The functional valency of dodecamannosylated fullerenes with Escherichia coli FimH - Towards novel bacterial antiadhesives

Article abstract of DOI:10.1039/c0cc04468g

Fullerene hexakis-adducts bearing 12 peripheral mannose moieties have been prepared by grafting sugar derivatives onto the fullerene core and assayed as inhibitors of FimH, a bacterial adhesin, using isothermal titration calorimetry, surface plasmon reson

Full text of DOI:10.1039/c0cc04468g

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The functional valency of dodecamannosylated fullerenes with Escherichia
coli FimH—towards novel bacterial antiadhesivesw
Maxime Durka,a Kevin Buffet,a Julien Iehl,b Michel Holler,b Jean-Francois Nierengarten,*b
¸
c
c
a
´
Joemar Taganna, Julie Bouckaert* and Stephane P. Vincent*
Received 17th October 2010, Accepted 28th October 2010
DOI: 10.1039/c0cc04468g
Fullerene hexakis-adducts bearing 12 peripheral mannose
moieties have been prepared by grafting sugar derivatives onto
the fullerene core and assayed as inhibitors of FimH, a bacterial
adhesin, using isothermal titration calorimetry, surface plasmon
resonance and hemagglutination assays.
inhibition of hemagglutination and bladder cell assay.11 The
level of inhibition or binding was found to be highly dependent
on the number of mannoside residues and the nature of the
spacer. A direct relationship between these factors has however
not been clearly addressed so far. In this communication, we
now elucidate this critical point by a unique combination of
three experimental techniques: isothermal titration calorimetry
(ITC), SPR and hemagglutination assays. We also show
the ability of the fullerene-based globular glycoconjugates to
aggregate FimH, a key issue in view of developing antiadhesive
molecules. One of the most important questions that we
addressed was thus to determine the functional valency of
the multimers we synthesized, in other words the number of
adhesins that can bind the dodecaglycosylated fullerenes.
For this study, we designed fullerene sugar balls 1–3 (Fig. 1)
displaying twelve peripheral mannose residues. Fullerene
hexakis-adducts 1 and 2 were prepared in four steps using
the methodology we recently described.4
The emerging resistance of most pathogenic bacteria to
clinically relevant antibiotics urges the scientific community
to find a new class of molecules or a new mode of actions to
treat infectious diseases. Antivirulence has recently emerged as
an alternative antibacterial chemotherapeutic strategy to the
discovery of new bactericides or bacteriostatic molecules.1
Indeed, innovative approaches should arise from targeting
the bacterial functions that contribute to the infection and
lead the pathogen to cause damage in a host. Different
virulence mechanisms that allow pathogenic bacteria to
invade, to disseminate and adapt in the host have been
evidenced. With the antivirulence concept in mind, we have
recently reported on the inhibition of heptosyltransferases as a
novel approach to inhibit the bacterial cell wall resistance to
innate immune response.2,3 As part of this research, we now
report on fullerene-sugar balls4 designed for a complementary
antivirulence approach: the prevention of colonization and
bacterial adhesion to host cells. Indeed, bacterial adhesion to
cell surface tissues is one of the very first steps of the infective
process and is often mediated by carbohydrate–protein
interactions. This is the case of the uropathogenic strain of
E. coli that causes urinary tract infections.5 This bacterium
exploits FimH, an adhesin overexpressed at the tip of pili,
to bind terminal mannose moieties found at the surface of
the host epithelial cells.6 The multivalent presentation of
carbohydrates is generally an essential point for achieving
high affinity interactions with lectins7,8 and different
synthetic scaffolds allowing a multivalent presentation of
mannosyl ligands have been already assayed against FimH
using either surface plasmon resonance (SPR),9 ELISA10 or
As demonstrated by Roy et al., the presence of an aromatic
residue properly positioned in the spacer between the
carbohydrate unit and the central scaffold of the multivalent
platform may dramatically affect the binding properties of
the multimers towards adhesins.9 Thus, we designed and
synthesized fullerene 3 which bears a benzylic aromatic ring
a
´
University of Namur (FUNDP), Departement de Chimie,
Laboratoire de Chimie Bio-Organique, rue de Bruxelles 61, B-5000
Namur, Belgium. E-mail: stephane.vincent@fundp.ac.be
b
´
Laboratoire de Chimie des Materiaux Moleculaires, Universite de
Strasbourg et CNRS (UMR 7509), Ecole Europeenne de Chimie,
´
´
´
`
´
Polymeres et Materiaux (ECPM), 25 rue Becquerel, 67087
Strasbourg Cedex 2, France.
E-mail: nierengarten@chimie.u-strasbg.fr
c Structural Biology Brussels at the Vrije Universiteit Brussel, VIB,
Pleinlaan 2, 1050 Brussels, Belgium. E-mail: bouckaej@vub.ac.be
w Electronic supplementary information (ESI) available: Experimental
details for the ITC experiments, the hemagglutination assays, and for
the preparation of the new compounds. See DOI: 10.1039/c0cc04468g
Fig. 1 Fullerene sugar balls 1–3 and the corresponding monovalent
model compounds 4–6.
c
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Chem. Commun., 2011, 47, 1321–1323 1321

