FOCUS REVIEW

DOI: 10.1002/asia.201400133

Fullerene Sugar Balls: A New Class of Biologically Active Fullerene

Derivatives

Iwona Nierengarten and Jean-FranÅois Nierengarten*[a]

Dedicated to Prof. Thomas Ebbesen on the occasion of his 60th birthday

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Abstract: Among the large variety of bioactive C60 derivatives, fullerene derivatives substituted
with sugar residues, that is, glycofullerenes, are of particular interest. The sugar residues are not
only solubilizing groups; their intrinsic biological properties also provide additional appealing fea-
tures to the conjugates. The most recent advances in the synthesis and the biological applications of
glycofullerenes are summarized in the present review article with special emphasis on globular gly-
cofullerenes, that is, fullerene sugar balls, constructed on a hexa-substituted fullerene scaffold. The
high local concentration of carbohydrates around the C60 core in fullerene sugar balls is perfectly
suited to the binding of lectins through the “glycoside cluster effect”, and these compounds are po-
tential anti-adhesive agents against bacterial infection. Moreover, mannosylated fullerene sugar
balls have shown antiviral activity in an Ebola pseudotyped infection model. Finally, when substitut-
ed with peripheral iminosugars, dramatic multivalent effects have been observed for glycosidase in-
hibition. These unexpected observations have been rationalized by the interplay of interactions in-
volving the catalytic site of the enzyme and non-glycone binding sites with lectin-like abilities.

Keywords: fullerenes · inhibitors · lectins · multivalent ligands · sugar

Introduction their intrinsic biological properties also provide additional

appealing features to the conjugates. As part of this re-
Following the discovery of the macroscopic-scale C60 synthe- search, we have recently developed globular glycofullerenes,
sis by Krtschmer, Huffman, and co-workers,[1] advances in that is, fullerene sugar balls, constructed on a hexa-substitut-
fullerene synthetic chemistry rapidly moved towards the cre- ed fullerene scaffold.[15] The peculiar spherical distribution
ation of functional systems with increased attention to po- of the sugar residues around the fullerene core gave rise to
tential applications.[2] This has given impetus to make fuller- unprecedented globular polytopic ligands with remarkable
ene research a truly interdisciplinary branch of science lo- biological properties. These results are summarized in the
cated at the interfaces between chemistry, physics, and biol- present review.
ogy.[2] Fullerene derivatives have shown a wide range of
physical properties that make them attractive for applica-
tions in materials science.[3] Examples include solar energy 1. From Glycofullerenes to Fullerene Sugar Balls
conversion,[4] optical limiting,[5] and optoelectronics.[6] On
the other hand, fullerenes had also a significant impact in In the early 1990s, Vasella and Diederich reported the first
the field of biology.[7] However, biological applications of synthesis of a glycofullerene (compound 1, Figure 1).[16] The
fullerenes have been somehow limited by the absence of sol- protected carbohydrate residue was introduced on the
ubility of most fullerene derivatives in water, and the devel- carbon cage by reaction of a nucleophilic glycosylidene car-
opment of efficient strategies to obtain biocompatible ful- bene precursor with C60.[16] Several other examples of mono-
lerenes has been a central aspect of this research.[7] A large and divalent glycofullerenes have been then prepared either
variety of water-soluble fullerene derivatives have been pre-
pared over the years, and spectacular findings have been re-
ported about their biological properties. For example, func-
tionalized fullerenes were found to be competitive inhibitors
of human immunodeficiency virus (HIV) protease[8] or to be
appealing sensitizers for DNA photocleavage[9] and photo-
dynamic therapy.[10] Furthermore, Nakamura and co-workers
have shown that cationic fullerene derivatives exhibit a prac-
tically useful level of transfection.[11–13]
Among the bioactive fullerene derivatives developed
during the last two decades, C60 derivatives substituted with
sugar residues, that is, glycofullerenes, are of particular inter-
est.[14] The sugar residues are not only solubilizing groups,

[a] Dr. I. Nierengarten, Dr. J.-F. Nierengarten

Laboratoire de Chimie des Matriaux Molculaires
Universit de Strasbourg et CNRS (UMR 7509)
Ecole Europenne de Chimie, Polymres et Matriaux
25 rue Becquerel, 67087 Strasbourg Cedex 2 (France) Figure 1. Typical examples of glycofullerenes bearing one or two sugar
E-mail: nierengarten@unistra.fr residues.

