Clustering of Escherichia coli type-1 fimbrial adhesins by using multimeric heptyl α- D -mannoside probes with a carbohydrate core

Article abstract of DOI:10.1002/chem.201100515

Heptyl α-D-mannoside (HM) is a strong inhibitor of the FimH lectin that mediates the initial adhesion of the uropathogenic Escherichia coli (E. coli) to the bladder cells. We designed a set of multivalent HM ligands based on carbohydrate cores with struct

Full text of DOI:10.1002/chem.201100515

FULL PAPER
DOI: 10.1002/chem.201100515
Clustering of Escherichia coli Type-1 Fimbrial Adhesins by Using Multimeric
Heptyl a-d-Mannoside Probes with a Carbohydrate Core
Mehdi Almant,[a] Vincent Moreau,[a] Josꢀ Kovensky,[a] Julie Bouckaert,*[b, c] and
Sꢀbastien G. Gouin*[a, d]
Abstract: Heptyl a-d-mannoside (HM)
is a strong inhibitor of the FimH lectin
that mediates the initial adhesion of
the uropathogenic Escherichia coli (E.
coli) to the bladder cells. We designed
a set of multivalent HM ligands based
on carbohydrate cores with structural
valencies that range from 1 to 7. The
chemical strategy used to construct the
regular hydrophilic structures consisted
of the repetition of a critical glucoside
fragment. A primary amino group was
grafted at the sugar reducing end to
couple the multimers to a fluorescent
label. A one-pot synthetic approach
was developed to tether the ligands
and the fluorescein isothiocyanate
(FITC) probe to the scaffold simulta-
neously. Isothermal calorimetry with
the monomeric FimH lectin revealed
nanomolar affinities and saturation of
all structurally available binding sites
on the multivalent HM ligands. Direct
titrations domain showed almost strict
correlation of enthalpy–entropy com-
pensation with increasing valency of
the ligand, whereas reverse titration
calorimetry demonstrated negative co-
operativity between the first and the
second binding site of the divalent
heptyl mannoside.
A
multivalency
effect was nevertheless observed by in-
hibiting the haemagglutination of type-
1 piliated UTI89 E. coli, with a titer as
low as 60 nm for the heptavalent HM
ligand. An FITC-labeled HM trimer
showed capture and cross-linking of
living bacteria in solution, a phenom-
enon not previously described with
low-valency ligands.
Keywords: calorimetry · carbohy-
drates · click chemistry · clusters ·
multivalency
Introduction
scriptions of antibiotics and their use in animals and agricul-
ture.[1] In parallel, the antimicrobial developments continue
to stagnate and the actual research programs may be unable
to provide an efficient solution in the next decade.[2] Nowa-
days, at least one mechanism of resistance has been identi-
fied for each class of antibiotics, and we are currently wit-
nessing highly resistant strains emerging around the world.[3]
There is an urgent need for the development of antibiotics
with new mechanisms of action and of alternative therapeu-
tics that are less prone to give rise to antibiotic resistance.
An appealing strategy envisioned by a part of the scientific
community is the development of anti-adhesive drugs that
are able to prevent bacterial attachment to the host cells.
Bacterial adhesion is generally promoted by proteins named
adhesins that bind to the carbohydrates displayed on the
surface of the host cells. Interfering with pathogen adhesion
is an attractive alternative, because the bacteria are not
killed and thus are less prone to develop resistance. A large
set of glycomimetics with different structures have been de-
signed by chemists to interfere with early stages of bacterial
infection.[4] Glycomimetics generally display several copies
of the ligands for the lectin on a common scaffold. The af-
finity enhancement observed for such systems can be much
higher than expected from the sum of the constitutive inter-
actions, a phenomenon referred to as the “multivalent or
glycoside cluster effect.”[5]
The development of bacterial strains that are multiresistant
to antibiotic treatments is a serious health problem that is
getting worse. This is partially due to the widespread pre-
[a] Dr. M. Almant, Dr. V. Moreau, Prof. J. Kovensky, Dr. S. G. Gouin
Laboratoire des Glucides UMR CNRS 6219
Institut de Chimie de Picardie, UFR des Sciences
Universitꢀ de Picardie Jules Verne
33 rue Saint-Leu, 80039 Amiens Cedex 1 (France)
[b] Dr. J. Bouckaert
Unitꢀ de Glycobiologie Structurale et Fonctionnelle
UMR CNRS 8576
Universitꢀ des Sciences et Technologies de Lille 1
59, 655 Villeneuve d’Ascq (France)
Fax : (+33)320436555
E-mail: Julie.Bouckaert@univ-lille1.fr
[c] Dr. J. Bouckaert
Molecular recognition, Vrije Universiteit Brussel
Pleinlaan 2, 1050 Brussels (Belgium)
[d] Dr. S. G. Gouin
Present address: Universitꢀ de Nantes
Chimie et Interdisciplinaritꢀ, Synthꢁse, Analyse,
Modꢀlisation (CEISAM), UMR CNRS 6230
UFR des Sciences et des Techniques
2, rue de la Houssiniꢁre, BP 92208
44322 Nantes Cedex 3 (France)
Fax : (+33)251125712
We recently focused our efforts on the development of in-
hibitors of virulent strains of the Gram-negative bacterium
Escherichia coli, associated with urinary tract infections
E-mail: sebastien.gouin@univ-nantes.fr
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201100515.
