Optimizing saccharide-directed molecular delivery to biological receptors: Design, synthesis, and biological evaluation of glycodendrimer-cyclodextrin conjugates

Article abstract of DOI:10.1021/ja047864v

Dendritic β-cyclodextrin (βCD) derivatives bearing multivalent mannosyl ligands have been prepared and assessed for their binding efficiency toward the tetrameric plant lectin concanavalin A (Con A) and a mammalian mannose/fucose specific cell surface receptor from macrophages. The synthetic strategy exploits the reactivity between isothiocyanate and amine functionalities for the high-yielding assembly via thioureido links of the various building blocks, including host, spacer, branching, and carbohydrate ligand elements. The methodology has been applied to the preparation of a series of βCD-polymannoside scaffolds differing in the ligand valency and geometry. This series allowed us to explore: (i) The effects of the glycodendritic architecture on the binding efficiency; (ii) the mutual influence between the cyclodextrin core and the glycodendritic moieties on the molecular inclusion and lectin-binding properties; and (iii) the consequence of inclusion complex formation, using the anticancer drug docetaxel (Taxotere) as a target guest, on biological recognition. Our results confirm the high drug solubilization capability of this new type of βCD-dendrimer construct and indicate that subtle changes in the architecture of the conjugate may have important consequences on receptor affinity. Interestingly, the host-guest interaction can be monitored to build up supramolecular dynamic glycoclusters with increased lectin affinity. Alternatively, the information obtained from the structure-lectin-binding avidity-inclusion capability studies has been put forward in the design of very efficient molecular transporters for docetaxel based on glycodendritic CD dimers.

Full text of DOI:10.1021/ja047864v

Published on Web 08/04/2004
Optimizing Saccharide-Directed Molecular Delivery to
Biological Receptors: Design, Synthesis, and Biological
Evaluation of Glycodendrimer-Cyclodextrin Conjugates
Juan M. Benito,† Marta Go´mez-Garc´ıa,‡ Carmen Ortiz Mellet,*,‡
Isabelle Baussanne,§ Jacques Defaye,*,§ and Jose´ M. Garc´ıa Ferna´ndez*,†
Contribution from the Instituto de InVestigaciones Qu´ımicas, CSIC, Ame´rico Vespucio 49, Isla
de la Cartuja, E-41092 SeVilla, Spain, Departamento de Qu´ımica Orga´nica, Facultad de
Qu´ımica, UniVersidad de SeVilla, Aptdo. 553, E-41071 SeVilla, Spain, and CNRS - UniVersite´
Joseph Fourier-Grenoble 1, De´partement de Pharmacochimie Mole´culaire (UMR 5063, FR 2607
ICMG), BP 138, F-38243 Meylan, France
Received April 14, 2004; E-mail: jogarcia@iiq.csic.es; mellet@us.es; jacques.defaye@ujf-grenoble.fr
Abstract: Dendritic â-cyclodextrin (âCD) derivatives bearing multivalent mannosyl ligands have been
prepared and assessed for their binding efficiency toward the tetrameric plant lectin concanavalin A (Con
A) and a mammalian mannose/fucose specific cell surface receptor from macrophages. The synthetic
strategy exploits the reactivity between isothiocyanate and amine functionalities for the high-yielding
assembly via thioureido links of the various building blocks, including host, spacer, branching, and
carbohydrate ligand elements. The methodology has been applied to the preparation of a series of âCD-
polymannoside scaffolds differing in the ligand valency and geometry. This series allowed us to explore:
(i) The effects of the glycodendritic architecture on the binding efficiency; (ii) the mutual influence between
the cyclodextrin core and the glycodendritic moieties on the molecular inclusion and lectin-binding properties;
and (iii) the consequence of inclusion complex formation, using the anticancer drug docetaxel (Taxote`re)
as a target guest, on biological recognition. Our results confirm the high drug solubilization capability of
this new type of âCD-dendrimer construct and indicate that subtle changes in the architecture of the
conjugate may have important consequences on receptor affinity. Interestingly, the host-guest interaction
can be monitored to build up supramolecular dynamic glycoclusters with increased lectin affinity. Alternatively,
the information obtained from the structure-lectin-binding avidity-inclusion capability studies has been
put forward in the design of very efficient molecular transporters for docetaxel based on glycodendritic CD
dimers.
Multivalent interactions between carbohydrate-binding pro-
teins (lectins) and the oligosaccharide moieties of glycoprotein
and glycolipid components of extracellular matrices and cell
surfaces are involved in extensive cellular recognition processes
including development, differentiation, morphogenesis, fertiliza-
tion, the immune response, implantation, cell migration, and
cancer metastasis.1 The high specificity of oligosaccharide-
lectin interactions has already been exploited in the development
of multivalent sugar-based therapeutic agents2 designed to
interfere with carbohydrate molecular recognition3 and carbo-
hydrate-based anticancer drugs.4 The targeted aggregation and
clearance of pathogenic species is another promising application
of this concept.5 The synthesis and characterization of com-
pounds that not only possess multivalent carbohydrate recogni-
tion sites, but also the potential to act as carriers of drugs, is
gaining an increased amount of attention. Glycoligands with
(2) Multivalency is a prerequisite to attain biologically useful affinities between
carbohydrate ligands and their protein receptors. Presentation of the sugar
epitopes as multiple copies on an appropriate scaffold (molecular, dendritic,
polymeric) creates a multivalent display that can efficiently mimic the nature
mode of affinity enhancement, resulting in higher affinities than expected
from the addition of the individual interactions. For selected literature on
this concept, termed generically the cluster effect, see: (a) Kiessling, L.
