TJ.
E-mail: susan.matthews@uea.ac.uk; Fax: +44 1603-592003; Tel: +44 1603
595986
bInstitute of Materials and Research, Department of Metallurgy and
Materials, Dawood Collge of Engineering and Technology, Karachi, Pakistan
cInstitut de Chimie et Biochimie Mole´culaires et Supramole´culaires, Labora-
toire de Chimie Organique 2 – Glycochimie, UMR 5246, CNRS, Universite´
Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918, F-69622,
Villeurbanne, France. E-mail: sebastien.vidal@univ-lyon1.fr
dCERMAV – CNRS, affiliated with Universite´ Joseph Fourier and ICMG,
BP 53, 38041, Grenoble, France
eInstitute for Glycomics, Gold Coast Campus, Griffith University, Queens-
land, 4222, Australia
† Electronic supplementary information (ESI) available: 1H, 13C NMR and
mass spectra for all new compounds. See DOI: 10.1039/c1ob05676j
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Fig. 1 Sugar functionalised resorcin[4]arenes.
and lactose-specific galectin-1 (Gal-1). The synthetic route allows
introduction of lower-rim sugar motifs through the synthesis of
resorcin[4]arenes bearing alkynyl groups suitable for reaction with
azide functionalised sugar moieties through Cu-catalyzed azide–
alkyne click chemistry (CuAAC).14
Fig. 2 rccc boat and rctt chair diastereoisomers of resorcin[4]arenes a) flat
representation; b) 3-dimensional representation; c) calculated structures of
the diastereoisomers for a simple octa-O-alkyl resorcin[4]arene.15
Results and discussion
We have recently developed methods for the direct preparation
of octaalkyl resorcin[4]arenes15 from 1,3-dimethoxybenzene and a
number of different aldehydes. Such methods offer the potential
of selective synthesis of one of the two major diastereoisomers of
resorcin[4]arenes; the rccc boat and the rctt chair, with additional
functionality at the lower rim. These two diastereoisomers (Fig. 2)
allow an evaluation of the topological role in binding to lectins
as the rccc boat enables clustering of the sugar residues on one
face of the core and the rctt chair provides two discrete binding
regions on opposite faces. Interestingly, few alkyne derivatives
of resorcin[4]arenes have been described and to date these have
exclusively been upper-rim derivatives and have been used for
the preparation of organometallic complexes, particularly highly
valent cobalt complexes, rather than for cycloadditions.16
A two step procedure was devised for the preparation of alkyne
bearing resorcin[4]arenes, involving pre-functionalisation of a suit-
able aldehyde prior to cyclisation. 4-(Propargyloxy)benzaldehyde
was synthesised according to literature procedures17 and treated
with 1,3-dimethoxybenzene in the presence of concentrated sul-
phuric acid in an acetic acid medium (Scheme 1). Similarly to our
other reported derivatives, treatment at room temperature and for
short time periods at elevated temperature (80 ◦C) resulted in a
mixture of the two diastereoisomers with selectivity towards the
rctt chair. However, full stereoselectivity could be obtained, with
high macrocyclisation yield, over a 7 day reaction time period.
This elongated reaction time prompted an investigation of addi-
tional routes to the rctt diastereoisomer focusing on microwave
irradiation. Using the same reaction solvent and catalyst, a 90%
yield of the single rctt diastereoisomer was obtained in 5 min
heating at 70 ◦C, 100 W power and 20 bar pressure. However,
on changing the solvent to dichloromethane, no macrocyclisation
was achieved, a result also seen when using dichloromethane and
Scheme 1 Direct synthesis of tetrapropargylated resorcin[4]arenes using
a pre-functionalised aldehyde.
a Lewis acid catalyst (SnCl4), conditions previously developed
by Iwanek18 for the synthesis of O-alkyl resorcin[4]arenes under
standard conditions.
The rccc boat diastereoisomer can be isolated, from the mixture
obtained at room temperature, via column chromatography using
9 : 1 CHCl3 : EtOAc, following precipitation of the majority chair
diastereoisomer (45 : 55 boat : chair) from dichloromethane.
The 1H NMR (DMSO-d6) for the reaction at room temperature
clearly shows the presence of the two isomers through the ap-
pearance of two non-equivalent singlets in the characteristic peak
range (d 4.9–5.5) for the methylene bridge hydrogens (Fig. 3a and
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using CuI, DIPEA in DMF, to afford the acetylated glycoclusters
5 in high yields (58–85%). The reaction was performed under
microwave activation and the cycloaddition was complete within
15 min. The unmasking of the acetate protecting groups was
then achieved under smooth solvolysis conditions to obtain the
desired resorcin[4]arene-based hydroxylated glycoclusters in near
quantitative yields 6.
The stereochemistry, including the flattened boat of the rccc
boat, of the propargylated resorcin[4]arene-based scaffolds 3a-b
was conserved in the conjugated macromolecules. This is clearly
1
demonstrated in the H NMR spectra, where for the rctt chair
diastereoisomers 5aG, 5aL, 6aG and 6aL (Fig. 4 a,b,e,f respectively),
the characteristic high field signal (d ~5.7) of one of the aromatic
residues is observed whereas for the rccc boat diastereoisomers
(Fig. 4 c,d,g,h) the same proton is downfield (d ~5.9). Interestingly
in the case of the rctt chair diastereoisomers the signals for the
pendant aromatic groups have fully coalesced in the protected
sugar derivatives whereas in the rccc boat diastereoisomers the
coalescence is less pronounced.
Fig. 3 Expansion of the aromatic region of the 1H NMR spectra
(400 MHz, DMSO-d6) of 3: a) Products isolated from reaction at 25 ◦C in
acetic acid using H2SO4 catalysis, showing presence of 3a and 3b; b) purified
rccc boat diastereoisomer 3b; c) purified rctt chair diastereoisomer 3a.
Supplementary Information). Additionally, two series of peaks,
with integrals corresponding to the two individual bridge signals
can be identified, leading to the conclusion that, as expected, the
mixture contains both boat and chair isomers. However, for the
same reaction performed at 80 ◦C or under microwave conditions,
a single diastereoisomer is isolated (Fig. 3c). This shows the
expected C2h symmetry of the rctt chair, as indicated by the
presence of two signals for the OMe protons (d 3.58, 3.62, see
Supplementary Information), four signals for the protons in the
aromatic core of the resorcin[4]arene (d 5.51, 6.09, 6.43, 6.50), but
only a single pair of doublets for the identical pendant aromatic
groups (d 6.40, 6.50) and the expected triplet and doublet for
the alkynes (d 3.48, 4.60, see Supplementary Information). The
1H NMR of the second diastereoisomer, isolated after column
chromatography, shows that, as previously observed for large
pendant groups,15b the rccc isomer exists in the C2v flattened
boat form, with characteristically broadened signals for the four
inequivalent protons of the aromatic core of the resorcin[4]arene
(d 5.56, 5.96, 6.31, 6.46) and two signals for the OMe protons
(d 3.41, 3.56) (Fig. 3b and Supplementary Information).
Fig. 4 Partial 1H NMR spectra (aromatic region) of acetylated glyco-
clusters 5 (CDCl3, 400 MHz, 298 K) a: rctt chair diastereoisomer 5aG, b:
rctt chair diastereoisomer 5aL, c: rccc boat diastereoisomer 5bG, d: rccc
boat diastereoisomer 5bL) and hydroxylated glycoclusters 6 (DMSO-d6 +
eD2O, 400 MHz, 298 K) e: rctt chair diastereoisomer 6aGf: rctt chair
diastereoisomer 6aL, g: rccc boat diastereoisomer 6bG, h: rccc boat
diastereoisomer 6bL).