Chem. Eur. J. 2011, 17, 10029 – 10038
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(UTIs). Initial attachment of the pathogens to the bladder
cells is mediated by a mannose-specific adhesin named
FimH, situated at the edge of the tip fibrillum of type-1 fim-
briae. Simultaneous binding of several fimbriae to manno-
sides displayed by the glycoprotein uroplakin Ia allows for a
tight binding of the bacteria to the uroepithelial cells. Previ-
ous results have also shown that inhibition of type-1 fimbri-
ated E. coli can be efficiently achieved with synthetic mono-
valent mannosides that bear hydrophobic aglycons in the
anomeric position such as aryl groups.[6–8] Hultgren and co-
workers have recently identified a biaryl mannoside that dis-
plays low nanomolar binding affinity and submicromolar
cellular activity in a fluorescence polarization and haemag-
glutination assay, respectively.[9] Interestingly, simpler alkyl
mannosides were also shown to be strong binders of the
FimH protein.[10] With a binding affinity of 5 nm recorded by
surface plasmon resonance (SPR), and the ability to reduce
the bacterial level in vivo, heptyl a-d-mannoside (HM) was
identified as a promising potential anti-adhesive drug.[11]
Synthetic multimeric mannosides were also designed to
generate more potent anti-adhesive drugs through the glyco-
side cluster effect. The chemical strategies reported general-
ly consist of tethering simple mannoside ligands to function-
alized cores such as dendrimers,[12] nanoparticles[13,14] and
polymeric materials,[15,16] thereby leading to glycoclusters
with specific valencies, architectures, and epitopes densities.
The observed multivalent effects and enhanced affinities
with type-1 fimbriated E. coli are generally limited com-
pared to the gain reported when grafting anomeric lipophilic
aglycons.[17] Such a trend was observed by Roy and col-
leagues with mannosylated neoglycoproteins and dendrim-
ers.[12]
We also explored in our group the possibility of making
potent anti-adhesive drugs by applying the concept of multi-
valency to strong monovalent FimH inhibitors. We designed
multimeric HM with structural valencies that ranged from 1
to 4 on a carbohydrate scaffold and identified a tetramer
that inhibited the bacterial bladder cell binding at a 64-fold
lower concentration than HM.[18] The derivative was the
most potent anti-adhesive compound evaluated by this assay
toward uropathogenic E. coli. Although the synthetic strat-
egy was straightforward, it was not applicable to the design
of higher-valency compounds. Binding assays were also lim-
ited, as the set of multimers lacked an additional functional
group for grafting onto a surface or chemical coupling to a
fluorescent probe.
Scheme 1. Chemical structure of the HM glycoconjugates.
This reference is more appropriate than generally used
methyl mannoside to take into account direct interactions of
the aglycon groups with FimH. In such a regular system, the
modulation of valency does not introduce new functional
groups or affect the hydrophilicity or the spatial presenta-
tion of HM epitopes, because a critical fragment is repeated
n times. This is of importance to strictly assess how the va-
lency of the ligand influences lectin and bacterial binding.
The binding mode of the multimers with FimH was mea-
sured by isothermal titration calorimetry (ITC). Binding af-
finities of synthetic glycoclusters toward type-1 piliated E.
coli were evaluated by inhibition of haemagglutination
(HAI). Fluoresceine isothiocyanate (FITC) was grafted onto
a trimer to assess whether a low-valency ligand can aggre-
gate bacteria in solution.
Results and Discussion
Glucose 1, maltose 3, and maltotriose 4 were used as scaf-
folds to generate the mono-, di-, and trivalent HM ligands.
The copper-catalyzed azide–alkyne cyclization (CuAAC)
was selected to graft alkynyl-armed HM epitopes onto
azido-functionalized carbohydrate cores.[23] This reaction
owes its usefulness due to its high efficiency and selectivity
in different solvent systems including water.[24] Furthermore,
numerous recent examples successfully report the use of
CuAAC to design glycoclusters and other sugar mimet-
ics.[25,26] Compound 2 was obtained in three synthetic steps
as previously described in the literature (Scheme 2).[27] Tosy-
lation of unprotected glucose in the C-6 position followed
by acetylation of the hydroxyl groups and substitution by an
azide efficiently provided gram-scale quantities of 2. Mal-
tose 3 and maltotriose 4 were functionalized with a proce-
dure that we previously developed for the selective azida-
tion of unprotected carbohydrates.[28] The protocol used a
Carbohydrates are particularly appealing scaffolds for the
design of glycoclusters. The hydroxyl groups can be selec-
tively functionalized, and we and others have used this fea-
ture to modulate the topology of the ligands of carbohy-
drate-centered glycosides.[19–22] The highly hydrophilic core
also prevents potential solubility problems due to the pres-
ence of hydrophobic substituents. We present here a proto-
col for the synthesis of mono, di-, tri-, and heptavalent HM
based on a carbohydrate scaffold (Scheme 1). Differences in
affinity will be reported relative to the monovalent deriva-
tive that bears the glucoside fragment and the triazole ring.
[29]
PPh3/CBr4/NaN3 mixture in a 2:2:10 ratio relative to the
sugar unit, followed by acetylation of the crude mixture. An
alternative consists of a selective chloration of the C-6 car-
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E. coli Fimbral Adhesins
FULL PAPER
due to the oxidation of thiol 15 to the corresponding di-
sulfide. Compounds 2, 5, 6, and 10 were then converted
in two steps to the corresponding trichloroacetamidates
11, 12, 13, and 14 with an overall yield of 80, 73, 80, and
65%, respectively. Thioglycosylation occurred with the
Schmidt procedure by using an excess amount of protect-
ed cysteamine and TMSOTf as catalyst. Expected com-
pounds 16, 17, 18, and 19 were obtained with fair yields
of 75, 65, 55, and 35%, respectively. The reactions should
be conducted at low temperature (ꢀ208C) to avoid the
partial formation of a anomers. The CuAAC reactions
were performed under microwave irradiation with the
Scheme 2. Azidation of saccharides.
bons, acetylation of the hydroxyl groups, and two successive
nucleophilic displacements by sodium iodide and azide.[30]
Although this last procedure is much longer and requires
additional purification steps, it allows the preparation of 5
and 6 in higher scales.
Carbohydrate cores with additional numbers of sugar
units are particularly difficult to functionalize in a selective
and quantitative way. Development of an efficient method-
ology is also hampered by the high cost of the correspond-
ing commercial sugars. To our knowledge, there are no re-
ports of the direct and selective halogenation or tosylation
on the C-6 position of linear oligosaccharides.