L.; Pontrello; Schuster, M. C. Synthetic Multivalent Carbohydrate Ligands
as Effectors or Inhibitors of Biological Processes. In Carbohydrate-based
Drug DiscoVery; Wong, C.-H., Ed.; Wiley-VCH: Weinheim, Germany,
2003; Vol. 2, pp 575-608. (b) Lundquist, J. J.; Toone, E. J. Chem. ReV.
2002, 102, 555-578. (c) Lee, R. T.; Lee, Y. C. Glycoconjugate J. 2000,
17, 543-551. (d) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew.
Chem., Int. Ed. 1998, 37, 2754-2794. (e) Lee, Y. C.; Lee, R. T. Acc. Chem.
Res. 1995, 28, 321-327.
† Instituto de Investigaciones Qu´ımicas, CSIC.
(3) (a) Fan, E.; Zhang, Z.; Minke, W. E.; Hou, Z.; Verlinde, C. L. M. J.; Hol,
W. G. J. J. Am. Chem. Soc. 2000, 122, 2663-2664. (b) Kitov, P. I.;
Sadowska, J. M.; Mulvey, G.; Armstrong, G. D.; Ling, H.; Pannu, N. S.;
Read, R. J.; Bundle, D. R. Nature 2000, 403, 669-672.
‡ Universidad de Sevilla
§ CNRS and Universite´ Joseph Fourier.
(1) (a) Dam, T. K.; Brewer, C. F. Chem. ReV. 2002, 102, 387-429. (b) Bertozzi,
C. R.; Kiessling, L. L. Science 2001, 291, 2357-2364. (c) Koeller, K. M.;
Wong, C.-H. Nat. Biotechnol. 2000, 18, 835-841. (d) Sears, P.; Wong,
C.-H. Angew. Chem., Int. Ed. 1999, 38, 1875-1917. (e) Singh, R. S.;
Tiwary, A. K.; Kennedy, J. F. Crit. ReV. Biotechnol. 1999, 19, 145-178.
(f) Lis, H.; Sharon, N. Chem. ReV. 1998, 98, 637-674. (g) Dwek, R. Chem.
ReV. 1996, 96, 683-720. (h) Gabius, H.-H.; Siebert, H.-C.; Andre´, S.;
Jime´nez-Barbero, J.; Ru¨diger, H. ChemBioChem 2004, 5, 740-764.
(4) (a) Roy, R.; Baek, M.-G. ReV. Mol. Biotechnol. 2002, 90, 291-309. (b)
Allen, J. R.; Harris, C. R.; Danishefsky, S. J. J. Am. Chem. Soc. 2001,
123, 1890-1897. (c) Danishefsky, S. J.; Allen, J. R. Angew. Chem., Int.
Ed. 2000, 39, 836-863. (d) Hakomori, S.; Zang, Y. Chem. Biol. 1997, 4,
97-104.
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9
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J. AM. CHEM. SOC. 2004, 126, 10355-10363
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A R T I C L E S
Benito et al.
this added value could act as “intelligent” vectors for site-
specific delivery of therapeutics. For this purpose, various
components, which are designed for encapsulation, solubiliza-
tion, stabilization, targeting, reduction of immunogenicity and
toxicity, and pharmacological activity, among others, have to
be combined in a sophisticated manner. Some of the frequently
used methods include conjugation of carbohydrate ligands with
proteins or other polymers, polymerization of glycosylated
monomers, and formation of liposomes using glycolipids or
neoglycolipids.6 Although these approaches have been successful
to some extent, the products thus obtained are generally
ambiguous in composition and structure, which may be trouble-
some regarding accurate reproducibility of the biological results.
Moreover, this heterogeneity frequently impairs a systematic
study of the consequences of structural modifications of the
different components of the system in the final properties.
Grafting biorecognizable saccharide epitopes onto macrocy-
clic molecular hosts to produce homogeneous compounds with
a well-defined structure and a precise number of carbohydrate
ligands is an interesting alternative for receptor-mediated
glycotargeting.7 Cyclodextrins (cyclomaltooligosaccharides, CDs)
offer unique features toward this goal because they are es-
sentially nonimmunogenic carbohydrates with inherent low
pharmacological activity and high biocompatibility, and a variety
of selectively functionalized derivatives are accessible through
recent methodologies for manipulation of their polyfunctional
framework. They are well known to encapsulate various organic
molecules of appropriate size within their truncated cone-shaped
hydrophobic cavity to afford host-guest supramolecular species
in aqueous solution, a property that has already been exploited
in pharmaceutical applications.8 Multivalent cyclodextrin con-
jugates may, therefore, form ternary lectin-carbohydrate ligand-
guest complexes through simultaneous host-guest complexation
at the CD cavity and carbohydrate-protein interaction at the
carbohydrate ligand moiety (Figure 1).