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by direct functionalization of C60 with reactive sugar deriva-
tives[17, 18] or by reaction of fullerene derivatives bearing suit-
able functional groups, thud allowing conjugation of the sac-
charides with complementary functions in a final synthetic
step.[19, 20] Typical examples are depicted in Figure 1.
Whereas synthetic aspects for the preparation of glycoful-
lerenes have been intensively investigated, only a few bio-
logical applications have been reported with such com-
pounds.[14, 16–20] For example, the ability of fullerene deriva-
tive 2 (Figure 1) to efficiently generate reactive oxygen spe-
cies (ROS) upon photoirradiation in the presence of O2 has
been exploited to selectively degrade HIV protease owing
to the good affinity of this protein for fullerenes.[19] The pho-
totoxicity of glycoconjugates 3 and 4 (Figure 1) was evaluat-
ed for potential applications in photodynamic therapy.[18]
While substantial production of singlet oxygen (1O2) under
irradiation with a 355 nm laser has been observed for both
compounds, the monovalent derivative 3 showed a better
photosensitizing ability. In addition, in vitro studies using
HeLa cells further confirmed their photocytotoxicity. It has
also been found that glycofullerenes 3 and 4 form stable
self-assembled nanostructures in water.[18] These spherical
supramolecular structures with diameters of about 100–
300 nm are capable of encapsulating organic molecules such
as acridine red, thus constituting, for instance, promising
candidates for slow drug delivery. The ability of mannoful-
lerenes 3 and 4 to bind to concanavalin A (Con A), a man-
noside-recognizing lectin, has been also investigated.[18] Col-
loidal suspensions of the stable self-assembled nanostruc-
tures obtained from 4 in water presented a higher blocking
activity than monoadduct 3, with a submicromolar MIC
(minimum inhibitory concentration) value in a lectin-in-
duced hemagglutination assay.
The water solubility of glycofullerenes substituted with
one or two sugar residues is, however, generally quite limit-
ed. A convenient strategy to improve their solubility in
aqueous media is to further increase the number of sugar
residues grafted onto the fullerene core. A powerful meth-
odology developed by Nakamura[21] allows for the introduc-
tion of five carbohydrate moieties onto a C60 scaffold to
yield highly water-soluble glycofullerenes such as 5
(Figure 2). The copper-catalyzed alkyne–azide cycloaddition
(CuAAC) reaction allowed efficient conjugation of unpro-
tected sugar moieties with the terminal alkyne groups of the
pentaalkynylated C60 core.[21b] Another synthetic approach

Iwona Nierengarten studied chemistry at

the University of Wroclaw, Poland, and
received her doctoral degree under the su-
pervision of Krystyna Bukietynska
(2005). She worked as a postdoctoral
fellow with Anne-Marie Albrecht-Gary at
the University of Strasbourg, France
(2005–2008). She is currently a post-doc-
toral fellow in the group of Jean-FranÅois
Nierengarten. Her research focuses on the
chemistry of fullerenes and pillar[n]ar-
enes for applications in materials science
and biology.

Jean-FranÅois Nierengarten studied bio-

chemistry and chemistry at the University
of Strasbourg, France, and received his
doctoral degree under the supervision of
Jean-Pierre Sauvage and Christiane Die-
trich-Buchecker in Strasbourg (1994). He
worked as a postdoctoral fellow with
FranÅois Diederich at the ETH-Zrich,
Switzerland (1994–1996). He then ob-
tained a CNRS researcher position in
1996. Currently, he is Directeur de Re-
cherche at the CNRS (DR1) and head of
the Laboratoire de Chimie des Matriaux
Molculaires (University of Strasbourg
and CNRS). His research focuses on the chemistry of fullerenes for appli-
cations in materials science and biology. Other research interests are in the
area of dendrimers, luminescent transition metal complexes, porphyrins,
Figure 2. Typical examples of glycofullerenes bearing five or six sugar res-
and p-conjugated systems.
idues.