On the contrary, the selective halogenation of the lower
rim of cyclomaltooligosaccharides (a-, b-, and g-cyclodex-
trins, CDs) is relatively straightforward and has been previ-
ously reported.[31–33] The per-6-bromo-b-CD 7 was first pre-
pared in good yields with the Vilsmeier–Haack reagent
[(CH3)2NCHBr]+Brꢀ.[33] Subsequent per-acetylation with
acetic anhydride and per-benzoylation with benzoyl bromide
followed by nucleophilic substitution with sodium azide al-
lowed isolation of the compounds 8 and 9, respectively.[34]
Several methodologies are reported to form the linear
malto-oligosaccharide from the corresponding CDs with
Lewis or Brønsted acids such as H2SO4/Ac2O,[35] FeCl3/
Ac2O,[36] ZnBr2/PhSSiMe3,[37] HClO4/Ac2O,[38,39] TiCl4, or
BF3/MeOCH2CO2H.[40] These different protocols were eval-
uated on the acetylated derivative 8 without any success.
The expected linear heptasaccharide was only formed with
H2SO4/Ac2O[35] and in a low yield (less than 10%) due to
the formation of shorter linear oligomers by a depolymeriza-
tion process. Finally, the ring opening was performed with a
good yield of 85% starting from the benzoylated derivative
9 (Scheme 3). The reaction was performed with the HClO4/
Ac2O mixture and at a low temperature of ꢀ208C to avoid
the formation of shorter oligosaccharides.
Scheme 3. Ring opening of cyclodextrin 9.
previously described mannoside 20.[18] The protocol allowed
the fast formation of the expected cycloadducts 21, 22, and
23 with yields of 80, 73, and 76% respectively. The large
D(dCꢀ4ꢀdCꢀ5) values (d>20 ppm) observed by 13C NMR
spectroscopy for the different structures indicated the exclu-
sive formation of the 1,4-cycloadducts, as smaller values are
expected for 1,5-disubstitued regioisomers.[43]
Quantitative cleavage of the Fmoc, acetyl, or benzoyl
groups were finally performed in a one-step procedure with
a 7n solution of ammonia in methanol. Heptasaccharide 19
was subjected to the cyclization step and the crude product
directly deprotected to 27. The set of deprotected com-
pounds 24, 25, 26, and 27 (Scheme 4) were purified by
HPLC on a C18 column.
We decided to graft a fluorescent dye onto 26 to assess its
capacity to aggregate E. coli in solution. Commercially
available fluorescein isothiocyanate was grafted on the triva-
lent ligand 26 in DMF, and subsequent purification by
HPLC allowed isolation of the expected fluorescent ligand
30 (Scheme 5).
In parallel, we wanted to assess the possibility of perform-
ing an unprecedented one-pot coupling of the HM ligands
and FITC probe to the carbohydrate scaffolds. Fmoc and
acetyl groups of compound 18 were then deprotected at
With the azido compounds 2, 5, 6, and 10 in hand, we
next focused on the introduction of the protected amino
group in the anomeric position. A direct thioglycosylation
with previously described 9-fluorenylmethoxycarbonyl
(Fmoc)-protected cysteamine 15[41] and 2, 5, 6, and 10 was
first considered. Unfortunately, no reaction occurred with
the set of Lewis acids tested (BF3·OEt2, trimethylsilyl tri-
fluoromethanesulfonate (TMSOTf), SnCl4, [MoO2Cl2][42]
)
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10031

J. Bouckaert, S. G. Gouin et al.
ited (Table 1). Due to serial di-
lutions, the maximal error is ꢁ1
well, or a factor of two. HM,
one of the most potent FimH
inhibitors reported to date, was
used as a reference compound.
Monovalent derivate 24 was
identified as a strong inhibitor
within the same concentration
range as HM (Table 2). More
importantly,
a significant en-
hancement was observed with
the multimers 25, 26, and 27.
Potency increases with valency,
and the heptavalent compound
27 is a 64-fold better inhibitor
than HM, which means a nine-
fold improvement when report-
ed on a mannose molar basis.
Thus, there is a enhancement of
the valency of the ligand on
bacteria-mediated red blood ag-
glutination. This can only be ex-
plained by the capacity of mul-
tivalent 25, 26, and 27 to cross-
link different FimH molecules
on pili of adjacent E. coli. Com-
pared to our previously de-
scribed multivalent HM deriva-
tives based on oligoethylene
glycol scaffolds, 25, 26, and 27
displayed higher affinity en-
hancements by HAI when re-
ported on a mannose molar
basis. This highlights the impor-
tance of a careful core selection
for the design of FimH inhibi-
tors. With the very low titer of
60 nm, 27 is to our knowledge
the most potent FimH inhibitor
Scheme 4. Synthesis of cycloadducts 24–27.
identified by this assay.
room temperature in methanolic ammonia to lead to crude
compound 28. Dissolution of the unprotected scaffold 28 in
a DMF/water mixture with unprotected HM ligand 29,
FITC, and a catalytic quantity of copper sulfate and sodium
ascorbate afforded the expected probe 30 within 35% yield
after HPLC purification (Scheme 6).
Finally, the increasing titer potency and multivalent effect
relative to mannosides epitopes from 24 to 27 also suggest
that ligands with higher valencies based on a similar archi-
tecture would be even more potent inhibitors.
Table 1. HAI of HM and synthetic glycoconjugates 27–30.
Compound
Valency
Titer [mm]
Ratio [L/HM]
Pot [m]
Inhibition of haemagglutination (HAI): Binding affinities of
the glycoconjugates 24–27 for type-1 piliated UTI89 clinical
isolate E. coli were evaluated by HAI. Interaction of E. coli
FimH adhesins with the glycocalyx of guinea pig erythro-
cytes formed a cross-linked network into the wells. Subse-
quent additions of HM or glycoconjugates 24–27 in a two-
fold dilution series prevented the agglutination reaction.