These two tetravalent propargylated scaffolds were then conju-
gated with two carbohydrate epitopes (galactose and lactose) in
order to study the potential interactions with biologically relevant
lectins. The galactose-functionalised glycoclusters were evaluated
against PA-IL and the lactose-functionalized derivatives against
Gal-1, a human lectin implicated in cancer, inflammation and
host-pathogen interactions.19
The synthetic route employed was determined based on a
number of factors. Conjugation by CuAAC was preferred to
amide bond conjugation to avoid the use of excess of either the
resorcin[4]arenes or functionalized sugars. The CuAAC reaction
The conjugation of the topoisomeric propargylated
resorcin[4]arene 3a–b was achieved by CuAAC with azido-
5f
20
functionalised galactoside 4G and lactoside 4L (Scheme 2),
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Scheme 2 Synthesis of four topoisomeric resorcin[4]arene-based glycoclusters.
is fast and high yielding (usually >95% for a single reaction)
and therefore avoids the potential of incomplete reaction of the
alkynyl groups which may lead to mono-, di- or tri-functionalised
resorcin[4]arene byproducts. In addition, acetylated carbohydrate
derivatives were employed rather than unprotected sugars in
order to obtain glycoclusters which are readily soluble in organic
solvents and can be easily purified using standard silica gel
chromatography. The subsequent deprotection step is facile and
gives the desired glycoclusters in high yield and purity.
In order to assess the role of multivalency on lectin binding
and to ascertain the influence of the linker on binding, two
monovalent carbohydrate probes bearing only the linker arm
terminated by a triazole ring were also synthesised (Scheme
3). Glycosylation of peracetyl galactopyranose 7G or lactose 7L
with 2-[2-(2-chloroethoxy)ethoxy]ethanol followed by azidation
of the chloroethyl moiety afforded the acetylated glycosides 4G
and 4L. 1,3-Dipolar cycloaddition under CuAAC conditions with
propargyl acetate provided the acetylated cycloadducts 8G and
5f
8L and subsequent deprotection of the acetate groups afforded the
5f
hydroxylated monovalent carbohydrate probes 9G and 9L.
The binding affinity of the glycoclusters was investigated
towards PA-IL and Gal-1 using a range of techniques including
a haemagglutination inhibition assay (HIA), an enzyme-linked
lectin assay (ELLA) and surface plasmon resonance (SPR).
Haemagglutination assays (HIA) were performed for both
5h
lectins. The monovalent probes 9G and 9L displayed minimum
inhibitory concentrations (MIC) in the millimolar range towards
PA-IL and Gal-1 respectively (Table 1). The multivalent gly-
coclusters 6 when evaluated under the same conditions were
poorly soluble in the buffer solutions used, however they were
of acceptable solubility under the conditions used. Nevertheless
they caused the degradation of the erythrocytes and a haemolytic
solution of the cells was obtained and thus MIC values could
not be determined. Similarly, surface plasmon resonance (SPR)
experiments could be readily performed with the monovalent
5d
probes 9G and 9L (Figure 27 supporting information) towards
PA-IL and Gal-1 but the limited solubility of the glycoclusters 6
did not allow the determination of IC50 values for these compounds
(Table 1). The enzyme-linked lectin assay (ELLA) could be
performed only with PA-IL and the galactosylated derivatives
9G,5d 6aG and 6bG. Due to the experimental conditions required
for the assay with Gal-1, IC50 values could not be determined. The
IC50 value measured for the monovalent probe 9G was in the high
micromolar range (Table 1). In contrast, the tetravalent glyco-
clusters 6aG and 6bG displayed much stronger inhibition potency
with IC50 values in the sub-micromolar range corresponding to
a 314-fold and 244-fold improvement respectively (Fig. 5). It is
Scheme 3 Synthesis of the monovalent carbohydrate probes.
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Table 1 Measurement of inhibition for the monovalent probes 9G and 9L and for the resorcin[4]arene-based glycoclusters 6
PA-IL
HIA
Gal-1
HIA
ELLA
SPR
SPR
Ligand (Valency)
MICa (mM)
IC50 (mM)
IC50 (mM)
MICa (mM)
IC50 (mM)
9G (1)
105h
2205f
-/-
0.7
0.9
-/-
-/-
63.55d
-/-
n.s.c
n.s.c
-/-
-/-
-/-
-/-
229
-/-
-/-
n.s.c
n.s.c
9L (1)
-/-
1.25
6aG (4)
6bG (4)
6aL (4)
6bL (4)
Haemol.b
Haemol.b
-/-
-/-
-/-
Haemol.b
Haemol.b
-/-
a Minimum inhibitory concentration. b Haemolysis of erythrocytes. c Not soluble under the experimental conditions required.
possible for the two carbohydrates to be orientated to bind to two
neighbouring binding sites on the tetrameric protein (Fig. 6) which
may account for the highly similar inhibition potencies observed
for the two diastereoisomers Modelling of the resorcin[4]arenes
in the presence of the protein indicates in both cases that the
glycocluster can span two binding sites on a single protein (Fig. 7)
and as proposed with the 1,3 alternate calixarene molecule a
chelate-like binding may be possible in which the remaining two
carbohydrates are available to bind to an additional protein.
Fig. 5 Inhibition of PA-IL adhesion to a galactosylated surface measured
by ELLA for compounds 9 (᭡), 6a ( ) and 6b (ꢀ). Error bars represent
᭹
G
G
G
standard deviation over triplicates, lines are sigmoidal fits.
interesting to note, that in this case, the influence of topology is
less clear than previously observed for calix[4]arene scaffolds.5d
Nevertheless, these improved binding properties towards PA-
IL are still significant in the series of multivalent glycoclusters
previously studied5f,5h by ELLA.
Fig. 6 Low energy conformations of compounds 6bg (A), 6aG (B) and
1,3 alternate galactosyalted calix[4]arene (C)5d with the linkers built in a
extended conformation. Distances have been measured from atom C1 of
galactose units.
A number of our previously reported calixarene-,5d,5h porphyrin-
5h
,
b-peptoid-5h or fullerene-based5e,5f glycoclusters showed better
affinities towards PA-IL using SPR analysis (IC50 in the sub-
micromolar range). The galactosylated glycoclusters 6G also
displayed IC50 values (measured by ELLA) in the sub-micromolar
range but these are rather higher values in comparison. Thus
their binding properties towards PA-IL play a highly important
role in further developing this family of multivalent ligands for
PA-IL. Rationalisation of this slightly reduced affinity is difficult
since isothermal titration microcalorimetry (ITC) measurements
could not be performed due to solubility problems and thus the
thermodynamic aspects of the interactions and their stoichiometry
could not be determined.
However, in the light of the good binding abilities observed,
we performed a molecular modelling study of the two new
resorcin[4]arene glycoclusters 6aG and 6bG in comparison to
the most efficient PA-IL ligand identified to-date, a calixarene
fixed in the 1,3 alternate conformation that features identical
carbohydrate arms.5d Comparison of the minimised structures
for the three ligands indicates that similarly to the calixarene
ligand, in both the rccc boat and rctt chair diastereoisomers it is
Fig. 7 Molecular modeling of the interaction between two adjacent
PA-IL monomers and compound 6bG. The protein is represented with
its accessible surface, calcium ions are represented as green spheres.
