Glycosidase inhibition with fullerene iminosugar balls: A dramatic multivalent effect

Article abstract of DOI:10.1002/anie.201002802

(Figure Presented) Superball ! A dodecavalent iminosugar derivative with a fullerene core (see picture) shows a binding enhancement of up to three orders of magnitude over the corresponding monovalent ligand in glycosidase inhibition assays. This is the first evidence of a significant multivalent effect in glycosidase inhibition.

Full text of DOI:10.1002/anie.201002802

Angewandte
Chemie
DOI: 10.1002/anie.201002802
Fullerenes
Glycosidase Inhibition with Fullerene Iminosugar Balls: A Dramatic
Multivalent Effect**
Philippe Compain,* Camille Decroocq, Julien Iehl, Michel Holler, Damien Hazelard,
Teresa Mena Barragꢀn, Carmen Ortiz Mellet,* and Jean-Franꢁois Nierengarten*
The electronic and structural properties of fullerene deriva-
tives make them very attractive candidates for the construc-
tion of nanostructures that are potentially useful for applica-
tions in materials science and biological chemistry.[1] In
particular, the C60 hexakis adducts with a Th-symmetrical
octahedral addition pattern initially developed by Hirsch and
co-workers[2] are unique organic molecules with an appealing
compact spherical scaffold for the construction of multifunc-
tional nanomaterials.[3] However, the synthesis of functional-
ized fullerene hexakis adducts from malonates and C60 is
difficult.[3,4] This major problem limits the applications of such
systems and has been recently solved by the development of
synthetic methodologies based on the postfunctionalization
of easily accessible building blocks of fullerene hexakis
adducts.[5,6] It has been shown that fullerene hexakis adducts
that bear 12 peripheral carbohydrate moieties can be
prepared in excellent yields by grafting unprotected sugar
derivatives onto the fullerene core.[7] Although these full-
erene sugar balls are obviously perfectly suited for applica-
tions in the field of carbohydrate–lectin interactions,[8] the
evaluation of carbohydrate-processing enzyme inhibition
with such multivalent derivatives is less obvious. Indeed,
among the possible strategies to attain specific potent
glycosidase inhibition, the concept of multivalent design has
been clearly overlooked.[9] Most enzymes actually have a
single, deep active site that is usually less accessible than the
shallow binding pockets or grooves on the lectin surfaces.[10]
Consequently, a limited number of binding mechanisms,
including statistical rebinding, are possible, whereas multi-
valent ligands may interact with multiple receptors by addi-
tional mechanistic options (e.g., the chelate effect, receptor
clustering).[9,11] It is likely that these factors may have
hampered interest in projects directed towards the design of
multivalent glycosidase inhibitors. In addition, the experi-
mental results obtained to date were not particularly encour-
aging.[12] Di- to tetravalent analogues of 1-deoxynojirimycin,
which is a well-known glycosidase inhibitor,[13] generally
displayed comparable if not decreased inhibition compared
with their monomeric counterparts. The best result reported
to date was found for a trivalent iminosugar that showed a
sixfold affinity enhancement towards Jack bean a-mannosi-
dase.[14] Herein we report the synthesis of a fullerene hexakis
adduct decorated with 12 iminosugar residues. The inhibition
profile of this fullerene iminosugar ball has been systemati-
cally evaluated against various glycosidases,[15] and dramatic
multivalent effects have been observed for the first time.