The inhibition titer is defined as the lowest concentration of
the glycoconjugate at which haemagglutination is still inhib-
HM
24
25
26
27
1
1
2
3
7
4
4
0.5
0.25
0.06
1
1
8
16
64
1
1
4
5
9
Isothermal titration calorimetry: The affinities of the multi-
valent mannosides 24, 25, 26, and 27 towards the FimH ad-
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E. coli Fimbral Adhesins
FULL PAPER
lerenes that were shown to be
able to accommodates up to
seven FimH molecules relative
to the twelve mannosides dis-
played on the scaffold.[44] Com-
pared to the mannose-function-
alized fullerenes, the topologi-
cal distribution of the manno-
side residues in 27 seems more
favorable
for
cross-linking
FimH molecules. We expect
this distribution to follow a left-
handed helix with regards to
the nature of the carbohydrate
core that consists of glucose
residue with a
N
bonds, similarly to amylose.[45]
The dissociation constants,
Kd, in Table 2 indicated that the
synthetic glycoconjugates are
nanomolar inhibitors of FimH.
There is an initial improvement
in affinity for FimH going from
the monomeric compound 24 to
the dimeric ligand 25 and tri-
meric compound 26. The tri-
meric ligand 26 displayed the
steepest transition in the direct
titration curve, which was relat-
ed to a maximal affinity meas-
ureable by a normal direct titra-
tion. The affinity decreases
again for the heptavalent 27, in
apparent contradiction with the
potency of 27 in preventing the
haemagglutination by the uro-
Scheme 5. Synthesis of probe 30.
Table 2. ITC data for glycoconjugates 24–27 to the soluble, monomeric FimH lectin.
[a]
Cpd
nstr
Kd
DG8
DH8[a]
DS8
c2/DOF[c]
nfun
[nm]
[kcalmolꢀ1
]
[kcalmolꢀ1
]
[calmolꢀ1 Kꢀ1
]
24
25
1
2
1/2
2/2
3
14.4 (ꢁ3.4)
2.6 (ꢁ1.7)
ꢀ10.6
ꢀ11.6
ꢀ11.0
ꢀ7.7
ꢀ10.1 (ꢁ0.1)
ꢀ16.6 (ꢁ0.2)
ꢀ14.3 (ꢁ0.7)
ꢀ3.3 (ꢁ5.9)
ꢀ43.6 (ꢁ1.3)
ꢀ68.3 (ꢁ4.2)
1.5
ꢀ16.9
ꢀ11.1
14.8
7.2ꢃ104
1.5ꢃ105
3.5ꢃ106
3.5ꢃ106
7.2ꢃ106
1.3ꢃ108
0.97
0.46
1.00
1.00
0.31
0.15
25[d]
25[d]
26
7.6 (ꢁ14.4)
2128 (ꢁ7696)
2.4 (ꢁ2.6)
ꢀ11.8
ꢀ10.2
ꢀ108
27[b]
7
36.2 (ꢁ22.7)
ꢀ197
[a] Standard deviations are in parentheses. [b] Average over two direct titration measurements. [c] Degree of
fitting. [d] Divalent compound 25 was measured both using direct and reverse titration, the reverse tritration
displaying the individual binding parameters for binding to the first (1/2) and the second (2/2) mannoside on
25. The nstr value is the structural valency of the compound; nfun is the molar ratio of monomeric FimH lectin
domain over the compound, or the inverse of the functional valency of the glycoconjugates. The Gibbs free pathogenic E. coli (Table 1).
energy for the interaction is calculated as DG8=DH8ꢀTDS8, with T being the absolute temperature during
However, contrary to haemag-
glutination, strong affinity en-
hancements were not expected
the ITC measurement.
hesin have been assessed using isothermal titration calorim-
etry (VP-ITC instrument, Microcal). In a series of direct ti-
trations, the FimH lectin domain was placed in the cell and
the ligands were titrated into the cell through the needle
(Figure 1).
As can be seen from Table 2, the number of FimH lectins
bound per molecule, or the functional valency nfun, increases
congruently with the structural valency nstr of the glycocon-
jugates. Approximately two FimH lectin domains bind to
the divalent ligand 25, three FimH molecules per trivalent
derivative 26 and seven FimHs were found to bind to the
heptavalent 27. It therefore seems that the close proximity
of the clustered ligands does not prevent the simultaneous
binding of FimH lectins and that ultimately all structural
binding sites on the multivalent ligand can be saturated by
FimH. These results are in accordance with ITC experi-
ments recently reported with dodecamannosylated ful-
in the ITC measurements because of the monomeric nature
of the FimH lectin. In bacterial cross-linking on the multiva-
lent ligands, the FimH adhesin binds tightly to HM and ag-
gregates closely together with the other FimH lectins onto
the soluble carbohydrate scaffold. The larger the functional
valency of the ligand, the larger these microclusters of FimH
grow. Increasing noise levels were indeed observed in the
ITC experiments from the monovalent compound 24, over
the bi- (25) and trivalent (26) ligands, to the heptavalent 27
(Figure 1 and Table 2 (c2/DOF)). Successive protein–protein
attractions and repulsions within these microclusters could
cause small heat releases and heat uptakes, respectively. The
integration of the heat release upon each injection over the
equilibration period may thus vary more considerably for
compounds with higher ligand valencies. However, here
these signals are not indicative of significant protein precipi-
tation and the overall integrated enthalpy of the reaction is
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10033

J. Bouckaert, S. G. Gouin et al.
ting of FimH binding to se-
quential binding sites on the
multivalent mannosidic ligand
renders the same thermody-
namic parameters of entropy
and enthalpy as in direct titra-
tion; however, this time can be
measured per individual bind-
ing site. Ideally, this delivers a
microscopic affinity constant
per binding site on the
ligand.[46] Reverse titration can
as such reveal positive, negative
or noncooperativity between se-
quential binding sites.[47]
The reverse titration experi-
ments yielded reliable data only
for the divalent HM ligand 25
(Figure 3). It was possible to
derive interesting thermody-
namic information from the re-
verse titration experiment with
the low valency compound 25.