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unit (HU) was obtained by the addition of 25 mL of the 4%
erythrocyte solution to 25 mL aliquots of sequential (2-fold) lectin
dilutions. The mixture was incubated at 25 ◦C for 60 min. The
HU was measured as the minimum lectin concentration required
to observe haemagglutination. For the lectin-inhibition assays,
lectin concentrations of 4 HU were used. This concentration was
found to be 8 mg mL-1 for PA-IL and 6 mg mL-1 for Gal-1.
Subsequent inhibition assays were then carried out by the addition
of 12.5 mL lectin solution to 25 mL of sequential dilutions of
glycoclusters, monovalent probes and controls. These solutions
were then incubated at 25 ◦C for 2 h then 12.5 mL of 4% erythrocyte
solution was added followed by an additional incubation at 25 ◦C
for 30 min. The minimum inhibitory concentration for each
molecule was determined for each duplicate.
Conclusion
Two new resorcin[4]arenes suitably functionalised for ‘click’
chemistry, by the inclusion of alkynes at the lower-rim, were
synthesised in a short synthetic sequence. A suitable choice of
reaction conditions allowed for the selective formation of the
rctt chair diastereoisomer through either prolonged treatment
at elevated temperature or the use of microwave heating. The
conjugation of these propargylated resorcin[4]arene scaffolds
with azido-functionalized carbohydrate probes allowed for the
efficient preparation of topological isomers of galactosylated
and lactosylated glycoclusters. These cyclic architectures offer a
general platform for the design of conformationally restricted
isomers and the display of binding moieties in a specific spatial
arrangement. Although the range of techniques available for
measurement of affinity are limited due to solubility problems,
the ELLA results obtained for the galactosylated glycoclusters
demonstrated a strong affinity for PA-IL. However, no difference
in binding properties could be observed due to the topology of
the glycoclusters. Our efforts are now focused on the design of
more water soluble resorcin[4]arene-based glycoclusters in order
to improve their binding properties towards biologically relevant
lectins.
Enzyme-linked lectin assays (ELLA)
ELLA were conducted by using 96-well microtitre plates (Nunc
Maxisorb) coated with PAA-a-D-Gal: 100 mL of a 5 mg mL-1
solution in carbonate buffer, pH 9.6 for 1 h at 37 ◦C then
blocking at 37 ◦C for 1 h with 100 mL per well of 3% (w/v)
BSA in PBS. Inhibitor solutions (50 mL) were subjected to serial
dilutions (3-fold) with PBS-BSA 0.3% (w/v). Then, biotinylated
PA-IL solution (50 mL, 0.5 mg mL-1) was added to each well
and the plates were incubated for 1 h at 37 ◦C. After three
washings with T-PBS (50 mL; PBS containing 0,05% Tween 20)
for 5 min, horseradish streptavidin-peroxidase conjugate (HRP,
100 mL) (dilution 2 : 5000; Boehringer-Mannheim) was added and
left for 1 h at 37 ◦C. After three more washings, colouration was
developed by using 100 mL per well of 0.05 M phosphate/citrate
buffer containing o-phenylenediamine dihydrochloride (0.4 mg
mL-1) and urea hydrogen peroxide (0.4 mg mL-1; OPD kit, Sigma-
Aldrich) for 15 min and stopped with 30% sulfuric acid (50 mL).
Absorbance was then read at 490 nm by using a microtitre plate
reader (BioRad 680) and transformed into inhibition percentages
with the help of positive and negative controls. Plots (inhibition
percentage versus inhibitor concentration) and sigmoidal fitting
provided IC50 values.
Experimental
All reagents for synthesis were commercial (highest purity avail-
able for reagent grade compounds) and used without further
purification. Solvents were distilled over CaH2 (CH2Cl2), Mg/I2
(MeOH) or purchased dry. All reactions were performed under
an argon atmosphere. Reactions under microwave activation were
performed on a Biotage Initiator system. NMR solvents were
purchased from Euriso-Top (Saint Aubin, France) or Apollo.
NMR spectra were recorded at 293 K, except where stated, using
a 300 MHz or a 400 MHz spectrometer. Shifts are referenced
relative to deuterated solvent residual peaks. Complete signal
assignments from 1D and 2D NMR were based on COSY,
HSQC and HMBC correlations. High resolution (HR-ESI-QToF)
mass spectra were recorded using a Bruker MicroTOF-Q II XL
spectrometer. Optical rotation was measured using a Perkin Elmer
polarimeter. Melting points were determined using IA9000 digital
melting point apparatus and are uncorrected. Compounds 4G,5d
Surface plasmon resonance (SPR)
8G,5d 9G and 4L were prepared as previously described.
5d
20
SPR inhibition experiments were performed on a Biacore 3000
instrument at 25 ◦C. Measurements were carried out on 3 channels
with 3 immobilised sugars: a-L-fucose (channel 1), b-D-galactose
(channel 2) and a-D-lactose (channel 3). Immobilization of sugars
was performed at 25 ◦C using running buffer (HBS) at 5 mL min-1.
Immobilization on each channel (CM5 Chip) was performed
independently. First, the channel was activated by injecting a
fresh mixture of EDC/NHS (35 mL, 420 s). Then, a solution of
streptavidin (100 mg mL-1 in AcONa pH 5 buffer) was injected
(50 mL, 600 s). The remaining reactive species were quenched by
injecting ethanolamine (1 M, 35 mL, 420 s). Finally a solution
of the desired biotinylated PAA–sugar (Lectinity, 200 mg mL-1)
was coated onto the surface (50 mL, 600 s) through streptavidin–
biotin interaction. This procedure led to 804 RU (resonance unit)
(fucoside), 796 RU (galactoside) and 1022 RU (lactoside) of
immobilized sugars on channel 1, 2 and 3 respectively. Inhibition
experiments were performed with the galactosylated channel 2
Recombinant lectins
Recombinant PA-IL was produced in E. coli according to our pre-
viously reported protocols.21 Human galectin-1 was generated via
transformation of pET-3a-gal-1 plasmid, that encodes non-tagged
gene LGALS1, into E. coli strain BL21 DE3 and expressed and
purified following protocols that we have reported previously.22
Haemagglutination inhibition assays (HIA)
Haemagglutination inhibition assays (HIA) were performed in
U-shaped 96-well microtitre plates. Rabbit erythrocytes were
purchased from Biome´rieux and used without further washing.
Erythrocytes were diluted to a 4% solution in NaCl (150 mM).
Lectin solutions of 2 mg mL-1 were prepared in Tris-HCl 20 mM,
NaCl 100 mM and CaCl2 100 mM. The haemagglutination
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and the lactosylated channel 3 and plots represent subtracted data
(channel 2–channel 1 or channel 3–channel 1).
NMR (100 MHz, DMSO-d6 + eDMF 363 K) 156.5, 155.9, 155.8,
136.3, 131.6, 130.1, 128.3, 125.9, 124.7, 114.4, 98.1, 97.4, 80.1,
77.6, 56.6, 56.3, 56.3, 42.2. IR 3265, 3006, 2941, 2830, 1608, 1581,
1504, 1463, 1450, 1438, 1396, 1382, 1291, 1228, 1219, 1200, 1182,
1162, 1097, 1032. HR-ESI-MS (positive mode): m/z calcd for
C72H68NO12 [M + NH4]+ 1138.4736, found 1138.4742. Mp 270 ◦C
decomp.