Figure 1. Schematic representation of cyclodextrin-glycodendrimer
conjugates considered in this study. Polysubstituted derivatives bearing
multiple biorecognizable saccharide ligands show better lectin-binding
properties due to the multivalent effect; yet, monosubstituted conjugates
exhibit superior inclusion capabilities. CD-glycodendrimer architectures
are now proposed to combine both favorable features.
conclusions stem from the literature data: (i) the CD scaffold
is particularly well-suited for glycocluster-lectin interaction
studies; (ii) polyglycosylated structures exhibit higher affinities
toward specific lectins than do monosubstituted structures, as
expected from the cluster effect; and (iii) polysubstitution may,
however, seriously impair inclusion and complex stabilization
of potential guests as a result of limiting access to the
hydrophobic cavity, lack of stabilizing interactions of the
included guest, and distortion of the cavity due to steric
hindrance.9 Our own results indicated an about 10-fold decrease
in the solubilization capability of hydrophobic guests in water
for branched âCDs upon (C-6)-heptafunctionalization.11 Ideal
CD carriers for drug targeting should combine both the
advantages of monosubstitution with regard to efficient inclusion
capabilities and a multivalent display of the required saccharide
epitope to comply with the need of high biological receptor
binding efficiency. We assumed that this goal could be achieved
by conjugating a monosubstituted CD host moiety and a
glycodendritic domain12 (Figure 1). We have now developed
an efficient preparation of dendritic wedges suitable for both
external carbohydrate coating and covalent attachment to the
Several examples of oligosaccharide-branched cyclodextrins
have been reported in the past few years.9,10 Three main
(6) (a) Yamazaki, N.; Kojima, S.; Bovin, N. V.; Andre´, S.; Gabius, S.; Gabius,
H.-J. AdV. Drug DeliVery ReV. 2000, 43, 225-244. (b) Vyas, S. P.; Sihorkar,
V. AdV. Drug DeliVery ReV. 2000, 43, 249-271. (c) Forssen, E.; Willis,
M. AdV. Drug DeliVery ReV. 1998, 29, 249-271. (d) R´ıhova´, B. AdV. Drug
DeliVery ReV. 1998, 29, 273-289. (e) Rice, K. G. In Glycosciences: Status
and PerspectiVes; Gabius, H.-J., Gabius, S., Eds.; Chapman & Hall:
London, 1997; pp 471-483.
(7) (a) Aoyama, Y. Chem.-Eur. J. 2004, 10, 588-593. (b) Sansone, F.; Chierici,
E.; Casnati, A.; Ungaro, R. Org. Biomol. Chem. 2003, 1, 1802-1809. (c)
Casnati, A.; Sansone, F.; Ungaro, R. Acc. Chem. Res. 2003, 36, 246-254.
(d) Fulton, D. A.; Stoddart, J. F. Bioconjugate J. 2001, 12, 655-672. (e)
Fujimoto, K.; Miyata, T.; Aoyama, Y. J. Am. Chem. Soc. 2000, 122, 3558-
3559. (f) Roy, R.; Kim, J. M. Angew. Chem., Int. Ed. 1999, 38, 6767-
6770. (g) Aoyama, Y.; Masuda, Y.; Chuleeraruk, J.; Nishiyama, K.;
Fujimoto, K.; Fujimoto, T.; Shimizu, T.; Hayashida, O. Pure Appl. Chem.
1998, 70, 2379-2384.
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359. (b) Uekama, K.; Hirayama, F.; Irie, T. Chem. ReV. 1998, 98, 1741-
2076. (c) Fro¨mming, K.-H. In ComprehensiVe Supramolecular Chemistry;
Atwood, J. L., Davies, J. E. D., McNicol, D. C., Vo¨gtle, F., Eds.;
Pergamon: Oxford, 1996; Vol. 3, pp 57-188. (d) Loftsson, T.; Brewster,
M. E. J. Pharm. Sci. 1996, 85, 1017-1025. (e) Sejtli, J. Med. Res. ReV.
1994, 14, 353-386. (f) Wenz, G. Angew. Chem., Int. Ed. Engl. 1994, 33,
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(9) For a review, see: Ortiz Mellet, C.; Defaye, J.; Garc´ıa Ferna´ndez, J. M.
Chem.-Eur. J. 2002, 8, 1982-1990.
(11) (a) Ortiz Mellet, C.; Benito, J. M.; Garc´ıa Ferna´ndez; J. M.; Law, H.;
Chmurski, K.; Defaye, J.; O’Sullivan, M. L.; Caro, H. N. Chem.-Eur. J.
1998, 4, 2523-2531. (b) Baussanne, I.; Benito, J. M.; Ortiz Mellet, C.;
Garc´ıa Ferna´ndez; J. M.; Defaye, J. ChemBioChem 2001, 2, 777-783.
(12) For recent reviews on carbohydrate-coated dendrimers, see: (a) Cloninger,
M. J. Curr. Opin. Chem. Biol. 2002, 6, 742-748. (b) Turnbull, W. B.;
Stoddart, J. F. ReV. Mol. Biotechnol. 2002, 90, 231-255. (c) Lindhorst, T.
K. Top. Curr. Chem. 2002, 218, 201-235. (d) Bezouska, K. ReV. Mol.
Biotechnol. 2002, 90, 269-290. (e) Nepogodiev, S. A.; Stoddart, J. F. AdV.
Macromol. Carbohydr. Res. 2003, 2, 191-293.
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S.-I.; Gabius, H.-J. Bioconjugate Chem. 2004, 15, 87-98. (b) Ortega-
Caballero, F.; Gime´nez-Mart´ınez, J. J.; Vargas-Berenguel, A. Org. Lett.
2003, 5, 2389-2392. (c) Yockot, D.; Moreau, V.; Demailly, G.; Djeda¨ıni-
Pilard, F. Org. Biomol. Chem. 2003, 1, 1810-1818. (d) Vargas-Berenguel,
A.; Ortega-Caballero, F.; Santoyo-Gonza´lez, F.; Garc´ıa-Lo´pez, J. J.;
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Glycodendrimer−Cyclodextrin Conjugates
A R T I C L E S
â-cyclodextrin (âCD) core,13 the most readily available CD
representative. A critical advantage of the methodology is that
it allows sampling compounds with varied, yet perfectly defined,
structural characteristics. In a first series of compounds, we
explored the effect of the presence of the CD host moiety on
receptor binding as a function of the overall shape of the system
and of the density and valency of the receptor-binding elements.