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allows the binding of roughly 6 mannose moieties. Fullerenes
1 and 2 displayed similar dissociation constants than their
corresponding monomers 4 and 5, respectively. The analysis
of the thermodynamic data showed that displaying the mannose
residues around the fullerene core structure is favourable for the
enthalpic contribution. On the other hand, the binding affinities
were hampered by a significant entropic cost, as observed for
other lectins such as ConA.13
This entropic cost and the difference in binding properties of
sugar balls 1 and 2 could be explained by the structure of FimH
itself. Contrary to most other lectins, FimH displays a deep
and narrow binding pocket. The mannose residues have to
reach the bottom of this cavity to properly bind the lectin.
Thus, the fullerene core structure has to approach the protein
surface, as shown in the model structure provided in the ESI.w
We then turn our attention to fullerene 3 bearing a benzyl
moiety linked to each mannose unit. Due to its hydrophobic
nature, this fullerene was not fully soluble at the highest
concentrations required for the ITC experiments and the
hemagglutination assays. Therefore, all the ITC experiments
have been performed with 5% DMSO to allow a comparison
with fullerenes 1 and 2. An unexpected phenomenon occurred
during both the usual titration of FimH by 3 and the reverse
titration mode (titration of 3 with FimH). Instead of the typical
sigmoidal plot, we observed a first binding event followed by
a strong exothermic jump that could be attributed to
an aggregation of the protein during the experiment. Given
the fact that the fullerene was totally soluble under these
conditions and that FimH did not aggregate with compounds
1–2, 4–6 and 12, we can safely conclude that multimer 3 itself
triggered the aggregation of FimH. From the first binding event
observed in the ITC plot, the aggregation begins when 5 to 6
mannose residues are bound to FimH.
Scheme 1 Reagents and conditions: (i) 8, Pd(PPh3)2Cl2, CuI, NEt3,
DMF, m.w., 6 min; (ii) MsCl, DMAP, Py, 45 min, RT; (iii) NaN3,
DMF, 2 h, 60 1C; (iv) MeONa, MeOH, 15 min, 0 1C to RT.
at the anomeric position and an amide group that might
provide favorable contacts with the lectin binding cavity, as
expected from the FimH 3D structure.12 The key intermediate
13 bearing an azide functionality was prepared by
a
Sonogashira cross-coupling reaction between alkyne 8 and
mannoside 9 in 77% yield (Scheme 1). The former was easily
prepared from D-mannose peracetate and iodobenzyl alcohol
under standard conditions. Intermediate 10 was deprotected to
give the monomeric mannoside 12 which served as a reference
for the biological evaluations. The same intermediate 10 was
transformed into the desired deprotected azide 13, through a
sequence of mesylation, azidation and Zemplen deprotection.
´
Finally, we coupled azide 13 with the fullerene bearing twelve
alkyne functionalities. The structure of compound 3 was
1
confirmed by its H and 13C NMR spectra.
The binding affinities of compounds 1–6, and 12 for FimH
were determined by ITC. The latter is an important technique
because it provides the stoichiometry of the binding process
and a true view of the equilibrium affinity. To the best of our
knowledge, this study represents the first ITC experiments
reported on FimH to date. The results are summarized in
Table 1. The comparison of fullerenes 1 and 2 clearly shows
that increasing the distance between the mannose residues and
the fullerene core improved significantly the binding affinities.
Interestingly, the ITC allowed us to determine the number N
of carbohydrates that can bind the lectin. Therefore, the value
1/N (Table 1) represents the number of lectins that can bind to
the glycosylated fullerene. Here again, the spacer length plays a
significant role since around 4 mannose units from fullerene 1
effectively bind FimH whereas fullerene 2, with a longer spacer,
This specific phenomenon is indeed interesting since, from a
therapeutic point of view, such an aggregation process could be
advantageous in a strategy dedicated to preventing bacterial
colonizations. The fact that the molarity observed for the
aggregation of FimH with fullerene 3 is roughly the same as
the molar ratio calculated for fullerene 2 strongly suggests that
the two processes (binding and aggregation) are linked.
To determine the binding affinity of fullerene 3, we inhibited
in solution the binding of FimH to a mannose-functionalized
sensor chip by the addition of fullerene 3 and measured SPR on
a Biacore3000. The binding affinity of the whole series of
molecules is reported in Table 1. Importantly, the data
Table 1 FimH relative affinities of mannosylated fullerenes as determined by ITC and SPR
ITCd
SPR
DH/kJ molÀ1
DS/J molÀ1 KÀ1
Kd/nM
1/Na
Kd/nM
Nc
Ligand
1
2
3
4
5
6
12
À238.28 Æ 3.31
À444.92 Æ 6.64
NDb
À43.07 Æ 3.91
À61.19 Æ 0.28
À46.67 Æ 0.65