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has been reported by Hirsch and co-workers.[22] Dendrons

bearing an azido function at the focal point and six protect-
ed a-d-mannopyranosyl subunits at the periphery were
treated with C60 to afford a mixture of mono- and bisglyco-
dendron adducts. Separation and purification by column
chromatography gave protected 6 in 8 % yield. Deacetyla-
tion under Zempln conditions finally provided compound
6. Glycoconjugate 6 is highly water-soluble (> 40 mg mL 1),
but its amphiphilic nature still triggered the formation of
supramolecular aggregates in aqueous solutions, as deduced
from DOSY NMR spectroscopic and TEM investigations.
To the best of our knowledge, no biological applications of
compounds such as 5 or 6 have been reported so far.
All the glycofullerenes already mentioned in this section
are amphiphilic molecules, with a hydrophobic fullerene
subunit occupying a part of the outer architecture of the
molecule. Low solubility in aqueous media and/or aggrega-
tion phenomena remain major limitations for some biologi-
cal applications with such compounds. Recently, we have
shown that the amphiphilic character of glycofullerenes can
be avoided[15] by taking advantage of the unique globular
structure of fullerene hexakis-adducts with a Th-symmetrical
octahedral addition pattern.[23] As far as the synthesis of full-
erene hexakis-adducts with peripheral sugar residues, that is,
fullerene sugar balls, is concerned, the direct grafting of six
malonates bearing sugar residues to the fullerene core
would be quite limited. Indeed, fullerene hexakis-adducts
are generally obtained in very low yields from structurally
complicated malonates.[23] Furthermore, this strategy would
require the preparation of new malonates for each deriva-
tive as well as the use of protecting groups for the hydroxy
functions of the sugars. Indeed, the selected synthetic strat-
egy was based on post-functionalization of the readily avail-
able fullerene hexa-adduct building block 7 (Scheme 1).[24–26]
Fullerene sugar balls were efficiently prepared by grafting
unprotected sugar derivatives 8–14 bearing a terminal
alkyne group onto the fullerene core under classical
CuAAC conditions.[15, 27–33] The clicked derivatives C60(8)12
Scheme 1. Preparation of fullerene sugar balls from building block 7.
C60(14)12 were thus obtained in fair to excellent isolated
yields.
The chemical structure of compounds C60(8)12 C60(14)12
was confirmed by 1H and 13C NMR spectroscopic analyses. spectra of the cycloaddition product displayed minor traces
The 13C NMR spectra of fullerene hexakis-adducts C60(8)12 of azide groups (typically, a band was observed at ca.
C60(14)12 were particularly helpful to provide evidence of 2092 cm 1), the material became completely insoluble after
their T-symmetrical structure. Their characterization by a few days of storage.[29]
mass spectrometry was found particularly difficult due to Following the development of an efficient procedure for
a high degree of fragmentation and/or to the formation of the preparation of fullerene sugar balls from polyazide 7
matrix adducts. Despite these problems, evidence of the ex- and sugar alkyne building blocks, the reverse synthetic ap-
pected molecular ion peak could be obtained in most of the proach starting from fullerene derivative 15 bearing 12 ter-
cases. IR spectroscopy was also an important analytical tool minal alkyne groups was also investigated (Scheme 2).[15] It
for the characterization of the adducts. The IR spectra of was also found that fullerene sugar balls can be directly pre-
C60(8)12 C60(14)12 confirmed that no azide (2092 cm 1) resi- pared from fullerene derivative 16. Indeed, compound 16
dues remained in the final products. This was further con- can be desilylated in situ with tetrabutylammonium fluoride
firmed by the stability of the prepared compounds. Indeed, (TBAF) to generate 15,[24b] to which the sugar azide precur-
a slow polymerization was systematically observed when un- sors can be subsequently clicked. Sugar azide derivatives
reacted azide subunits were still present in the products pre- 17–21 were thus efficiently grafted onto polyalkyne 15 or
pared from 7 and alkyne derivatives. As soon as the IR 16[15, 27–33] but also glycodendrons 22–24, thus giving rise to