The model lectin we studied for this purpose is the tetrameric
plant lectin concanavalin A (Con A),14 that specifically recog-
nizes R-D-mannopyranosyl epitopes. The binding affinity was
evaluated by enzyme-linked lectin assay (ELLA),15 which
provides information on the intrinsic lectin-ligand affinity,
devoid of aggregation effects.2b,15a
Chart 1. Building Blocks Used To Build â-Cyclodextrin-Centered
Mannosyl-Coated Glycodendrimers
For the most efficient ligands in the above study, the influence
of CD complex formation on lectin affinity was regarded using
docetaxel (DTX, Taxote`re) - one of the most successful agents
currently in use for the chemotherapy of breast and ovarian
cancer16 - as the guest molecule. The drug carrier potential
was further investigated using macrophages expressing the well-
characterized mannose/fucose specific receptor,17 a lectin of
interest as a therapeutic target, at their surface. The information
obtained from structure-lectin-binding affinity-inclusion ca-
pability studies, which pointed for guest-induced clusterization
effects, may be exploited in the design of tailor-made molecular
transporters. As a rationale of this concept, we further report
here the extension of the CD-dendrimer approach to the
preparation of multitopic glycodendrimer-CD dimer conjugates
designed to form very strong sandwich-type complexes with
Taxote`re while retaining optimal biological receptor recognition
properties.
spacer, by using 6-azidohexanoyl chloride21 (6) as a bifunctional
bridging segment, has also been considered to ensure the
accessibility of the grafted bioactive components to molecular
recognition events. Alternatively, methyl 6-amino-6-deoxy-R-
D-glucopyranoside22 (7), a convenient surrogate for the substi-
tuted glucopyranosyl subunit in monofunctionalized cyclodextrin
derivatives, was used as the core element instead of 1 for
comparative purposes (Chart 1). The coupling methodology
exploits the connectivity of isothiocyanate and amine-function-
alized monomers to give thiourea adducts,23 taking advantage
of the efficient transformation of the terminal azido group in
the spacer element into an isothiocyanate functionality through
a tandem Staudinger-aza-Wittig-type reaction with triph-
enylphosphine and carbon disulfide.24 The amide bridging
reaction was also used to couple the spacer element to the
dendritic moiety.
Results and Discussion
Synthetic Strategy. To build the typical tree structure of
dendrimers onto the primary hydroxyl rim of the âCD torus, a
set of building blocks that can be combined in an iterative and
modular manner with a relatively low synthetic cost has been
devised. The key templates are 6I-amino-6I-deoxy-âCD (1), for
which an improved synthetic route has been recently elabo-
rated,13 the selectively protected 1,2,3-triaminopropane branch-
ing element 2,18 and the isothiocyanate-functionalized R-D-
mannopyranosyl derivatives 319 and 5. The latter was prepared
by reaction of the known tris(R-D-mannopyranosyloxymethyl)-
methylamine 420 with thiophosgene. Intercalation of a six-carbon
(13) Baussanne, I.; Benito, J. M.; Ortiz Mellet, C.; Garc´ıa Ferna´ndez, J. M.;
Defaye, J. Chem. Commun. 2000, 1489-1490.
(14) (a) Bouckaert, J.; Hamelryck, T.; Wyns, L.; Loris, R. Curr. Opin. Struct.
Biol. 2001, 11, 635-643. (b) Vijayan, M.; Chandra, N. Curr. Opin. Struct.
Biol. 1999, 9, 707-714.
Both convergent and divergent approaches have been imple-
mented for the preparation of this new type of hybrid neogly-
(15) Selected examples of lectin-binding affinity evaluation by ELLA: (a)
Corbell, J. B.; Lundquist, J. J.; Toone, E. J. Tetrahedron: Asymmetry 2000,
11, 95-111. (b) Roy, R.; Page´, D.; Figueroa Pe´rez, S.; Verez Bencomo,
V. Glycoconjugate J. 1998, 15, 251-263. (c) Page´, D.; Roy, R. Glyco-
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Med. Chem. 1996, 4, 1949-1961.
(20) Ashton, P. R.; Hounsell, E. F.; Jayaraman, N.; Nilsen, T. M.; Spencer, N.;
Stoddart, J. F.; Young, M. J. Org. Chem. 1998, 63, 3429-3437.
(21) Charon, D.; Mondange, M.; Pons, J.-F.; Blay, K. L.; Chaby, R. Bioorg.
Med. Chem. 1998, 6, 755-765.
(22) Garc´ıa Ferna´ndez, J. M.; Ortiz Mellet, C.; Fuentes, J. J. Org. Chem. 1993,
58, 5192-5199.
(23) For a recent review on carbohydrate-derived thioureas, see: (a) Garc´ıa
Ferna´ndez, J. M.; Ortiz Mellet, C. AdV. Carbohydr. Chem. Biochem. 2000,
55, 35-135. Recent examples of glycosylthiourea-coated glycoclusters: (b)
Andre´, S.; Pieters, R. J.; Vrasidas, I.; Kaltner, H.; Kuwabara, I.; Liu, F.-
T.; Liskamp, R. M. J.; Gabius, H.-J. ChemBioChem 2001, 2, 822-830. (c)
Vrasidas, I.; de Mol, N. J.; Liskamp, R. M. J.; Pieters, R. J. Eur. J. Org.