À47.76 Æ 0.33
À674
À1360
NDb
À11
95 Æ 8
26 Æ 2
NDb
92 Æ 6
20 Æ 2
30 Æ 1
40 Æ 3
3.89
6.53
NDb
1.04
1.06
0.95
0.97
39.8 Æ 2.0
12.3 Æ 1.2
8.9 Æ 1.1
70.0 Æ 2.7
10.7 Æ 1.6
9.2 Æ 1.1
32.9 Æ 3.2
3.4
7
7
—
—
—
—
À60
À30
À20
a
b
1/N represents the number of lectin molecules that can bind the fullerene. ND: not determined; the complete plot could not be obtained due to
c
the aggregation. Effective D-mannose units as evaluated from Fig. 2: best fitting of chi2 vs. n in SPR measurements. DH and DS values refer to
the global multimer/protein association.
d
c
1322 Chem. Commun., 2011, 47, 1321–1323
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deduced from the SPR measurements were in good agreement
with those obtained by ITC (Table 1). The Kd values of
fullerenes 1, 2 and 3 compare well with those of the best
multimeric mannosides reported to date in the literature
(0.45 to 273 nM), as measured by SPR.9
Table 2 Inhibition of hemagglutination (HAI)
Ligand
4
1
5
2
12
3
Titera/mM
100
3.4
30
6.6
1.5
4.4
29.5
10.4
2.8
Ratiob m/D
a
b
HAI were performed in phosphate buffer with 10% DMSO. Ratio:
monomer titer (m)/corresponding dodecamer titer (D).
For monomers 4, 5, 6 and 12, the fitting using the solution
affinity interaction model worked properly and provided Kd
values in the nanomolar range. However, the same equation did
not allow a correct fitting for fullerenes 1, 2 and 3 (see ESIw). To
obtain a better fit of the SPR solution affinity between FimH
and the fullerenes, we substituted the fullerene concentrations
by the ‘‘effective mannose concentration’’, by multiplying the
fullerene concentration with the number n of mannose units
accessible to the FimH adhesin, and evaluated the evolution of
chi2 versus n (Fig. 2). Excellent fits were achieved with a number
of effective mannose units, n, almost identical to the functional
valency, 1/N, from the calorimetric data (Table 1). The Kd
values reported in Table 1 thus correspond to the dissociation
constant of one functional mannose subunit as part of the whole
fullerene structure. SPR thus confirmed the ITC findings that
the fullerenes can accommodate four FimH molecules for the
shortest spacer and up to seven lectins for fullerenes 2 and 3. In
conclusion, the functional valencies of the fullerenes (1/N in
Table 1), directly measured using ITC, could be readily applied
to the molar concentrations of the multivalent glycoconjugates
to correspond to the SPR solution affinity model.
Therefore, the inhibitory capacities of the fullerenes with E.
coli UTI89 were evaluated in a hemagglutination inhibition
assay (HAI). Addition of bacteria to guinea pig erythrocytes
produces a cross-linked matrix due to interaction of FimH
with the glycocalyx of the red blood cells. Subsequent
additions of the fullerenes into the wells eventually prevented
the agglutination reaction. The inhibition titer is defined as the
lowest concentration of the glycoconjugate at which
hemagglutination is still inhibited (Table 2).
Under those conditions, all the fullerene derivatives displayed
a low micromolar inhibition level, in each case lower than their
corresponding monomer. A clear multivalent effect was only
evidenced with fullerene 1 which has an inhibition titer 30-fold
lower (ca. 3-fold per mannose residue) than its corresponding
monomer 4.
In conclusion, we have reported on the synthesis of fullerene
glycoconjugates that were assessed as ligand of the bacterial
adhesin FimH. Low nanomolar affinities levels were measured
by ITC and SPR. Most importantly, the number of possible
interactions between the multimers and the lectin and the
average binding strength per functional mannose unit could
be measured. Thus, we proved, for the first time, that a globular
C60 structure can accommodate up to 7 FimH molecules. The
functional valency of these fullerenes could be assessed by the
unique comparison of two complementary techniques.
This work was supported by FNRS (FRFC grant
2.4.625.08.F) and a RTN grant (2005–019561).
Contrary to many plant and bacterial lectins that are
multimeric and may give rise to huge multivalent effects, FimH
is a monomeric adhesin for which multivalent effects are not
expected as a purified protein. However, as mentioned by Roy
et al.9 and Heidecke and Lindhorst,10 the average binding
strength per mannose unit of multimers (such as fullerenes 1, 2
and 3) compared to methyl-a-D-mannose (Kd = 2.2 mM)12 is
apparently greatly enhanced. Our data however clearly show
that the length and the structure of the anomeric substituent are
the key factors to dramatically enhance the binding affinity of the
carbohydrate subunit rather than multivalent effects.
Notes and references
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Fig. 2 Evaluation of the number n of effective ligands per fullerene
using SPR. Fullerene 1 = n; 2 = &; 3 = J.
c
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Chem. Commun., 2011, 47, 1321–1323 1323