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Scheme 2. Preparation of fullerene sugar balls from alkylynated C60 building blocks 15 and 16.
glycoclusters with up to 36 peripheral sugar residues.[27–28] Fi-
nally, iminosugar derivatives (25–29) were also used to func-
tionalize the fullerene scaffold.[31, 33]
As all the fullerene sugar balls were obtained under
CuAAC conditions, the removal of the copper catalyst was
a critical issue in light of their biological applications. In
order to completely remove potential copper-based impuri-
ties, these glycoconjugates were systematically purified by
filtration on a Sephadex or on a QuadraSil Mercaptopropyl
column. Importantly, the apparent structural sophistication
of these compounds is not associated with complicated syn-
thetic routes. For example, compound C60(17)12 has been
prepared on a 500 mg scale in four synthetic steps from
commercially available compounds in an overall yield of
21 %. Applications of such compounds are therefore not
limited by their availability. In addition, hexa-substituted
fullerene derivatives are only weak electron acceptors[34] and
no longer efficient singlet oxygen sensitizers.[35] Therefore,
problems associated with the phototoxicity of mono- or di-
substituted fullerene derivatives[36] resulting from the pro-
duction of ROS are prevented in the case of hexasubstituted
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Iwona Nierengarten and Jean-FranÅois Nierengarten
derivatives. Additionally, classical MTT assays have been re-
cently performed with some fullerene sugar balls and no
particular cytotoxicity or cytostatic activity could be evi-
denced.[37]
2. Biological Properties of Fullerene Sugar Balls
2.1. High-Affinity Lectin Ligands: Towards Novel Bacterial
Anti-Adhesives
Carbohydrate–protein interactions play an essential role in
a large variety of biological processes.[38] The proteins in-
volved in such biomolecular interactions are called lectins.
The design of high-affinity ligands of such proteins has been
intensively investigated to obtain synthetic molecules capa-
ble of blocking or influencing some essential biological pro-
cesses.[38] A typical example is the prevention of colonization
and bacterial adhesion to host cells.[39] Bacterial adhesion to
cell surface tissues is actually one of the very first steps of
infective processes and is often mediated by carbohydrate–
protein interactions. A possible strategy for blocking the in-
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fection would consist in occupying the bacterial lectins
binding sites with high-affinity ligands to prevent the adhe-
sion event. Importantly, the multivalent presentation of car-
bohydrates is generally an essential point for achieving high-
affinity interactions with such lectins.[38] The high local con-
centration of carbohydrates around the C60 core in fullerene
sugar balls is indeed perfectly suited to the binding of lectins
through a “glycoside cluster effect”.[27–30] Initial binding stud-
ies with C60(21)12 and C60(22)12 using the classical model
lectin concanavalin A (Con A) and isothermal titration calo-
rimetry (ITC) demonstrated that mannoses on the surface
of these fullerene sugar balls were effectively recognized by
this lectin in a multivalent manner.[27] Binding studies have
been also performed with biologically relevant bacterial lec-
tins. For example, glycoclusters C60(8)12 and C60(18)12 have
been assayed towards PA-IL (LecA), a bacterial lectin from
Pseudomonas aeruginosa.[29] This bacterium is an opportun-
istic pathogen responsible for nosocomial infections and for
high morbidity in cystic fibrosis patients. Its virulence and
binding to host cells are mediated by lectin–carbohydrate in-
teractions involving PA-IL (LecA) and PA-IIL (LecB) that
are specific for galactose and fucose, respectively.[40] Fuller-
ene sugar balls C60(8)12 and C60(18)12 were tested as ligands
of PA-IL to evaluate their potential for competing with the
binding of this lectin to glycosylated surfaces. The affinities
measured by hemagglutination inhibition assay, enzyme-
linked lectin assay (ELLA), and surface plasmon resonance
(SPR) were very high. In addition, comparison of the dodec-
avalent fullerene-based glycoclusters with monovalent car-
bohydrate reference probes revealed multivalent effects
with spectacular increase in binding affinities (up to 12 000-
fold), albeit with some differences depending on the analyti-
cal technique. Fullerene sugar balls C60(8)12 and C60(18)12 are
among the most potent ligands of PA-IL measured to date