Chem. 2001, 4685-4692. (d) Dubber, M.; Lindhorst, T. K. Org. Lett. 2001,
3, 4019-4022. (e) Krist, P.; Vannucci, L.; Kuzma, M.; Man, P.; Sadalapure,
K.; Patel, A.; Bezouska, K.; Posp´ısil, M.; Petrus, L.; Lindhorst, T. K.; Kren,
V. ChemBioChem 2004, 5, 445-452.
(24) For a recent example of this transformation in the carbohydrate field, see:
Garc´ıa-Moreno, M. I.; D´ıaz-Pe´rez, P.; Benito, J. M.; Ortiz Mellet, C.;
Defaye, J.; Garc´ıa Ferna´ndez, J. M. Carbohydr. Res. 2002, 337, 2329-
2334.
(16) (a) Potier, P. Chem. Soc. ReV. 1992, 21, 113-119. (b) Fitzpatrick, F. A.;
Wheeler, R. Int. Immunopharmacol. 2003, 3, 1699-1714.
(17) (a) Wileman, T. E.; Lennartz, M. R.; Stahl, P. D. Proc. Natl. Acad. Sci.
U.S.A. 1986, 83, 2501-2505. (b) Taylor, M. E.; Conary, J. T.; Lennartz,
M. R.; Stahl, P. D.; Drickamer, K. J. Biol. Chem. 1990, 265, 12156-12162.
(c) Taylor, M. E. Glycobiology 1997, 7, v-viii. (d) Feinberg, H.; Park-
Snyder, S.; Kolatkar, A. R.; Heise, C. T.; Taylor, M. E.; Weiss, W. I. J.
Biol. Chem. 2000, 275, 21539-21548. (e) Nappaer, C. E.; Dyson, M. H.;
Taylor, M. E. J. Biol. Chem. 2001, 276, 14759-14766.
(18) Benoist, E.; Loussouarn, A.; Remaud, P.; Chatal, J.-C.; Gestin, J.-F.
Synthesis 1998, 1113-1118.
(19) (a) Camarasa, M. J.; Ferna´ndez-Resa, P.; Garc´ıa-Lo´pez, M. T.; de las Heras,
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9
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A R T I C L E S
Benito et al.
Scheme 1. Synthesis of Isothiocyanate-Armed Polymannoside
Dendrons
Scheme 2. Synthesis of Precursors for Divergent Preparations of
âCD-Glycodendrimer Conjugates
feasible,25 we encountered that purification, usually the most
delicate step in the preparation of such complex molecules, is
much more easily performed at this stage. Base-catalyzed
removal of the acetyl protecting groups afforded the target fully
unprotected compounds 27-32 in high purity and virtually
quantitative yield.26 Gel permeation chromatography of the crude
product allowed isolation of analytically pure samples that were
used in the biological evaluation tests. The monovalent ligand
27 and the model conjugates 33-38 served as control com-
pounds in the binding studies (Chart 2).
The structure of the new âCD conjugates was ascertained
from their microanalytical, FAB, or MALDI-TOF mass spec-
trometry and NMR data. The 1H and 13C NMR spectra recorded
at room temperature showed the typical line broadening
associated with restricted rotation at the pseudoamide NH-
(CdS) bonds.23a Line broadening was still evident at higher
temperatures (50-70 °C) for compounds having multiple
thiourea groups. Moreover, the unsymmetrical nature of the
monosubstituted âCD moiety and the occurrence of diastereoto-
pism at the carbon-centered branching points resulted in
extensive signal overlapping. Resonance attribution was ac-
complished by combining 1D and 2D NMR experiments and
comparison of the spectra with data for the corresponding methyl
R-D-glucopyranoside-derived model conjugates. Thus, the 13C
NMR resonances at 184-183 ppm (δCdS) and the high field
chemical shifts for the substituted primary hydroxyl carbon atom
C-6I, as well as for the N-linked carbon atoms in the spacer
and branching elements, confirmed the presence of the thiourea
tethers. The signals for C-4I and C-5I exhibited characteristic
low-field and high-field shifts, respectively, as compared to the
corresponding signals of the unsubstituted sugar units. The
resonances for the anomeric carbon atoms (δC-1′) of the coating
monosaccharides were found at 80-82 ppm for manno-
pyranosylthiourea units and 99-100 ppm for O-linked manno-
pyranosides (see Figure 2).
coconjugates. In the convergent approach, defined glycoden-
drons bearing a terminal isothiocyanate group (11, 13, 15, and
17) were used to generate the multivalent mannosyl displays.
Variation in the number and density of the mannose residues
was achieved by judicious combination of the different building
blocks as indicated in Scheme 1. The divergent approach started
from the thiourea bridging reaction between the host element 1
(or model core 7) and the spacer-armed branching element 18.
Acid-catalyzed hydrolysis of the Boc protecting groups in the
corresponding adduct 19 (f20) generated two amine function-
alities suitable for further nucleophilic addition to isothiocyanate-
functionalized glycodendrons (Scheme 2). Either of these two
routes led, after the final assembly of the mannosyl ligands and
the âCD (or model core) fragments, to the corresponding
hemiacetylated conjugates 21-26 (Chart 2) in satisfactory
overall yield. Albeit a synthesis without protecting groups is
Con A Binding Efficiency. The affinity of the prepared âCD
conjugates 27-32 toward Con A was measured by the enzyme-
linked lectin assay (ELLA).15 In addition, the analogous
polymannosides in which the âCD core has been replaced by a
single R-D-glucopyranoside subunit 33-38 were tested in the
binding studies to ascertain the influence of the CD moiety on
the protein-ligand recognition event. ELLA measures the ability
of a soluble saccharide to inhibit the association between a
labeled lectin and a polymeric ligand attached to the microtiter
(25) For an example on the preparation of carbohydrate-coated glycodendrimers
using unprotected sugar isothiocyanate conjugates, see: Kieburg, C.;
Lindhorst, T. K. Tetrahedron Lett. 1997, 38, 3885-3888.