Conditions for PA-IL. The running buffer for PA-IL experi-
ments is HEPES 10 mM, NaCl 150 mM, CaCl2 10 mM, Tween P20
0.005%, pH 7.4. Inhibition experiments with PA-IL are measured
with galactosylated channel (2). Inhibition studies consisted of the
injection (150 mL, 10 mL min-1, dissociation: 120 s) of incubated
(>1 h, r.t.) mixtures of PA-IL (5 mM) and various concentrations
of inhibitor (2-fold cascade dilutions). For each inhibition assay,
PA-IL (5 mM) without inhibitor was injected to observe the
full adhesion of the lectin onto the sugar-coated surface (0%
inhibition). The CM5 chip was fully regenerated by successive
injections of D-galactose (2 ¥ 30 mL, 100 mM in running buffer).
Compound 3b rccc diastereoisomer, traditional synthesis
1,3-Dimethoxybenzene (2.0 g, 14 mmol) and 4-(propargyloxy)-
benzaldehyde (2.3 g, 14 mmol) were stirred in acetic acid (76 mL)
in the presence of concentrated H2SO4 (0.85 mL) at 25 ◦C for
18 h. The reaction mixture was cooled and the resulting precipitate
isolated by filtration and washed with ether. Precipitation from
dichloromethane yielded pure rctt isomer. The filtrate was purified
by column chromatography (9 : 1 hexane : ethyl acetate) to yield the
rccc isomer (0.61 g, 16%).
Conditions for Gal-1. The running buffer for Gal-1 experi-
ments is HEPES 10 mM, NaCl 150 mM, Tween P20 0.005%,
pH 7.4. Inhibition experiments with Gal-1 are measured with
lactosylated channel (3). Inhibition studies consisted of the
injection (80 mL, 10 mL min-1) of incubated (>1 h, r.t.) mixtures
of Gal-1 (40 mM) and various concentrations of inhibitor (2-
fold cascade dilutions). For each inhibition assay, Gal-1 (40 mM)
without inhibitor was injected to observe the full adhesion of
the lectin onto the sugar-coated surface (0% inhibition). The CM5
chip was fully regenerated by successive injections of NaCl (10 mL,
1 M in running buffer) and D-galactose (10 mL, 100 mM in running
buffer).
For all experimental settings (PA-IL and Gal-1), binding was
measured as RU over time after blank subtraction, and data were
then evaluated using the BIAevaluation Software, version 4.1. For
IC50 evaluation, the response (Req – fitted) was considered as the
amount of lectin bound to the carbohydrate-coated surface at
equilibrium in the presence of a defined concentration of inhibitor.
Inhibition curves were obtained by plotting the percentage of
inhibition against the inhibitor concentration (on a logarithmic
scale) by using Origin 7.0 software (OriginLab Corp.) and IC50
values were extracted from sigmoidal fit of the inhibition curve.
1H NMR (400 MHz, DMSO-d6) d 6.57 (8H, d, J = 9 Hz,
ArHpendant), 6.50 (8H, d, J = 9 Hz, ArHpendant), 6.46 (2H, s, ArHresorc),
6.31 (2H, s, ArHresorc), 5.96 (2H, s, ArHresorc), 5.56 (2H, s, ArHresorc),
5.46 (4H, s, ArCH), 4.65 (8H, d, J = 2.5 Hz, OCH2C), 3.56 (12H, s,
OCH3), 3.52 (4H, t, J = 2.5 Hz C CH), 3.41 (12H, s, OCH3). 13
C
NMR (100 MHz, DMSO-d6) d 156.2, 155.9, 155.6, 137.0, 129.9,
124.1, 123.9, 114.2, 80.3, 78.6, 56.3, 56.1, 55.8, 42.3. IR 3276, 3002,
2941, 2833, 1609, 1583, 1506, 1465, 1438, 1360, 1375, 1294, 1217,
1200, 1178, 1159, 1097, 1031. HR-ESI-MS (positive mode): m/z
calcd for C72H68NO12 [M + NH4]+ 1138.4733, found 1138.4742.
Mp 242–244 ◦C
General procedure for 1,3-dipolar cycloaddition (Method A)
Unless otherwise stated, the alkyne-functionalized compound,
copper iodide, DIPEA and azido-derivative in DMF were intro-
duced into a Biotage Initiator 2–5 mL vial. The vial was flushed
with argon and the solution was sonicated for 30 s. The vial
was sealed with a septum cap and heated at 110 ◦C for 15 min
under microwave irradiation (solvent absorption level: high). After
uncapping the vial, the mixture was diluted with EtOAc (250 mL).
The organic layer was washed with 150 mL portions of 1 N HCl,
saturated NaHCO3, water, and brine successively. The organic
layer was dried (Na2SO4), filtered and evaporated. The crude
product was purified by flash silica gel column chromatography to
afford the desired cycloadducts.
Compound 3a rctt diastereoisomer, traditional synthesis
1,3-Dimethoxybenzene (2.0 g, 14 mmol) and 4-(propargyloxy)-
benzaldehyde (2.3 g, 14 mmol) were stirred in acetic acid (76 mL)
in the presence of concentrated H2SO4 (0.85 mL) at 80 ◦C for
7 d. The reaction mixture was cooled and the resulting precipitate
isolated by filtration and washed with Et2O to yield the desired
product (3.30 g, 85%).
Acetylated rctt glycocluster 5aG
Compound 3a rctt diastereoisomer, microwave synthesis
Obtained as a white foam (189 mg, 85%) following Method A:
Compound 3a (80 mg, 71 mmol, 1 eq.), 4G (225 mg, 430 mmol,
6 eq.), CuI (6.8 mg, 36 mmol, 0.5 eq.) and DIPEA (63 mL,
0.36 mmol, 5 eq.) in DMF (3 mL). Microwave irradiation: 15 min
at 110 ◦C. After workup, the residue was purified by silica gel
Concentrated H2SO4 (0.045 mL) was added to 1,3-dimethoxy-
benzene (0.1 g, 0.072 mmol) and 4-(propargyloxy)benzaldehyde
(0.11 g, 0.072 mmol) in acetic acid (5 mL) and irradiated in a CEM-
Discover single cavity microwave under stirring for 5 min at 70 ◦C
under 100 W power and 20 Bar pressure. The reaction mixture
was cooled and the resulting precipitate isolated by filtration and
washed with ether to yield the desired product (3.49 g, 90%).