and can be considered as potential anti-adhesive agents
against infection by Pseudomonas aeruginosa.
Mannosylated fullerene derivatives C60(9)12, C60(10)12, and
C60(19)12 have been assayed as inhibitors of FimH,[30] a bacte-
rial adhesin involved in the adhesion of uropathogenic Es-
cherichia coli bacteria to host cells. Remarkably, low nano-
molar affinities were measured by ITC and SPR. However,
no multivalent effects could be verified in this particular
case. Indeed, contrary to many plant and bacterial lectins
that are multimeric and may give rise to multivalent effects,
FimH is a monomeric adhesin for which such effects are not
observed.[41] However, a single fullerene sugar ball molecule
can accommodate up to 7 FimH molecules and the manno-
sylated fullerene derivatives displayed significantly higher
inhibition levels than their corresponding monomers in he-
magglutination inhibition assays, thus showing that a multi-
valent effect can be achieved at the cellular level. Such re-
versed multivalent effects, that is, resulting from the binding
of several proteins to the same polytopic inhibitor, have
been also observed for other mannosylated glycoclusters.[42]
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2.2. Antiviral Activity
Interactions of glycoconjugates present on the surface of
several pathogens with cellular receptors play a major role
in the initial stages of viral infections.[43] For example, DC-
SIGN, a C-type lectin, binds various microorganisms by rec-
ognizing high-mannose-containing glycoproteins on their en-
velope and functions as a receptor for several viruses like
HIV or Ebola.[43] This initial interaction causes co-internali-
zation of the DC-SIGN receptor and the viral particle. Due
to the important role of DC-SIGN in infection processes,
the discovery of new compounds with an adequate affinity
for this receptor is of great importance. With the aim to
evaluate the efficiency of mannosylated fullerene sugar balls
C60(21)12, C60(23)12, and C60(24)12 in cellular experiments,
Rojo, Delgado, and Martin et al. have addressed an infec-
tion study assay using Ebola virus glycoprotein (EboGP)
pseudotyped viral particles as infectious agents.[28] Infection
was performed on Jurkat cells expressing DC-SIGN. Be-
cause the Ebola virus does not infect T-lymphocytes, its
entry is entirely dependent on DC-SIGN for infection of
Jurkat cells. The cells were incubated at room temperature
for 20 min with the carbohydrate-based compounds and
then challenged with recombinant viruses. After 48 hours of
incubation, the inhibition of infection was evaluated for
each compound. Mannosylated fullerene sugar ball C60(21)12
showed a remarkable IC50 of 2 mm (IC50 : concentration of
compound needed to inhibit the infection by half). The IC50
found for glycodendrofullerene C60(23)12 with 36 peripheral
mannose subunits was 68 mm. In this particular case, the in-
crease of the valency produced a decrease in the activity,
probably as a result of steric congestions. These negative
steric effects could be suppressed, at least in part, by the in-
troduction of a longer spacer between the glycodendron and
the fullerene scaffold. When going from glycodendrofuller-
ene C60(23)12 to C60(24)12, a 200-fold increase in activity was
observed. With an IC50 of 0.3 mm, compound C60(24)12 can be
considered as a very active inhibitor of the DC-SIGN-de-
pendent infection process.
2.3. Enzyme Multivalent Inhibitory Effects
Whereas spectacular results have been obtained with multi-
meric carbohydrates exhibiting an affinity enhancement of
several orders of magnitude over the corresponding mono-
valent ligands in the field of carbohydrate–lectin interac-
tions,[38] only a few studies have been reported on the evalu-
ation of the inhibition of carbohydrate-processing enzymes
with multivalent inhibitors.[44] Most enzymes have a single,
deep active site, which is usually less accessible than the
shallow binding pockets or grooves on the lectin surface.
Accordingly, multivalent inhibitory effects are obviously not
expected, and such considerations may have hampered the
interest in projects directed towards the design of multiva-
lent enzyme inhibitors. Furthermore, experimental results
obtained from the few studies reported to date were not par-
ticularly encouraging.[44, 45] Di- to tetravalent analogues of 1-
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deoxynojirimycin, a well-known glycosidase inhibitor,[46]
generally displayed comparable if not decreased inhibition
as compared to their monomeric counterparts.[44] The first
positive effect was observed for trivalent iminosugar 30