In order to explore the potential of multivalency on
glycosidase inhibition with a globular polytopic ligand con-
structed around the fullerene scaffold, an N-alkyl analogue of
1-deoxynojirimycin was selected as the peripheral ligand. This
class of compounds is indeed poorly selective and displays
modest to good glycosidase inhibition.[13] It was thus antici-
pated that these compounds could be excellent models for the
examination of the influence of multivalency on inhibition
selectivity over a large range of glycosidases. In addition, the
alkyl chain on the endocyclic nitrogen atom of the iminosugar
is an ideal spacer that may allow for easy grafting onto the
central C60 core by means of a cycloaddition reaction.[16] The
synthesis of the azide building block is based on the
optimization of a strategy reported independently by Over-
kleeft et al.[17] and Vasella and co-workers.[18] As shown in
Scheme 1, the d-hydroxy amide 2 was obtained directly from
commercially available tetra-O-benzyl d-glucopyranose (1)
in 78% yield by oxidative amidation with iodine in 30%
aqueous ammonia (30%).[19] The main advantage of this one-
[*] Prof. P. Compain, C. Decroocq, Dr. D. Hazelard
Laboratoire de Synthꢀse Organique et Molꢁcules Bioactives
Universitꢁ de Strasbourg et CNRS (UMR 7509)
Ecole Europꢁenne de Chimie, Polymꢀres et Matꢁriaux
25 rue Becquerel, 67087 Strasbourg (France)
Fax: (+33)3-6885-2754
E-mail: philippe.compain@unistra.fr
J. Iehl, Dr. M. Holler, Prof. J.-F. Nierengarten
Laboratoire de Chimie des Matꢁriaux Molꢁculaires
Universitꢁ de Strasbourg et CNRS (UMR 7509)
Ecole Europꢁenne de Chimie
Polymꢀres et Matꢁriaux
25 rue Becquerel, 67087 Strasbourg (France)
Fax: (+33)3-6885-2774
E-mail: nierengarten@chimie.u-strasbg.fr
T. Mena Barragꢂn, Prof. C. Ortiz Mellet
Departamento de Quꢃmica Orgꢂnica, Facultad de Quꢃmica
Universidad de Sevilla
Profesor Garcꢃa Gonzꢂlez 1, 41012 Sevilla (Spain)
E-mail: mellet@us.es
[**] This work was supported by the CNRS (UMR 7509), the Centre
International de Recherche aux Frontiꢀres de la Chimie (FRC), the
Agence National de la Recherche (ANR, grant number 08JC-0094-
01), the Spanish Ministerio de Ciencia e Innovaciꢄn (contract
numbers CTQ2007-61180/PPQ), the Fundaciꢄn Ramꢄn Areces and
the Junta de Andalucꢃa, and doctoral fellowships from the French
Department of Research to C.D. and J.I. We further thank A. Schifrin
and I. Pfeifer for assistance with synthetic work, Dr. D. Rodriguez-
Lucena for helpful comments, and M. Schmitt for NMR measure-
ments.
ꢀ
pot process is that aldehyde oxidation and C N bond
formation are performed in a single synthetic step. Oxidation
of the hydroxy group at C5 followed by intramolecular
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201002802.
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Communications
Scheme 1. a) aq NH3 (30%), I2, THF, 16 h, 78%; b) DMSO, Ac2O;
c) NaBH3CN, HCOOH, MeCN, 808C, 60% over two steps; d) LAH,
THF, reflux, 90%; e) Br(CH2)6N3, Et3N, 4-dimethylaminopyridine
(DMAP), DMF, 1208C, 38% (51% based on recovered starting
material); f) BCl3, CH2Cl2, ꢀ608C!08C, 77%. g) Pent-1-yne, sodium
ascorbate, CuSO4·5H2O, DMF/H2O (1:1), 61%.
reductive amination afforded the expected lactam 3. Reduc-
tion with lithium aluminium hydride (LAH) and subsequent
alkylation of the corresponding amine by using 1-azido-6-
bromo-hexane afforded the O-benzylated iminosugar 4.
Selective cleavage of the benzyl protecting groups of 4
without interfering with the azide moiety was performed
successfully by treatment with an excess of BCl3 in CH2Cl2 at
low temperatures (ꢀ60–08C).[20] Reaction of the resulting
compound 5[21] with 1-pentyne in the presence of
CuSO4·5H2O and sodium ascorbate gave the derivative 6,
which was used as the monovalent model compound in the
inhibition assay.