FimH displays a higher affinity
for the first binding event on 25
(Kd =7.6 nm, Figure 2) than for
the monovalent 24 (14.4 nm,
Table 1). A higher microscopic
affinity is indeed expected for
the binding of the first ligand
on a multivalent glycoconjugate
than for the same ligand on a
monovalent glycoconjugate be-
cause of an increased probabili-
Scheme 6. Synthesis of probe 30.
ty of binding.[47] On the other
hand, the affinity for the second
well averaged out and has a small standard error (Table 2).
Dynamic protein–protein contacts could thus be the cause
of variations in the baseline at equilibrium (Figure 1, com-
pound 27) and could potentially trigger bacterial clustering
as well.
Thermodynamic parameters revealed an almost perfect
addition of enthalpies for FimH occupation upon the addi-
tion of every other mannoside going from the monovalent
24 to the heptavalent 27 (Table 2). The gain in enthalpy
with every other binding site in the compounds with increas-
ing valencies (n=1, 2, 3, or 7) is almost perfectly compen-
sated for the loss of entropy by partial enthalpy–entropy
compensation (Figure 2). The affinities of the four com-
pounds 24–27 thus appear highly correlated (Table 2,
Figure 2).
binding site on a multivalent glycoconjugate is by definition
lower than for the first one in the absence of cooperativity
due to the decreased number of potential binding sites.[47]
The large dump in affinity of FimH for the second binding
site of 25 is more significant than this statistical effect. Fit-
ting or the reverse titration indicates negative cooperativity
between the two sequential binding sites of 25 (Table 2, Fig-
ures 2 and 3).
Fluorescence microscopy: We decided to modify trivalent 26
for the microscopy experiments, as this compound was a
good compromise in view of its high inhibitory potency in
both ITC and HAI. Fluorescent microscopy experiments
were performed with FITC-labeled compound 30 and fluo-
rescently labeled bacteria with Hoechst 33258. A homoge-
nous solution of disperse bacteria was observed in the ab-
sence of glycoconjugate 30 (Figure 4a). When the sugar 30
was added at a concentration of 4 mm, clusters of bacteria
were formed (Figure 4b). The green in the cluster unambig-
uously indicated the presence of 30 in the middle of the bac-
terial cluster. These results showed that the formation of the
To understand this effect and discern the individual con-
tributions to affinity of the subsequently bound mannosides
on the multivalent ligands, we set up reverse titrations. In
our reverse titration method, FimH is titrated through the
needle in high concentrations and small volumes into the
measurement cell that contains the diluted compound. Fit-
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Chem. Eur. J. 2011, 17, 10029 – 10038

E. coli Fimbral Adhesins
FULL PAPER
lent ligand with short spacers
can promote cross-linking of
FimH that belong to different
bacteria.
Conclusion
We have developed an efficient
synthetic
methodology
to
design water-soluble ligands
with valencies that range from
1 to 7. We repeated a critical
glucoside fragment to build reg-
ular structures for a strict as-
sessment of potential multiva-
lent effects. An amino group
was also introduced in the
anomeric position of the scaf-
folds for further grafting of a
fluorescent probe. Alternative-
ly, we also developed a rapid
one-pot procedure to tether si-
multaneously the HM epitopes
and FITC onto the carbohy-
drate core. The set of ligands
designed are strong inhibitors
of FimH and display nanomolar
affinity for the isolated FimH
lectin domain in solution, as
evidenced by ITC experiments.
Insights into the binding-
mode operation were also pro-
vided by ITC experiments. Con-
sidering that a chelate effect is
not expected due to the mono-
meric nature of FimH, the af-
finity enhancement observed
can be either ascribed to intrin-
sic multivalent effects,[20b,48] in
which the proximity of the teth-
ered epitopes allow their subse-
Figure 1. ITC raw data and fittings of the multivalent ligands.
quent binding and recapture by
a unique FimH molecule, or to
bacterial clusters is promoted by the trivalent glycoconju-
gate 30. Previous papers reported on the possibility to clus-
ter bacteria with polymeric glycoconjugates that bear a large
number of lectin ligands.[15,16]
Seeberger and co-workers have grafted mannosides on a
rigid poly(p-phenylene ethynylene) (PPE) core for the ag-
gregation and fluorescent detection of bacteria in solu-
tion.[15] Alexander and co-workers have shown the possibili-
ty to control bacterial aggregation with thermoresponsive
polymer chains.[16] As far as we know, the possibility to pro-
mote E. coli clustering with low valency ligands, such as the
trimeric compound 30 here, has not been previously report-
ed. Thus, our results indicate for the first time that a triva-
an aggregative process, in which a multivalent ligand can
bind to several receptors simultaneously. ITC data are clear-
ly indicative of the second mechanism by showing that all
the structural binding epitopes of the multivalent ligands
were saturated by FimH molecules. The strong binding af-
finity of HM for the FimH receptor may limit the recapture
mechanism from operating during intrinsic multivalent ef-
fects.
Negative cooperativity between the sequential binding
sites on the multivalent HM ligands reduces the affinity of
FimH in the ITC experiments. This may be caused by steric
hindrance between the FimH lectin domains that are bound
tightly and closely together onto a relatively small carbohy-
Chem. Eur. J. 2011, 17, 10029 – 10038
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10035

J. Bouckaert, S. G. Gouin et al.
Figure 4. Laser scanning microscopy images of A) Uropathogenic E. coli
in solution fluorescently labeled with Hoechst stain 33258. Individual
bacteria are observed without clustering; scale bar 8.00 mm. B) Bacterial
cluster (in blue) co-localizes with FITC-labeled 30 (green); scale bar
3.60 mm.