(Figure 3), which showed a 6-fold affinity enhancement to-
wards Jack bean a-mannosidase.[45]
Figure 3. Trivalent iminosugar 30 and model compound 31.
As part of this research, the inhibition profile of fullerene
hexakis-adduct C60(25)12 decorated with 12 iminosugar resi-
dues was systematically evaluated towards various glycosi-
dases.[31] Remarkably, evidence of significant multivalent ef-
fects has been obtained for the first time. The most impres-
sive results were obtained for three a-glycosidases. Multiva-
lent iminosugar C60(25)12 was found to be a good inhibitor
of a-galactosidase of green coffee, whereas its monomeric
analog 31 was completely inactive. Stronger measurable
multivalent effects were obtained with isomaltase of bakers
yeast and a-mannosidase of Jack bean, with inhibition con-
stant values Ki improved by two and three orders of magni-
tude, respectively, over that of the corresponding monomer-
ic iminosugar (31). Analysis of the valency-corrected poten-
cy of the two multivalent systems toward a-mannosidase of
Jack bean revealed a 2-fold and 179-fold increase for the tri-
valent and dodecavalent systems 30 and C60(25)12, respec-
tively. These results nicely highlight the impact of the cen-
tral scaffold on the inhibition activity of the multivalent ar-
chitecture.
The concept of multivalent glycosidase inhibition was fur-
ther investigated with another family of glycomimetic conju-
gates, namely fullerene-sp2-iminosugar balls C60ACHTUNGRE(26–29)12.[33]
Glycosidase recognition processes were found to be sensi-
tive to multivalency as observed for C60(25)12. Interestingly,
compounds C60ACHTUNGRE(26–27)12 were also able to bind to peanut
(Arachis hypogaea) agglutinin (PNA), a lectin broadly used
in fundamental carbohydrate–protein interaction studies.[47]
The unique dual lectin-glycosidase ligand feature of the 1-
amino-5N,6O-oxomethylydenenojirimycin (1N-ONJ)–fuller-
ene conjugates was exploited to develop a modified two-site
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ELLA test in which PNA labeled with horseradish perox-
idase (HRP-PNA) and a-Manase compete to bind to the do-
decavalent sp2-iminosugar balls C60(26)12 or C60(27)12 in solu-
tion. By performing the assay in the absence and presence
of a potent active-site-directed monovalent inhibitor, the in-
volvement of the glycone catalytic site in the multivalent in-
hibitory effect can be mapped. Alternatively, a multivalent
inhibitor of the enzyme can be used to assess the contribu-
tion of non-glycone sites to the binding process (Figure 4).
The ensemble of results obtained in these studies strongly
suggests that the binding modes for monovalent and multi-
valent iminosugar-type glycomimetics towards a-Manase are
starkly different. Monovalent competitive inhibitors sit at
the glycone binding site in the catalytic pocket, whereas gly-
cosidase inhibition by multivalent inhibitors seems to
depend critically on interactions involving non-glycone bind-
ing sites. In other words, the interplay of interactions involv-
ing simultaneously the catalytic site and non-glycone bind-
ing sites enables positive cooperativity between inhitopes
and is responsible for the multivalency (Figure 4 B).
Figure 4. (A) Schematic representation of the HRP-PNA/a-Manase com-
petitive ELLA test developed to assess the contribution of the active
(glycone) site and of non-glycon sites in the enzyme to the a-Manase-sp2-
iminosugar-fullerene balls C60(26)12 or C60(27)12. At constant concentra-
tions of HRP-PNA and sp2-iminosugar conjugate, the proportion of
cross-linked lectin decreases with increasing concentrations of the
enzyme. This process is affected in the presence of an active-site-directed
monovalent inhibitor and of a multivalent inhibitor to an extent that de-
pends on the implication of the catalytic and non-glycon sites in the bind-
ing event. (B) and (C) Schematic representations of the shift in binding
modes after multivalent presentation of inhitopes for enzymes having ac-
cessible catalytic as well as aglyconic sites (B) or a deep catalytic site but
accessible lectin-like aglycone sites (C).
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A second set of competitive lectin/enzyme experiments in
which a-Manase was replaced by a-Glcase maltase provided
evidence of a significantly different scenario. In this case,
the active site for the enzyme is only marginally involved in
the binding of the multivalent fullerene iminosugar balls
and non-glycone binding sites with lectin-like abilities are
probably involved in this process, which ultimately lead to