(26) It should be noted that deacetylation must be conducted at 0 °C in the case
of conjugates bearing mannopyranosyltioureido substituents (21, 22, 25,
33, 34, and 37) to avoid undesired anomerization reactions. See: Benito,
J. M.; Ortiz Mellet, C.; Sadalapure, K.; Lindhorst, T. K.; Defaye, J.; Garc´ıa
Ferna´ndez, J. M. Carbohydr. Res. 1999, 320, 37-48.
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10358 J. AM. CHEM. SOC. VOL. 126, NO. 33, 2004

Glycodendrimer−Cyclodextrin Conjugates
A R T I C L E S
Chart 2. Chemical Structures of the âCD-Glycodendrimer Conjugates Prepared in This Study and of the Corresponding Model
Compounds in Which the âCD Host Moiety Has Been Replaced by a Methyl R-D-Glucopyranosyl Subunit
Table 1. Results from the Binding Inhibition of Horseradish
Peroxidase-Labeled Con A to Yeast Mannan by 6I-Mannosylated
âCD-Dendrimer and Methyl R-D-Glucopyranoside-Dendrimer
Conjugates
valency
IC50 (µM)
relative efficiencya
27
33
28
34
29
30
35
36
31
37
32
38
1
1
2
2
3
3
3
3
4
4
6
6
800
1360
780
710
91
180
190
186
95
1.7 (1.7)
1.0 (1.0)
1.75 (0.87)
1.9 (0.96)
14.9 (4.98)
7.6 (2.52)
7.2 (2.39)
7.3 (2.44)
14.3 (3.58)
12.4 (3.09)
136 (22.7)
113 (18.8)
110
10
12
Figure 2. 13C NMR spectrum (125.7 MHz, D2O, 343 K) of the hexavalent
âCD-dendrimer conjugate 32 showing atom notation and diagnostic signals.
a Taking the value for methyl 6-deoxy-6-(N′-R-D-mannopyranosylthio-
ureido)-R-D-glucopyranoside 33 (1360 µM) as the reference. Values in
parentheses are valency-corrected.
well (horseradish peroxidase-labeled Con A and yeast mannan,
respectively, in the present case). The concentration needed to
achieve 50% inhibition (IC50) is then assumed to be inversely
proportional to the lectin-saccharide binding free energy. The
results are collected in Table 1.
basis for hexavalent as compared to monovalent derivatives),
indicative of a strong cluster effect.2 The Con A binding capacity
of the monosubstituted hexavalent âCD conjugate 32 (IC50
)
10 µM) actually surpassed that previously encountered for per-
(C-6)-substituted âCD heptamannoside derivatives (IC50 > 70
µM),11b thus illustrating the strong dependence of carbohydrate-
lectin recognition processes upon the presentation mode of
Effect of Valency and Density. The corresponding IC50
values for inhibition of Con A-yeast mannan binding (Table
1) reflected the expected amplification of lectin-binding strength
for the higher-valent representatives (up to 22-fold in a molar
9
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A R T I C L E S
Benito et al.
receptor-binding epitopes in multivalent ligands.27 Notwith-
standing, virtually no difference in binding efficiency was
detected between mono- and divalent or between tri- and
tetravalent ligands. In fact, from the present data it may be
inferred that the observed affinity increases are mainly associated
with incorporation of triads of mannopyranosyl ligands, that
is, a density effect. Because, presumably, ELLA provides
information on the interaction between the glycoclusters and a
single binding site in the lectin, without contributions from
chelating or cross-linking effects,28 it can be concluded that the
observed density effect operates at the level of the so-called
microcluster effect.29 While optimizing the macrocluster effect
is of great importance when looking for inhibitors or effectors
of particular biological functions, optimizing the intrinsic affinity
for a biological receptor may be particularly relevant for
targeting purposes. This microcluster effect can reach biologi-
cally relevant values at a relatively low number of binding
motifs, but is very sensitive to structural (orientational, density,
geometrical) considerations.30 Although the mechanism by
which it operates is still unclear,31 our data highlight the
importance of flexible, diversity-oriented synthetic strategies for
the identification and optimization of good ligand candidates.
Effect of the Host in Lectin Binding. Comparison of ELLA
data for âCD (27-32) and methyl R-D-mannopyranoside
conjugates (33-38) allows the evaluation of the impact of the
presence of the host moiety in lectin binding. No significant
differences were observed between both series of compounds
when the six-carbon spacer element was present in the structure.