^
Figure 2. ( ) Direct titration experiments showed a strong enthalpy–en-
tropy compensation (R2 =0.9993) for the multivalent interaction of the
monomeric FimH lectin domain with compounds with structural and
uropathogenic E. coli. Multivalent inhibitors that surpass
the potency of HM were observed in this assay.
Furthermore, fluorescence microscopy evidenced that the
trivalent ligand 30 was able to promote bacterial clustering,
a phenomenon that is not accessible to monovalent ligands,
but that has previously been reported with polymeric glyco-
conjugates.
&
functional valencies (n) of 1 (24), 2 (25), 3 (26), and 7 (27). ( ) Reverse
titration of 25 shows a deviation from the thermodynamic enthalpy–en-
tropy compensation axis for the second binding site of the divalent glyco-
conjugate 25, thus demonstrating negative cooperativity between sequen-
tial mannose epitopes (see Table 2).
These results show that rather simple ligands with low va-
lencies are efficient chemical tools for capturing living bac-
teria in solution.
Experimental Section
General procedures: All purchased materials were used without further
purification. Dichloromethane and DMF were distilled from calcium hy-
dride, pyridine over KOH, and tetrahydrofuran over sodium and benzo-
phenone. Analytical thin-layer chromatography (TLC) was carried out
using Merck D.C.-Alufolien Kieselgel 60 F254. Flash chromatography
(FC) was performed using GEDURAN SI 60, 0.040–0.060 mm pore size,
using distilled solvents. 1H and 13C nuclear magnetic resonance (NMR)
spectra were respectively recorded at 300 and 75.5 MHz using a Bruker
AC-300 or at 600 and 150 MHz using a Bruker AC-600 spectrometer,
and chemical shifts are reported in parts per million relative to tetrame-
1
thylsilane or a residual solvent peak peak (CHCl3: H: d=7.26 ppm, 13C:
d=77.2 ppm; [D6]DMSO: 1H: d=2.54 ppm, 13C: d=40.4 ppm). Peak
multiplicity is reported as: singlet (s), doublet (d), triplet (t), quartet (q),
pentet (p), sixtet (s), multiplet (m), and broad (br). High-resolution mass
spectra HRMS were obtained by electrospray ionization (ESI) using a
Micromass-Waters Q-TOF Ultima Global instrument. Optical rotations
were measured using a 343 Perkin–Elmer instrument at 208C in a 1 cm
cell in the stated solvent; [a]D values are given in 10ꢀ1 8cmꢀ1 gꢀ1 (concen-
tration c given as g100 mLꢀ1). Microwave irradiation was performed
using a CEM Discover apparatus (300 W). Preparative reversed-phase
HPLC was carried out using a Waters PREP LC 4000 chromatography
system with a (DEDL) PL-ELS 1000 photodiode array detector. All
HPLC samples were purified using a preparative Prevail C-18 column
(2.2ꢃ25 cm). The mobile phase was H2O (solvent A) and MeOH (sol-
vent B). For method A: The mobile phase was H2O (solvent A) and
MeOH (solvent B). The gradient consisted of 5% A for 5 min to 100%
B in 55 min (22.0 mLminꢀ1 flow rate). For method B: The mobile phase
was H2O (solvent A) and MeOH (solvent B). The gradient consisted of
5% A for 5 min to 100% B in 55 min (22.0 mLminꢀ1 flow rate).
Figure 3. Reverse titration of FimH (21.3 mm) into divalent ligand 25
(2 mm) (22.15038C). From the previous observation that each of the two
binding sites on 25 can fully occupy a FimH molecule, the molar ratio
per sequential binding sites had been fixed to 1 in the fitting of sequential
binding to the first (Kd =(7.6ꢁ14.4) nm) and the second (Kd =(2.1ꢁ
7.7) mm) mannoside epitope.
drate scaffold. We could confirm that these apparent FimH
lectin microaggregates grow according to the structural va-
lency of the HM ligands by the determination of functional
stoichiometric ratios in direct titration calorimetry. A signifi-
cant binding improvement due to valency of the ligand was,
however, observed in the inhibition of haemagglutination by
Procedure for the thioglycosylations: Compound 13 (1.20 g, 1.18 mmol),
compound 15 (1.76 g, 5.9 mmol), and activated 4 ꢄ molecular sieves
10036
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ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 10029 – 10038

E. coli Fimbral Adhesins
FULL PAPER
(2.00 g) were dissolved in dry dichloromethane (5 mL). The mixture was
stirred at ꢀ208C for 30 min under argon atmosphere, and TMSOTf
(71 mL, 0.39 mmol) was added. After 15 min of stirring, NaHCO3 was
added (70 mg), and the mixture filtered and evaporated under reduced
pressure. The residue was subjected to chromatography on silica gel with
ethyl acetate/cyclohexane (8:2 to 6:4) to afford 18 (700 mg, 55%).
70H, 35ꢃCH2); 13C NMR (150 MHz, D2O): d=144.0 (7ꢃC=CHtriazole),
126.1 (7ꢃCH=Ctriazole), 101.42, 101.05 (C-1II–VII), 99.7 (7ꢃC-1HM), 85.6 (C-
1I), 81.4, 76.8, 72.6, 71.6, 71.2, 70.7, 70.6, 70.5, 70.2, 69.7, 67.6, 66.6 (C-2I–
VII, C-3I–VII, C-4I–VII, C-5I–VII, C-2HM, C-3HM, C-4HM, C-5HM, CH2O), 62.4,
61.7, 60.8 (7ꢃO-CH2-triazole, 7ꢃC-6HM); 51.6, 50.6 (C-6I–VII), 39.2, 36.9,
31.4, 28.7, 28.7, 28.5, 28.4, 27.4, 25.5, 25.1, 25.0 (CH2-NH, CH2-S, CH2);
HRMS (ES+): m/z calcd for C156H263N22O77SNa3F: 3734.7192; found:
3734.7242.