1H NMR (400 MHz, DMSO-d6) d 6.57 (2H, s, ArHresorc), 6.50
(8H, d, J = 9 Hz, ArHpendant), 6.43 (2H, s, ArHresorc), 6.40 (8H d, J =
9 Hz, ArHpendant), 6.09 (2H, s, ArHresorc), 5.51 (2H, s, ArHresorc), 5.48
(4H, s, ArCH), 4.60 (8H, d, J = 2.5 Hz, OCH2C), 3.62 (12H, s,
OCH3), 3.58 (12H, s, OCH3), 3.48 (4H, t, J = 2.5 Hz C CH).13C
flash chromatography (EtOAc then EtOAc/MeOH, 9 : 1). [a]D
=
1
-5.3 (c 0.75, CH2Cl2). H NMR (400 MHz, CDCl3) d 7.69 (s,
4H, H-triaz), 6.57 (s, 16H, exo-CH-ar), 6.46, 6.39, 6.18, 5.69 (4
s, 4 ¥ 2H, endo-CH-ar), 5.67 (s, 4H, ArCHRAr), 5.37 (d, J =
3.3 Hz, 4H, H-4), 5.18 (dd, J = 10.3 Hz, J = 8.2 Hz, 4H, H-
2), 5.04 (s, 8H, PhOCH2C-triaz), 5.00 (dd, J = 10.3 Hz, J =
3.3 Hz, 4H, H-3), 4.54–4.45 (m, 12H, H-1, OCH2CH2N), 4.17–
1
2
4.10 (m, 8H, H-6a, H-6b), 3.96–3.81 (m, 16H, H-5, GalOCH2,
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1
2
1
2
OCH2CH2N), 3.72–3.64 (m, 28H, OCH3, GalOCH2), 3.60–
3.90–3.80 (m, 16H, H-5, LacOCH2, OCH2CH2N), 3.77 (t, J =
1
3.52 (m, 24H, GalOCH2CH2OCH2CH2O), 2.12, 2.03, 2.01, 1.96
(4 s, 12H, CH3CO). 13C NMR (100 MHz, CDCl3) d 170.5,
170.4, 170.2, 169.5 (4 s, 4¥CH3CO), 156.2, 156.1, 155.7 (3 s,
CIVOCH3, CIVOCH2Ctriaz), 144.5 (CIV-triaz), 136.06, 136.04 (2 s,
exo-CIV-ar), 131.8 (endo-CH-ar), 130.1 (exo-CH-ar), 128.5 (endo-
CH-ar), 125.3, 124.6 (2 s, endo-CH-CIV-ar), 123.9 (CH-triaz),
113.8 (exo-CH-ar), 101.4 (C-1), 96.6, 95.3 (endo-CH-ar), 71.0,
70.8 (C-3, C-5), 70.7, 70.6, 70.3 (GalOCH2CH2OCH2CH2O),
69.5, 69.2 (GalOCH2-, OCH2CH2N), 68.9 (C-2), 67.2 (C-4), 62.2
(PhOCH2C-triaz), 61.4 (C-6), 56.40, 56.37, 55.9 (OCH3), 50.2
(OCH2CH2N), 42.1 (ArCHRAr), 20.9, 20.8, 20.7 (3 s, 4¥CH3CO).
HR-ESI-QToF MS (positive mode): m/z calcd for C152H190N12O60
[M + 2H]++ 1571.6087, found 1571.6112.
9.4 Hz, 4H, H-4), 3.71–3.56 (m, 28H, H-5¢, OCH3, LacOCH2),
2
3.52 (bs, 24H, GalOCH2CH2OCH2CH2O), 2.12, 2.07, 2.02, 2.01
1.98, 1.94 (6 s, 21H, 7¥CH3CO). 13C NMR (100 MHz, CDCl3) d
170.40, 170.39, 170.2, 170.1, 169.8, 169.7, 169.1 (7 s, 7¥CH3CO),
156.14, 156.08, 155.6 (3 s, CIVOCH3, CIVOCH2C-triaz), 144.4 (CIV-
triaz), 136.0 (exo-CIV-ar), 131.8 (endo-CH-ar), 130.1 (exo-CH-ar),
128.5 (endo-CH-ar), 125.2, 124.5 (2 s, endo-CH-CIV-ar), 123.8
(CH-triaz), 113.8 (exo-CH-ar), 101.4, 100.7 (C-1, C-1¢), 96.6, 95.3
(endo-CH-ar), 76.3 (C-4), 72.9, 72.7 (C-3, C-5¢), 71.7 (C-2), 71.0
(C-3¢), 70.7 (C-5), 70.62, 70.56, 70.2 (GalOCH2CH2OCH2CH2O),
69.4, 69.19 (GalOCH2, OCH2CH2N), 69.17 (C-2¢), 66.7 (C-4¢),
62.1, 62.0 (C-6, PhOCH2C-triaz), 60.8 (C-6¢), 56.3, 55.93, 55.90
(OCH3), 50.2 (OCH2CH2N), 42.0 (ArCHRAr), 20.92, 20.86,
20.75, 20.69, 20.68, 20.6 (6 s, 7¥CH3CO). HR-ESI-QToF MS
(positive mode): m/z calcd for C200H252N12Na2O92 [M + 2Na]++
2169.7597, found 2169.7651.
Acetylated rccc glycocluster 5bG
Obtained as a white foam (185 mg, 83%) following Method A:
Compound 3b (80 mg, 71 mmol, 1 eq.), 4G (225 mg, 430 mmol,
6 eq.), CuI (6.8 mg, 36 mmol, 0.5 eq.) and DIPEA (63 mL,
0.36 mmol, 5 eq.) in DMF (3 mL). Microwave irradiation: 15 min
at 110 ◦C. After workup, the residue was purified by silica gel flash
chromatography (EtOAc then EtOAc/MeOH, 9 : 1). [a]D= -3.3 (c
0.9, CH2Cl2). 1H NMR (400 MHz, CDCl3) d 7.75 (s, 4H, H-triaz),
6.77–6.62 (m, 16H, exo-CH-ar), 6.42, 6.19, 6.10, 5.85 (4 s, 4 ¥ 2H,
endo-CH-ar), 5.66 (s, 4H, ArCHRAr), 5.37 (d, J = 3.0 Hz, 4H,
H-4), 5.18 (dd, J = 10.4 Hz, J = 8.0 Hz, 4H, H-2), 5.11, 5.10 (2 s,
8H, PhOCH2C-triaz), 5.00 (dd, J = 10.4 Hz, J = 3.0 Hz, 4H, H-3),
4.55–4.45 (m, 12H, H-1, OCH2CH2N), 4.18–4.06 (m, 8H, H-6a,
Acetylated rccc glycocluster 5bL
Obtained as a white foam (82 mg, 61%) following Method A:
Compound 3b (35 mg, 31 mmol, 1 eq.), 4L (150 mg, 189 mmol, 6 eq.),
CuI (3 mg, 16 mmol, 0.5 eq.) and DIPEA (27 mL, 0.16 mmol, 5 eq.)
in DMF (3 mL). Microwave irradiation: 15 min at 110 ◦C. After
workup, the residue was purified by silica gel flash chromatography
(EtOAc/MeOH, 95 : 5 then 9 : 1). [a]D = -2.7 (c 0.7, CH2Cl2). 1H
NMR (400 MHz, CDCl3) d 7.74 (s, 4H, H-triaz), 6.72–6.66 (m,
16H, exo-CH-ar), 6.42, 6.20, 6.10, 5.85 (4 s, 4 ¥ 2H, endo-CH-
ar), 5.65 (s, 4H, ArCHRAr), 5.37 (d, J = 2.6 Hz, 4H, H-4¢), 5.17
(t, J = 9.4 Hz, 4H, H-3), 5.13–5.04 (m, 12H, H-2¢, PhOCH2C-
triaz), 4.95 (dd, J = 10.4 Hz, J = 3.4 Hz, 4H, H-3¢), 4.87 (dd,
J = 9.4 Hz, J = 8.0 Hz, 4H, H-2¢), 4.54–4.42 (m, 24H, H-1,
H-1¢, H-6a, H-6b, OCH2CH2N), 4.17–4.02 (m, 8H, H-6¢a, H-
1
H-6b), 3.96–3.81 (m, 16H, H-5, GalOCH2, OCH2CH2N), 3.73–
2
1
3.43 (m, 52H, OCH3, GalOCH2, GalOCH2CH2OCH2CH2O),
2
2.12, 2.03, 2.01, 1.96 (4 s, 12H, CH3CO). 13C NMR (100 MHz,
CDCl3) d 170.5, 170.4, 170.2, 169.6 (4 s, 4¥CH3CO), 156.3, 156.14,
156.07 (3 s, CIVOCH3, CIVOCH2C-triaz), 144.5 (CIV-triaz), 136.8
(exo-CIV-ar), 132.8 (endo-CH-ar), 129.9 (exo-CH-ar), 128.4 (endo-
CH-ar), 124.9 (endo-CH-CIV-ar), 124.0 (CH-triaz), 114.0 (exo-
CH-ar), 101.5 (C-1), 96.6, 95.6 (endo-CH-ar), 71.0, 70.75 (C-3,
C-5), 70.72, 70.6, 70.3 (GalOCH2CH2OCH2CH2O), 69.5, 69.3
(GalOCH2, OCH2CH2N), 68.9 (C-2), 67.2 (C-4), 62.2 (PhOCH2C-
triaz), 61.4 (C-6), 56.3, 56.1 (OCH3), 50.2 (OCH2CH2N), 42.3
(ArCHRAr), 20.9, 20.8, 20.7 (3 s, 4¥CH3CO). HR-ESI-QToF
MS (positive mode): m/z calcd for C152H190N12O60 [M + 2H]++
1571.6087, found 1571.6142.