However, the IC50 values for the monovalent and trivalent
adducts 27 and 29 (800 and 91 µM, respectively), in which the
carrier and the mannosyl ligand are directly linked through a
thiourea group, were indicative of binding efficiencies 85-90%
higher as compared to the homologous methyl R-D-glucopyra-
noside derivatives 33 and 35 (IC50 values 1360 and 180 µM,
respectively). This must be essentially ascribed to an additional
stabilizing interaction involving the cyclic heptasaccharide
carrier. It is probable that once the mannosyl ligand has been
accommodated in the Con A recognition site, the âCD
framework is close enough to interact with residues at the protein
surface. A similar situation has been found in the complex
formed by a âCD-gastrin conjugate and the human CCK-B
Figure 3. Inhibition of binding of horseradish peroxidase-labeled Con A
to yeast mannan by increasing concentrations of the monosubstituted
trivalent mannose-âCD conjugates 29 and 30 (dotted lines) and of their
corresponding inclusion complexes with docetaxel (solid lines). The âCD
conjugate-docetaxel molar ratio was 5:1.
receptor.32 Comparison of ELLA data for 29 and 30, differing
exclusively in the presence of the six-carbon spacer, unequivo-
cally shows that this interaction is dependent on the proximity
between the CD host and the biorecognizable saccharide
epitopes. The contribution of the âCD-Con A interaction to
the total free energy of binding can be estimated, from the
comparative IC50 values,15b at about -0.35 kcal mol-1 under
the particular conditions of the ELLA test. This result may be
relevant as the CD-receptor interaction might involve the CD
cavity, inducing the release of an included guest just after
delivery to the target site.
Drug Carrier Capabilities. The prepared cyclodextrin
conjugates exhibited extremely high water solubilities, above
20-fold higher as compared to the parent âCD (15 mM).
Moreover, docetaxel solubilization experiments in water showed
solubility values similar to those obtained previously for
monovalent CDs, significantly higher than those reported for
per-(C-6)-substituted derivatives.11b For instance, up to 4.5 and
4.7 g L-1 of Taxote`re was solubilized in 25 mM aqueous
solutions of 29 and 30 at 25 °C, respectively, that is, above
1000-fold solubility enhancements as compared to the water
solubility of the drug (0.004 g L-1).16a This already supports
the hypothesis depicted in Figure 1 and illustrates the superior
potential of dendritic cyclodextrins as drug carriers as compared
to monovalent or polysubstituted conjugates.
(27) For recent studies on the dependence of Con A-polymannoside binding
modes on multivalent ligand architecture and density, see: (a) Gestwicki,
J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K. A.; Kiessling, L. L. J. Am.
Chem. Soc. 2002, 124, 14922-14933. (b) Cairo, C. W.; Gestwicki, J. E.;
Kanai, M.; Kiessling, L. L. J. Am. Chem. Soc. 2002, 124, 1615-1619.
(28) The short distances between the mannopyranosyl binding epitopes in the
prepared conjugates cannot expand the 50 Å distance between two binding
sites in tetrameric Con A, precluding a chelate effect. On the other hand,
the presence of the relatively voluminous HRP protein as a label in the
lectin used for ELLA experiments probably prevents cross-linking processes
for such short intermannosyl spacers. Actually, no precipitation was
observed during the assays, supporting this assumption. For a discussion
on the significance of lectin-binding evaluation by ELLA, in comparison
with other techniques, see ref 15a.
(29) The terms mini cluster (microcluster) and macrocluster effect have been
previously used in the literature to refer to the observed increases on lectin-
binding affinities for small glycoclusters and large glycopolymers, respec-
tively. See: (a) Quesenberry, M. S.; Lee, R. T.; Lee, Y. C. Biochemistry
1997, 36, 2724-2732. (b) Fan, J.-Q.; Quesenberry, M. S.; Takegawa, K.;
Iwahara, S.; Kondo, A.; Kato, I.; Lee, Y. C. J. Biol. Chem. 1995, 270,
17730-17735.
(30) For recent evidence on the existence of an aggregation-independent
microcluster effect, see: Ko¨hn, M.; Benito, J. M.; Ortiz Mellet, C.;
Lindhorst, T. K.; Garc´ıa Ferna´ndez, J. M. ChemBioChem 2004, 5, 771-
777.
The ability of the docetaxel:29 and docetaxel:30 complexes
to bind Con A was evaluated by ELLA using lyophilizates
containing 1:5 guest:host molar ratios. It was established that
the docetaxel:âCD complex at the same molar ratio did not
interact with the lectin. The plots for the inhibition of the Con
A-yeast mannan association by the âCD polymannosides 29
and 30 and the corresponding complexes with docetaxel (DTX)
are shown in Figure 3. No precipitation was observed under
the experimental conditions, discarding decomplexation of the
(31) A sliding mechanism or the existence of extending binding sites in the
protein has been postulated previously to account for enhanced binding
affinities between lectins and small glycoclusters that do not fit either to
the chelate or to the aggregation model. See cf. ref 1a.
(32) Schaschke, N.; Fiori, S.; Wehyher, E.; Escrient, C.; Fourmy, D.; Mu¨ller,
G.; Moroder, L. J. Am. Chem. Soc. 1998, 120, 7030-7038.
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Glycodendrimer−Cyclodextrin Conjugates
A R T I C L E S
Figure 4. Chemical structure of docetaxel (DTX) and of the related drug
paclitaxel (taxol, PTX).
insoluble drug upon lectin binding. The results were indicative
of 2-fold higher Con A affinities in the case of the complexes
(IC50 values 43 and 51 µM) as compared to the individual hosts
(91 and 190 µM, respectively). An identical effect was observed
for the hexavalent derivative 32 (IC50 ) 6 µM for 32:DTX
complex as compared to 10 µM for 32).