Procedure for the microwave-assisted cyclization: Compound 18 (100 mg,
0.09 mmol), compound 20 (140 mg, 0.28 mmol), CuSO4 (21 mg,
0.13 mmol), and sodium ascorbate (21 mg, 0.26 mmol) were dissolved in
a mixture of dioxane (2 mL) and water (0.5 mL) The mixture was stirred
under microwave irradiation at 708C for 30 min. After evaporation to
dryness, the residue was dissolved in dichloromethane (5 mL) and fil-
tered. The filtrate was evaporated to dryness, and the mixture was sub-
jected to chromatography on a silica gel column with ethyl acetate/cyclo-
hexane (8:2) to methanol/ethyl acetate (1:9) as eluent to afford 23
(181 mg, 76%).
Procedure for the one-pot synthesis of 30: Compound 18 (20 mg,
0.02 mmol) was dissolved in a 7n solution of ammonia in methanol
(10 mL) and the mixture was stirred at room temperature for 24 h. After
evaporation under reduced pressure, the residue was dissolved in diethyl
ether (10 mL) and water (10 mL). The aqueous phase was extracted with
diethyl ether (2ꢃ20 mL) and evaporated under reduced pressure. Com-
pound 20 (30 mg, 0.06 mmol) was dissolved in a 7n solution of ammonia
in methanol (10 mL), and the mixture was stirred at room temperature
for 24 h. After evaporation under reduced pressure, the residue was dis-
solved in diethyl ether (10 mL) and water (10 mL). The aqueous phase
was extracted with diethyl ether (2ꢃ20 mL) and evaporated under re-
duced pressure. The two crude products, fluorescein isothiocyanate
(12 mg, 0.03 mmol), CuSO4 (7 mg, 0.015 mmol), and sodium ascorbate
(10 mg, 0.030 mmol) were dissolved in DMF (4 mL) and water (1 mL).
The mixture was stirred in a dark room at room temperature for 24 h.
After evaporation to dryness, the residue was diluted in water and exten-
sively washed with EtOAc. After lyophilization, the mixture was purified
by HPLC with B conditions to afford 30 (9 mg, 35%, tr =25 min).
Procedure for the deprotection of acetates and Fmoc groups: Compound
23 (150 mg, 0.05 mmol) was dissolved in a 7n solution of ammonia in
methanol (20 mL) and the mixture was stirred at room temperature for
24 h. After evaporation under reduced pressure, the residue was dis-
solved in diethyl ether (10 mL) and water (10 mL). The aqueous phase
was extracted with diethyl ether (2ꢃ20 mL) and evaporated under re-
duced pressure. The residue was purified by HPLC with conditions A to
afford 26 (62 mg, 76%).
Glycoconjugate 24: [a]D20 =+101 (c=1 in H2O); 1H NMR (600 MHz,
D2O): d=8.00 (s, 1H; Htriazole), 4.80 (d, J=1 Hz, 1H; H-1HM), 4.56 (m,
2H; O-CH2-triazole), 4.48 (d, J=10 Hz, 1H; H-1), 3.85–3.40 (m, 16H;
H-2, H-2HM, H-3, H-3HM, H-4, H-4HM, H-5, H-5HM, 2ꢃH-6, 2ꢃH-6HM, 2ꢃ
CH2O), 2.99–2.60 (m, 4H; CH2-S, CH2-NH2), 1.50–1.23 ppm (m, 10H; 5ꢃ
CH2); 13C NMR (150 MHz, D2O): d=142.3 (C=CHtriazole), 125.8 (CH=
Ctriazole), 99.6 (C-1HM), 85.3 (C-1), 77.5, 77.4, 76.9, 76.8, 72.7, 71.9, 70.9,
70.8, 70.6, 70.5, 70.3, 70.1, 67.8, 66.7 (C-2, C-3, C-4, C-5, C-2HM, C-3HM, C-
4HM, C-5HM, CH2O), 61.8 (O-CH2-triazole), 60.9 (C-6HM), 51.1 (C-6), 39.4,
39.3, 38.2, 38.1, 37.9, 37.7, 37.6, 36.9 (CH2-NH2, CH2), 31.4 ppm (CH2-S);
HRMS (ES+): m/z calcd for C24H44N4O11SH: 597.2806; found: 597.2820.
Isothermal titration calorimetry (ITC): The FimH adhesin lectin domain
was expressed and purified as previously described and finally dialyzed
against 20 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES) pH 7.4 with 150 mm NaCl.[11] The last change of dialysis buffer
was filter-sterilized and kept as buffer to dilute FimH and compounds for
the calorimetry. A VP-ITC (Microcal) instrument was used for both
direct and reverse titrations; 280 mL was always injected into the 1.4 mL
measurement cell. Respective concentrations and molar ratios in needle
and cell, injection volumes, and time intervals between injections were
varied to obtain 1) inflection and saturation about halfway through the
experiment, 2) sufficient heat production per injection to allow good
peak integration, and 3) sufficient time between the injections to allow a
return to equilibrium. The concentrations used in every ITC experiment
are given in the Supporting Information. Stirring speed of the needle was
always 307 rpm. Fitting was performed using the Origin software using
the equations for one set of binding sites in the direct titration fittings, or
for sequential binding sites for the reverse titration fittings.