1
2
6¢b), 3.91–3.81 (m, 16H, H-5, LacOCH2, OCH2CH2N), 3.78 (t,
1
2
J = 9.4 Hz, 4H, H-4), 3.71–3.59 (m, 16H, OCH3, LacOCH2),
3.59–3.49 (m, 28H, H-5¢, LacOCH2CH2OCH2CH2O), 3.47 (s,
12H, OCH3), 2.14, 2.08, 2.04, 2.03, 2.02, 1.99, 1.95 (7 s, 21H,
CH3CO). 13C NMR (100 MHz, CDCl3) d 170.48, 170.46, 170.3,
170.2, 169.9, 169.7, 169.2 (7 s, 7¥CH3CO), 156.3, 156.14, 156.06
(3 s, CIV-OCH3, CIV-OCH2C-triaz), 144.5 (CIV-triaz), 136.8 (exo-
CIV-ar), 132.8 (endo-CH-ar), 129.9 (exo-CH-ar), 128.4 (endo-CH-
ar), 124.9 (endo-CH-CIV-ar), 124.0 (CH-triaz), 114.0 (exo-CH-ar),
101.2, 100.8 (C-1, C-1¢), 96.6 (1 s, 2C, endo-CH-ar), 76.4 (C-4),
72.9, 72.7 (C-3, C-5¢), 71.7 (C-2), 71.1 (C-3¢), 70.8 (C-5), 70.7,
70.6, 70.3 (LacOCH2CH2OCH2CH2O), 69.5 (OCH2CH2N), 69.26
(LacOCH2), 69.22 (C-2¢), 66.7 (C-4¢), 62.2, 62.1 (C-6, PhOCH2C-
triaz), 60.9 (C-6¢), 56.3, 56.2, 56.1 (OCH3), 50.2 (OCH2CH2N),
42.3 (ArCHRAr), 21.0, 20.9, 20.82, 20.77, 20.75, 20.6 (6 s, 7C,
7¥CH3CO). HR-ESI-QToF MS (positive mode): m/z calcd for
C200H254N12O92 [M + 2H]++ 2147.7777, found 2147.7853.
Acetylated rctt glycocluster 5aL
Obtained as a white foam (94 mg, 58%) following Method A:
Compound 3a (42 mg, 37 mmol, 1 eq.), 4L (178 mg, 224 mmol, 6 eq.),
CuI (3 mg, 16 mmol, 0.5 eq.) and DIPEA (27 mL, 157 mmol, 5 eq.)
in DMF (3 mL). Microwave irradiation: 15 min at 110 ◦C. After
workup, the residue was purified by silica gel flash chromatography
(EtOAc/MeOH, 95 : 5 then 9 : 1). [a]D = -2.3 (c 0.43, CH2Cl2). 1H
NMR (400 MHz, CDCl3) d 7.67 (s, 4H, H-triaz), 6.55 (s, 16H, exo-
CH-ar), 6.45, 6.38, 6.17, 5.68 (4 s, 4 ¥ 2H, endo-CH-ar), 5.65 (s,
4H, ArCHRAr), 5.32 (d, J = 3.3 Hz, 4H, H-4¢), 5.16 (t, J = 9.3 Hz,
4H, H-3), 5.07 (dd, J = 10.4 Hz, J = 7.9 Hz, 4H, H-2¢), 5.03 (s, 8H,
PhOCH2C-triaz), 4.93 (dd, J = 10.4 Hz, J = 3.4 Hz, 4H, H-3¢),
4.86 (dd, J = 9.3 Hz, J = 8.0 Hz, 4H, H-2) 4.56–4.40 (m, 24H, H-1,
H-1¢, H-6a, H-6b, OCH2CH2N), 4.15–3.99 (m, 8H, H-6¢a, H-6¢b),
General procedure for deacetylation (Method B)
Unless otherwise stated, the acetylated glycoside or glycocluster
(1 eq.) was suspended in distilled MeOH, ultra-pure water and
ultra-pure triethylamine (5 : 1 : 1, v/v/v). The mixture was stirred
under argon at room temperature for 2 to 4 days. Solvents
were evaporated, co-evaporated with toluene three times and the
6594 | Org. Biomol. Chem., 2011, 9, 6587–6597
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resulting white foam was dissolved in ultra-pure water (5 mL) and
freeze-dried to afford pure glycoconjugates.
* Peaks not observed in the 13C NMR spectrum but clearly
identified from HSQC experiments.