blockage of the catalytic site entrance, thus impairing the
access and processing of the substrate (Figure 4 C).
Fullerene sugar balls were also evaluated as a new class
of multivalent inhibitors of a biologically relevant bacterial
glycosyltransferase (GT).[32] In this studies, compounds C60-
ACHTUNGRE(11–14)12 were assayed as inhibitors of heptosyltransferase
WaaC, a glycosyltransferase catalyzing the incorporation of
the first l-heptose into the lipopolysaccharide (LPS), which
is a key component of the outer membrane of Gram-nega-
tive bacteria.[48] From a pharmaceutical viewpoint, its bio-
synthesis has become a very important research field if one
considers that the mortality of many infectious diseases is
closely related to the amount of circulating LPS found in pa-
tient sera.[49] Interestingly, the inhibition levels of the fuller-
ene sugar ball derivatives C60ACHTUNGRE(11–14)12 were found to be in
the low micromolar range (IC50 7–45 mm), while the corre-
sponding monomeric glycosides displayed high micromolar
to low millimolar inhibition levels (IC50 values were always
above 400 mm). When evaluated on a “per-sugar” basis,
these inhibition data showed that, in each case, the average
affinity of a single glycoside of the fullerenes towards WaaC
was significantly enhanced when displayed as a multimer.
Conclusions
The most recent advances in the synthesis and the biological
applications of glycofullerenes have been summarized with
special emphasis on globular glycofullerenes, that is, fuller-
ene sugar balls, constructed on a hexa-substituted fullerene
scaffold. Their synthesis is based on the post-functionaliza-
tion of readily available C60 hexa-adduct building blocks.
This strategy allows for the easy preparation of a large vari-
ety of glycoclusters as illustrated by the structural diversity
of fullerene sugar balls depicted in Schemes 1 and 2. Such
compounds are displaying twelve or more functional groups
on their periphery in a unique globular topology. Fullerene
sugar balls have been assayed as ligands of bacterial lectins,
and the results obtained so far revealed that these com-
pounds are potential anti-adhesive agents against bacterial
infection. Moreover, fullerene sugar balls have shown inter-
esting antiviral activity in an Ebola pseudotyped infection
model, thus opening new perspectives for their applications.
Finally, when substituted with appropriate glycoside deriva-
tives, dramatic multivalent effects have been observed for
enzymatic inhibition. These unexpected observations have
been rationalized by the interplay of interactions involving
the catalytic site of the enzyme and non-glycone binding
sites with lectin-like abilities. The first evidence of signifi-
cant multivalent effects in enzymatic inhibition not only
& & Chem. Asian J. 2014, 00, 0 – 0
Iwona Nierengarten and Jean-FranÅois Nierengarten
opened a new field of research but also revealed that the
multivalent approach may be an appealing tool for innova-
tive drug discoveries.
Acknowledgements
The authors thank the Universit de Strasbourg, the CNRS and the
Agence National de la Recherche (ANR, Programme Blanc 2011,
Sweet60s) for financial support. We would like to warmly thank all our
co-workers for their outstanding contributions; their names are cited in
the references. This multidisciplinary research was only possible thanks
to collaborations with colleagues all around Europe: S. P. Vincent
(Namur, Belgium), C. Ortiz Mellet, J. M. Garca Fernndez and J. Rojo
(Sevilla, Spain), S. Vidal (Lyon, France), A. Imberty (Grenoble, France),
P. Compain (Strasbourg, France), J. Bouckaert (Lille, France), and N.
Martn (Madrid, Spain). We would like to thank all of them for their en-
thusiasm, support, and contributions.
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Received: January 28, 2014
Published online: && &&, 0000
9  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim & &
 www.chemasianj.org Iwona Nierengarten and Jean-FranÅois Nierengarten

FOCUS REVIEW
Fullerenes Sweet 60s: The most recent advances
in the synthesis and biological applica-
Iwona Nierengarten, tions of glycofullerenes are summar-
Jean-FranÅoisNierengarten* &&&&—&&&& ized in the present review article, with
special emphasis on globular glycoful-
Fullerene Sugar Balls: A New Class of
lerenes, that is, fullerene sugar balls,
Biologically Active Fullerene Deriva-
constructed on a hexa-substituted full-
tives
erene scaffold.

& & Chem. Asian J. 2014, 00, 0 – 0 10  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ÝÝ These are not the final page numbers!