The synthesis of fullerene iminosugar ball 8 is shown in
Scheme 2. Compound 7[6] was desilylated in situ with tetra-n-
butylammonium fluoride (TBAF) to form the corresponding
hexaadduct bearing 12 terminal alkyne units, with which the
azide precursor 5 was subsequently reacted. The functional-
ized fullerene 8 was thus obtained in 83% yield. The chemical
structure of compound 8 was confirmed by its 1H and
13C NMR spectra, which show the expected signals for the
12 equivalent peripheral iminosugar residues as well as the
characteristic features of the fullerene hexakis-adduct core
(see the Supporting Information). To further confirm the
structure of compound 8, mass spectra (MALDI-TOF and
ESI-MS) were recorded under different conditions. However,
as already observed in the case of related fullerene sugar
balls,[7] a high level of fragmentation prevented the observa-
tion of the expected molecular ion peak. This difficulty
prompted us to prepare compound 9 by reaction of the
fullerene derivative 7 with the benzylated iminosugar 4
Scheme 2. a) 5, TBAF, sodium ascorbate, CuSO4·5H2O, DMF/H2O
(1:1), 83%; b) 4, TBAF, sodium ascorbate, CuSO4·5H2O, CH2Cl2/H2O
(1:1), 78%.
compounds 8 and 9 show the characteristic features of
fullerene hexaadducts.[5]
Fullerene iminosugar 8 and its monovalent analogue 6
were tested against a range of commercially available
glycosidases and their inhibition constants (Ki) were eval-
uated (Table 1). The biological results may be divided
unevenly into three categories with regard to inhibition:
decreased, unaffected, and increased by multivalency. Only
one example of a significant affinity decrease (ninefold)
compared to the monovalent iminosugar was observed for
sweet almond b-glucosidase. For two other glucosidases
(amyloglucosidase and bovine liver b-glucosidase), the multi-
valent and monovalent iminosugars showed similar Ki values.
1
(Scheme 2). The H and 13C NMR spectra of compound 9
show the same characteristic features that were observed for
unprotected compound 8 (see the Supporting Information).
MALDI-TOF mass spectrometry also confirmed the struc-
ture of fullerene derivative 9. The level of fragmentation was
less dramatic in this case and the molecular ion peak could be
clearly observed at m/z 9911.02 ([M + H]+, calculated for
C
618H659N48O72: 9911.12). Finally, the UV/Vis spectra of
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Angewandte
Chemie
Table 1: Glycosidase inhibitory activities Ki.[a]
of mannojirimycin show little if any inhibition of mannosi-
dases.[13]
Further research will certainly be needed for the ration-
Enzyme
Monovalent
Multivalent
Relative inhibition
iminosugar 6 iminosugar 8 potency of 8 over 6[b]
alization of the significant binding enhancements observed
for glycosidase inhibition with multivalent inhibitors. When
considered on an inhibitor residue basis, our results unequiv-
ocally evidence the existence of a multivalent effect beyond
the expected statistical rebinding and local concentration
effect. A sliding mechanism of the iminosugars in the enzyme
active site or an additional binding at a close allosteric site
may account for the distinct specificity enhancement
observed.[9,11,22] Beyond its fundamental interest, our work
shows that, in addition to the modulation of selectivity,
multivalency is a promising tool for the design of potent
glycosidase inhibitors.
b-galactosidase
Bovine liver
262
34
7.7 (0.64)
a-galactosidase
Aspergillus niger
Green coffee
NI[c]
NI[c]
NI[c]
84
–
>23 (>1.9)
b-glucosidase
Almonds (pH 7.3) 11
Bovine liver
95
247
0.1 (0.01)
1.9 (0.16)
482
a-glucosidase
Amyloglucosidase 0.71
(Asp. Niger)
0.69
1.0 (0.08)
Baker’s yeast
Isomaltase
(Baker’s yeast)
152
943
18
10.5
8.7 (0.72)
89.8 (7.48)
Received: May 8, 2010
Published online: July 7, 2010
Keywords: enzymes · fullerenes · iminosugars · inhibitors ·
multivalency
Naringinase
Penicillium
decumbens
.
9.1
0.41
22.2 (1.84)
b-mannosidase
Helix pomatia
NI[c]
322
NI[c]
0.15
–
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