Hydroxylated rctt glycocluster 6aG
Hydroxylated rctt glycocluster 6aL
Obtained as a freeze-dried white solid (122 mg, 97%) following
Method B: Compound 5aG (160 mg) was suspended in methanol
(4 mL), water (1 mL) and triethylamine (1 mL). After stirring
at r.t. for 3 days and concentration, the mixture was dissolved
in ultra-pure water (5 mL) then freeze-dried to afford the pure
deacetylated glycocluster. [a]D = -6.7 (c 0.64, DMSO). 1H NMR
(400 MHz, DMSO-d6 + eD2O) d 8.04 (s, 4H, H-triaz), 6.69–
6.57 (m, 10H, exo-CH-ar, endo-CH-ar), 6.51–6.42 (m, 10H, exo-
CH-ar, endo-CH-ar), 6.17, 5.72 (2 s, 2 ¥ 2H, endo-CH-ar), 5.59
(s, 4H, ArCHRAr), 5.01 (s, 8H, PhOCH2C-triaz), 4.48 (t, J =
5.0 Hz, 8H, OCH2CH2N), 4.08 (d, J = 7.2 Hz, 4H, H-1), 3.85–
Obtained as a freeze-dried white solid (65 mg, 97%) following
Method B: Compound 5aL (92 mg) was suspended in methanol
(3 mL), water (0.5 mL) and triethylamine (0.5 mL). After stirring
at r.t. for 4 days and concentration, the mixture was dissolved
in ultra-pure water (5 mL) then freeze-dried to afford the pure
deacetylated glycocluster. [a]D = -8.6 (c 0.80, DMSO). 1H NMR
(400 MHz, DMSO-d6 + e D2O) d 8.04 (s, 4H, H-triaz), 6.69–
6.55 (m, 10H, exo-CH-ar, endo-CH-ar), 6.54–6.39 (m, 10H, exo-
CH-ar, endo-CH-ar), 6.17, 5.72 (2 s, 2 ¥ 2H, endo-CH-ar), 5.51
(s, 4H, ArCHRAr), 5.00 (s, 8H, PhOCH2C-triaz), 4.47 (bs, 8H,
OCH2CH2N), 4.24–4.09 (m, 8H, H-1, H-1¢), 3.88–3.76 (m, 12H,
1
3.74 (m, 12H, GalOCH2, OCH2CH2N), 3.64 (s, 12H, OCH3),
1
2
LacOCH2, OCH2CH2N), 3.76–3.36 (m, 40H, H-4, H-4¢, H-
6a, H-6b, H-6¢a, H-6¢b, LacOCH2, LacOCH2CH2OCH2CH2O,
1
2
2
3.62 (s, 12H, OCH3), 3.58–3.41 (m, 40H, H-3, H-6a, H-6b,
1
2
GalOCH2, GalOCH2CH2OCH2CH2O), 3.35–3.28 (m, 4H, H-
5), 3.28–3.20 (m, 8H, H-2, H-4). 13C NMR (100 MHz, DMSO-
d6 + e D2O) d 156.0, 155.9, 155.2 (3 s, CIVOCH3, CIVOCH2C-
triaz), 143.2 (CIV-triaz), 135.2 (exo-CIV-ar), 131.4* (endo-CH-ar),
130.0 (exo-CH-ar), 127.7* (endo-CH-ar), 125.0 (CH-triaz), 124.6,
123.6 (endo-CH-CIV-ar), 113.8 (exo-CH-ar), 103.7 (C-1), 96.0*
(endo-CH-ar), 75.3, 73.5, 70.6 (C-2, C-4, C-5), 69.9, 69.8, 69.7
(GalOCH2CH2OCH2CH2O), 68.8 (OCH2CH2N), 68.2 (C-3), 67.9
(GalOCH2), 61.2 (PhOCH2C-triaz), 60.6 (C-6), 56.0 (OCH3), 49.6
(OCH2CH2N), 41.7 (ArCHRAr). HR-ESI-QToF MS (positive
mode): m/z calcd for C120H156N12Na2O44 [M + 2Na]++ 1257.5061,
found 1257.5066.
OCH3), 3.36–3.22 (m, 20H, H-2¢, H-3, H-3¢, H-5, H-5¢), 3.00 (t,
J = 8.2 Hz, 4H, H-2). 13C NMR (100 MHz, DMSO-d6 + e D2O) d
156.0, 155.9, 155.2 (3 s, CIVOCH3, CIVOCH2C-triaz), 143.2 (CIV-
triaz), 135.2 (exo-CIV-ar), 131.3 (endo-CH-ar), 130.0 (exo-CH-ar),
127.8 (endo-CH-ar), 124.8 (CH-triaz), 124.6, 123.6 (endo-CH-CIV-
ar), 113.8 (exo-CH-ar), 104.0, 102.8 (C-1, C-1¢), 96.4 (endo-CH-
ar), 80.8, 75.7, 75.04, 75.00, 73.3, 73.2, 70.7 (C-2, C-2¢, C-3, C-
3¢, C-4¢, C-5, C-5¢), 69.9, 69.8 (LacOCH2CH2OCH2CH2O), 68.8,
68.6 (OCH2CH2N, LacOCH2), 61.2 (PhOCH2C-triaz), 60.6 (C-6,
C-6¢), 55.9 (OCH3), 49.6 (OCH2CH2N), 41.7 (ArCHRAr). HR-
ESI-QToF MS (positive mode): calcd for C144H198N12O64 [M +
2H]++ 1559.6298, found 1559.6286.
* Peaks not observed in the 13C NMR spectrum but clearly
identified from HSQC experiments.
Hydroxylated rccc glycocluster 6bL
Hydroxylated rccc glycocluster 6bG
Obtained as a freeze-dried white solid (54 mg, 99%) following
Method B: Compound 5bL (75 mg) was suspended in methanol
(3 mL), water (0.5 mL) and triethylamine (0.5 mL). After stirring
at r.t. for 4 days and concentration, the mixture was suspended
in ultra-pure water (5 mL) then freeze-dried to afford the pure
deacetylated glycocluster. [a]D = -6.9 (c 0.75, DMSO). 1H NMR
(400 MHz, DMSO-d6 + e D2O) d 8.06 (s, 4H, H-triaz), 6.70 (d,
J = 8.5 Hz, 8H, exo-CH-ar), 6.57 (d, J = 8.5 Hz, 8H, exo-CH-
ar), 6.48, 6.35, 6.03, 5.77 (4 s, 4 ¥ 2H, endo-CH-ar), 5.49 (s, 4H,
ArCHRAr), 5.04 (s, 8H, PhOCH2C-triaz), 4.47 (t, J = 4.7 Hz, 8H,
OCH2CH2N), 4.23-4.14 (m, 8H, H-1, H-1¢), 3.88–3.75 (m, 12H,
Obtained as a freeze-dried white solid (138 mg, 98%) following
Method B: Compound 5bG (180 mg, 5 mmol) was suspended in
methanol (4 mL), water (1 mL) and triethylamine (1 mL). After
stirring at r.t. for 3 days and concentration, the mixture was
dissolved in ultra-pure water (5 mL) then freeze-dried to afford
the pure deacetylated glycocluster. [a]D = -2.5 (c 1.3, DMSO). 1H
NMR (400 MHz, DMSO-d6 + e D2O) d 8.06 (s, 4H, H-triaz), 6.70
(d, J = 8.5 Hz, 8H, exo-CH-ar), 6.57 (d, J = 8.5 Hz, 8H, exo-CH-
ar), 6.49, 6.35, 6.03, 5.77 (4 s, 4 ¥ 2H, endo-CH-ar), 5.49 (s, 4H,
ArCHRAr), 5.04 (s, 8H, PhOCH2C-triaz), 4.47 (t, J = 5.0 Hz, 8H,
1
2
1
1
OCH2CH2N), 4.07 (d, J = 7.4 Hz, 4H, H-1), 3.87–3.69 (m, 12H,
GalOCH2, OCH2CH2N), 3.70–3.40 (m, 64H, H-3, H-6a, H-6b,
LacOCH2, OCH2CH2N), 3.75–3.39 (m, 76H, H-4, H-4¢, H-6,
H-6¢, OCH3, GalOCH2CH2OCH2CH2O, LacOCH2), 3.33–3.20
2
1
2
2
GalOCH2, GalOCH2CH2OCH2CH2O, OCH3), 3.34-3.28 (m, 4H,
H-5), 3.28–3.20 (m, 8H, H-2, H-4). 13C NMR (100 MHz, DMSO-
d6 + e D2O) d 156.1, 155.9, 155.7 (3 s, CIVOCH3, CIVOCH2C-
triaz), 143.2 (CIV-triaz), 136.2 (exo-CIV-ar), 132.1 (endo-CH-ar),
129.7 (exo-CH-ar), 127.7* (endo-CH-ar), 125.0 (CH-triaz), 124.1,
123.6 (endo-CH-CIV-ar), 114.0 (exo-CH-ar), 103.7 (C-1), 96.8,*
96.1* (endo-CH-ar), 75.3, 73.5, 70.7 (C-2, C-4, C-5), 69.95, 69.87,
69.7 (GalOCH2CH2OCH2CH2O), 68.9 (OCH2CH2N), 68.3 (C-
3), 68.0 (GalOCH2), 61.2 (PhOCH2C-triaz), 60.6 (C-6), 56.0, 55.0
(OCH3), 49.7 (OCH2CH2N), 42.0 (ArCHRAr). HR-ESI-QToF
MS (positive mode): m/z calcd for C120H156N12Na2O44 [M + 2Na]++
1257.5061, found 1257.5115.