Glycoconjugate 25: [a]D20 =+112 (c=1 in H2O); 1H NMR (600 MHz,
D2O): d=7.96, 7.82 (s, 2H; 2ꢃHtriazole), 5.25 (d, J=4.0 Hz, 1H; H-1II),
4.75 (d, J=1 Hz, 2H; 2ꢃH-1HM), 4.45 (m, 2H; O-CH2-triazole), 4.15 (m,
2H; O-CH2-triazole), 4.48 (d, J=10 Hz, 1H; H-1), 3.85–3.40 (m, 16H;
H-2I–II, 2ꢃH-2HM, H-3I–II, 2ꢃH-3HM, H-4I–II, 2ꢃH-4HM, H-5I–II, 2ꢃH-5HM
,
2ꢃH-6I–II, 4ꢃH-6HM, 4ꢃCH2O), 2.65–2.30 (m, 4H; CH2-S, CH2-NH2),
1.57–1.13 ppm (m, 20H; 10ꢃCH2); 13C NMR (150 MHz, D2O): d=144.1,
144.0 (2ꢃC=CHtriazole), 126.1, 125.7 (2ꢃCH=Ctriazole), 100.9, 99.6 (2ꢃC-
1HM), 85.2 (C-1), 80.7, 76.9, 75.4, 72.6, 72.4, 72.0, 71.7, 71.5, 70.9, 70.6,
Inhibition of haemagglutination (HAI): Inhibition of guinea pig red
blood cell haemagglutination by the type-1 piliated uropathogenic E. coli
strain UTI89 by the newly synthesized glycoconjugates 24–27. A twofold
dilution of glycoconjugates was prepared in HEPES (25 mL; 20 mm;
pH 7.4) with NaCl (150 mm), starting from 1 mm as the highest concentra-
tion. UTI89 E. coli were grown statically overnight in Luria-Bertani
medium at 378C, washed three times in ice-cold phosphate-buffered
saline, and resolubilized. The bacterial solution (25 mL) was added to the
twofold dilution series of the compound. Finally, guinea pig red blood
cells (50 mL), washed in buffer and diluted to 5%, were added to a final
100 mL and left on ice for 30 min before readout.
70.5, 70.0, 67.8, 66.6 (C-2I–II, C-3I–II, C-4I–II, C-5I–II, C-2HM, C-3HM, C-4HM
,
C-5HM, CH2O), 62.3, 62.2 (O-CH2-triazole), 61.7, 60.9 (C-6HM), 51.4, 50.4
(C-6I–II), 39.9, 31.4, 31.2, 28.6, 28.4, 28.3, 28.2, 25.3, 25.2, 25.1 ppm (CH2-
NH, CH2-S, CH2); HRMS (ES+): m/z calcd for C46H81N7O22SH:
1116.5233; found: 1116.5264.
Glycoconjugate 26: [a]D20 =+120 (c=1 in H2O); 1H NMR (600 MHz,
D2O) d=7.96, 7.89, 7.82 (s, 3H; 3ꢃHtriazole), 5.25, 5.15 (2d, J=4 Hz, 2H;
H-1II–III), 4.59 (d, J=1 Hz, 3H; 3ꢃH-1HM), 4.28 (d, J=10 Hz, 1H; H-1),
4.20–3.96 (m, 6H; 3ꢃO-CH2-triazole), 3.85–3.10 (m, 42H; H-2 I–III, 3ꢃH-
2HM, H-3I–III, 3ꢃH-3HM, H-4I–III, 3ꢃH-4HM, H-5I–III, 3ꢃH-5HM, 2ꢃH-6I–III
,
Fluorescence microscopy: UTI189 E. coli strains were grown statically
overnight in LB at 378C, washed and diluted in PBS buffer (NaCl:
137 mm, KCl: 2.7 mm, Na2HPO4: 10 mm, K2HPO4: 1.76 mm; pH 7.4). La-
beled trisaccharide 30 (4 mm) was diluted in buffer and poured into the
bacteria solution. Fluorescence microscopy and digital image acquisition
and analysis were performed using a Leica TCS SP5 AOBS fluorescence
microscope.
6ꢃH-6HM
, 6ꢃCH2O), 2.55–2.35 (m, 4H; CH2-S, CH2-NH2), 1.57–
1.13 ppm (m, 30H; 15ꢃCH2); 13C NMR (150 MHz, D2O): d=144.1,
144.0, 143.9 (C=CHtriazole), 125.7 (CH=Ctriazole), 99.6 (C-1HM), 85.2 (C-1I),
80.9, 76.8, 75.1, 72.6, 72.3, 72.2, 71.6, 71.5, 71.0, 70.9, 70.7, 70.6, 70.5, 70.1,
69.8, 67.7, 66.6 (C-2I–III, C-3I–III, C-4I–III, C-5I–III, C-2HM, C-3HM, C-4HM, C-
5HM, CH2O), 62.6, 62.4, 62.2, 61.7, 60.8 (O-CH2-triazole, C-6HM), 50.7, 50.2
(C-6I–III), 39.8, 36.8, 31.2, 28.6, 28.4, 28.4, 28.3, 28.2, 25.3, 25.3, 25.2,
25.1 ppm (CH2-NH, CH2-S, CH2); HRMS (ES+): m/z calcd for
C68H118N10O33SNa: 1657.7482; found: 1657.7470.
1
Glycoconjugate 27: [a]D20 =+98 (c=1 in H2O); H NMR (600 MHz, D2O)
d=7.90, 7.70 (s, 7H; 7ꢃHtriazole); 5.12 (7d, J=4 Hz, 7H; H-1II–VII), 5.00–
3.01 (m, 134H; H-1I, 7ꢃH-1HM, H-2I–VII, 7ꢃH-2HM, H-3I–VII, 7ꢃH-3HM, H-
4I–VII, 7ꢃH-4HM, H-5I–VII, 7ꢃH-5HM, 2ꢃH-6I–VII, 14ꢃH-6HM, 14ꢃCH2O, 7 ꢃ
O-CH2-triazole), 2.95–2.35 (m, 4H; CH2-S, CH2-NH2), 1.57–1.13 (m,
Acknowledgements
This work was carried out with financial support from the French
Agence Nationale de la Recherce (ANR JC07_183019), the Centre Na-
Chem. Eur. J. 2011, 17, 10029 – 10038
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10037

J. Bouckaert, S. G. Gouin et al.
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Received: February 15, 2011
Revised: June 16, 2011
Published online: July 19, 2011
10038
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