(m, 20H, H-2¢, H-3, H-3¢, H-5, H-5¢), 3.00 (t, J = 8.2 Hz, 4H, H-2).
13C NMR (100 MHz, DMSO-d6 + e D2O) d 156.0, 155.8, 155.6 (3
s, CIVOCH3, CIVOCH2C-triaz), 143.2 (CIV-triaz), 136.2 (exo-CIV-
ar), 132.1* (endo-CH-ar), 129.6 (exo-CH-ar), 127.8* (endo-CH-
ar), 124.9 (CH-triaz), 124.0, 123.5 (endo-CH-CIV-ar), 113.9 (exo-
CH-ar), 104.0, 102.8 (C-1, C-1¢), 96.7* (endo-CH-ar), 80.8, 75.7,
75.03, 74.98, 73.3, 73.2, 70.7 (C-2, C-2¢, C-3, C-3¢, C-4, C-5, C-
5¢), 69.8, 69.7 (LacOCH2CH2OCH2CH2O), 68.8 (OCH2CH2N),
68.2 (C-4¢, LacOCH2), 61.2 (PhOCH2C-triaz), 60.6 (C-6, C-6¢),
55.9 55.8 (OCH3), 49.6 (OCH2CH2N), 42.0 (ArCHRAr). HR-
ESI-QToF MS (positive mode): calcd for C144H198N12O64 [M +
2H]++ 1559.6298, found 1559.6288.
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* Peaks not observed in the 13C NMR spectrum but clearly
identified from HSQC experiments.
of calixarene glycoclusters.5d Scaffold and carbohydrates epitopes
were connected and the charges were derived and symmetrized
in order to obtain a neutral global charge. The resulting gly-
cocluster was then energy minimized using conjugate gradient
method and TRIPOS Force Field25 with addition of carbohydrate
parameters.26
1-[1,2,3-Triazol-4-yl-(acetoxy)methyl]-3,6-dioxaoct-8-yl
2,3,6,2¢,3¢,4¢,6¢-hepta-O-acetyl-b-D-lactoside 8L
Obtained as a colorless oil (95 mg, 73%) following Method A:
propargyl acetate (18.5 mg, 0.19 mmol, 1.3 eq.), 4L (115 mg,
0,145 mmol, 1 eq.), CuI (2.4 mg, 0.013 mmol, 0.1 eq.) and
DIPEA (66 mL, 0.435 mmol, 3 eq.). Purified by silica gel flash
chromatography (EtOAc). [a]D = +0.9 (c 1.07, CH2Cl2). 1H NMR
(400 MHz, CDCl3) d 7.76 (s, 1H, H-triaz), 5.33 (dd, J = 3.4 Hz,
J = 0.9 Hz, 1H, H-4¢), 5.19 (s, 2H, OCH2C-triaz), 5.16 (t, J =
9.2 Hz, 1H, H-3), 5.09 (dd, J = 10.4 Hz, J = 7.9 Hz, 1H, H-2),
4.94 (dd, J = 10.4 Hz, J = 3.4 Hz, 1H, H-3¢), 4.87 (dd, J = 9.6 Hz,
J = 8.0 Hz, 1H, H-2), 4.56–4.45 (m, 6H, H-1, H-1¢, H-6a, H-6b,
OCH2CH2N), 4.15–4.00 (m, 2H, H-6¢a, H-6¢b), 3.92–3.83 (m, 4H,
Two adjacent PA-IL monomers were considered for docking
compound 6b, using a procedure previously described.5d Briefly
one galactose epitope of glycocluster 6b was fitted into one of
the two binding sites of the dimer and a manual conformational
search was performed around the linker rotatable bonds for
bringing another galactose into the second binding site. Geometry
optimization was performed on the ligand, first with appropriate
constraints for determining the perfect orientation of the galactose
residues in the binding site, then with no constraints.
Acknowledgements
1
H-5, OCH2CH2N, LacOCH2), 3.78 (t, J = 9.4 Hz, 1H, H-4),
2
1
2
3.67 (ddd, J = 10.8 Hz, J = 6.4 Hz, J = 4.0 Hz, 1H, LacOCH2),
This work was funded by the University of East Anglia, The British
Council Higher Education Commission Pakistan Short Term PhD
Research Attachment (scholarship to ZHS), the Re´gion Rhoˆne-
Alpes (Cluster de Recherche Chimie) (additional funding for SC),
the Universite´ Claude Bernard Lyon 1 and the CNRS. Financial
help is acknowledged from Vaincre la Mucovisdicose and GDR
Pseudomonas. The authors would like to thank the EPSRC Mass
Spectrometry Service, University of Swansea and Dr D. Bouchu,
C. Duchamp, and N. Henriques for mass spectrometry analyses.
SPR experiments were performed with the help of Dr Wimmerova´,
(Masaryk University, Brno, Czech Republic) with funding from
the European Community 7th Framework Program under grant
agreement no. 205872. Associate Prof. Blanchard acknowledges
the financial support of the Cancer Council Queensland and
also the Australian Academy of Sciences for award of funds
for a research visit (2009) to CERMAV – CNRS under the
“International Science Linkage” program.
3.62–3.54 (m, 7H, LacOCH2CH2OCH2CH2, H-5¢), 2.13, 2.09,
2.06, 2.04, 2.03, 2.02, 2.00, 1.95 (8 s, 8 ¥ 3H, CH3CO). 13C NMR
(100 MHz, CDCl3) d 170.9, 170.4, 170.3, 170.2, 169.9, 169.7, 169.2
(7 s, 8¥CH3CO), 142.7 (CIV-triaz), 125.0 (CH-triaz), 101.2, 100.7
(C-1, C-1¢), 76.3 (C-4), 72.9 (C-3), 72.8 (C-5¢), 71.7 (C-2), 71.1 (C-
3¢), 70.8 (C-5), 70.7, 70.3 (2 s, LacOCH2CH2OCH2CH2O), 69.5,
69.2 (LacOCH2, OCH2CH2N), 69.2 (C-2¢), 66.7 (C-4¢), 62.1 (C-
6), 60.9 (C-6¢), 57.7 (OCH2C-triaz), 50.4 (OCH2CH2N), 21.00,
20.97, 20.9, 20.8, 20.7, 20.6 (6 s, 8¥CH3CO). HR-ESI-QToF MS
(positive mode): m/z calcd for C37H54N3O22 [M + H]+ 892.3193,
found 892.3168.
1-[1,2,3-Triazol-4-yl-(hydroxy)methyl]-3,6-dioxaoct-8-yl
b-D-lactoside 9L
Obtained as a lyophilized white powder (57 mg, 99%) following
Method B: 8L (93 mg, 0.1 mmol, 1 eq.), MeOH (2 mL), water
(0.5 mL) and triethylamine (0.5 mL). [a]D = -2.9 (c 0.31,
H2O). 1H NMR (400 MHz, DMSO-d6 + e D2O) d 7.92 (s,
1H, H-triaz), 4.53–4.43 (m, 4H, OCH2CH2N, OCH2C-triaz),
4.24–4.15 (m, 2H, H-1, H-1¢), 3.87–3.72 (m, 3H, OCH2CH2N,
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