The existence of additional interactions between the included
drug and the receptor protein might be at the origin of the above
effect. Notwithstanding, this observation seems to be indepen-
dent of the distance between the receptor-binding cluster and
the carrier moiety, suggesting a different mechanism. Probably,
the existence of two functional moieties in the docetaxel
molecule for inclusion in the âCD cavity (i.e., the two aromatic
rings; see Figure 4) results in the formation of species with a
1:2 guest-host stoichiometry, therefore with a formal hexavalent
character. The possibility of exploiting guest-promoted clus-
terization of multivalent carriers, a biomimetic mechanism, may
actually represent a new approach in active drug targeting. On
the other hand, this observation suggests that CD dimer
derivatives, capable of interacting simultaneously with both
aromatic rings in docetaxel to form 1:1 sandwich-type com-
plexes, could be still more efficient carriers for this particular
drug. A demonstration that the disclosed modular synthetic
strategy is flexible enough to respond to such design require-
ments is given hereinafter.
Figure 5. General architecture of CD dimer-glycodendrimer conjugates
and the chemical structure of compound 39, proposed in this study as
docetaxel carrier (the corresponding model compound 40 was also prepared
for control experiments).
Tailor-Made Dual-Cavity Hosts for Docetaxel. Recently,
the formation of sandwich-type complexes of the related drug
paclitaxel (PTX, taxol; Figure 4) with cyclodextrin dimers has
been reported.33 Interestingly, such dual-cavity hosts can either
fully sequester the drug, therefore acting as detoxifying agents,
or not affecting the cytotoxic activity at all, depending on the
CD dimer structure. The above commented results suggest a
similar ability to interact simultaneously with two âCD cavities
for docetaxel. The preparation of tailor-made multitopic hosts
able to satisfy the requirements for sandwich-type complexation
and biological receptor recognition was, therefore, appealing
(Figure 5). In fact, the possibility of generating molecular
diversity at the host end of the CD-glycodendritic architecture
to optimize host-guest interaction was already purposely
implicit in the modular strategy developed. Taking advantage
of our previous experience on the preparation of CD conjugates
through the thiourea bridging reaction, a convergent synthesis
of the glycodendritic host 39, the first example of a CD dimer
derivative endowed with the capability of biological receptor
recognition, has been devised.
In the above molecular design, we have kept the elements
that were already identified as optimal for lectin binding (i.e.,
a hexavalent dendritic presentation of the biorecognizable
mannosyl epitopes grouped in triads to account for the favorable
density effect) and inclusion capabilities (i.e., C-6 monosub-
stituted âCD elements). The synthetic strategy is depicted in
Scheme 3. Coupling reaction of the âCD monoamine 1 with
the selectively functionalized tris(2-aminoethyl)amine (TREN)-
derived branching element 41 provided the CD dimer moiety
42, which was activated for a further coupling process by
trifluoroacetic acid-catalyzed hydrolysis of the N-Boc protecting
group (f43). The reaction of diamine 44 and isothiocyanate 5
afforded the hexavalent bis(thiourea) 45 that was subsequently
armed by direct isothiocyanation of the terminal azido group
(f46).24 Conjugation of 43 and 46 followed by deacetylation
yielded the target TREN-based first-generation bidirectional
dendritic architecture 39. Similarly, by replacing the âCD
(33) (a) Liu, Y.; Chem, G.-S.; Li, L.; Zhang, H.-Y.; Cao, D.-X.; Yuan, Y.-J. J.
Med. Chem. 2003, 46, 4634-4637. (b) Moser, J. G.; Rose, I.; Wagner, B.;
Wieneke, T.; Vervoorts, A. J. Inclusion Phenom. Macrocycl. Chem. 2001,
39, 13-18. For additional examples on the synthesis and complexing
properties of cyclodextrin dimers, see: (c) Breslow, R.; Belvedere, S.;
Gershell, L.; Leung, D. Pure Appl. Chem. 2000, 72, 333-342. (d) de Jong,
M. R.; Engbersen, J. F. J.; Huskens, J.; Reinhoudt, D. N. Chem.-Eur. J.
2000, 6, 4034-4040. (e) Baugh, S. D. P.; Yang, Z.; Leung, D. K.; Wilson,
D. M.; Breslow, R. J. Am. Chem. Soc. 2001, 123, 12488-12494. (f) Liu,
Y.; Chen, Y.; Wada, T.; Inoue, Y. Chem.-Eur. J. 2001, 7, 2528-2535. (g)
Jiang, T.; Sukumaran, D. K.; Soni, S.-D. Lawrence, D. S. J. Org. Chem.
1994, 59, 5149-5155. (h) Modified Cyclodextrins: Scaffolds and Templates
for Supramolecular Chemistry; Easton, C. J., Lincoln, S. F., Eds.; Imperial
College Press: London, 1999; pp 191-226.
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A R T I C L E S
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Scheme 3. Synthesis of the Hexavalent âCD
Dimer-Polymannoside Conjugate 39 and Its Precursors
Figure 6. Molecular model of the sandwich-type complex between the
hexavalent âCD dimer conjugate 39 (light gray, dark red, white, light blue,
and yellow represent carbon, hydrogen, oxygen, nitrogen, and sulfur atoms,
respectively) and docetaxel (in green). According to docking experiments
(MACROMODEL 6.0, MM2*), the benzoyl ring (right side) would be
deeply included in one of the âCD cavities, while the benzyl group (left
side) would be only shallowly included in the second CD cavity due to the
steric hindrance imposed by the neighboring Boc group.
Actually, the dual-cavity system did not affect glycodendrimer-
lectin recognition at all as indicated by control experiments using
the model conjugate 40 (IC50 12 µM). Comparative evaluation
of the lectin-binding properties for the corresponding complexes
evidenced, however, significant differences. Thus, in the case
of the CD dimer derivative 39, the interaction with the protein
was not affected upon complex formation, in sharp contrast with
the 2-fold increase in Con A affinity observed for the monomeric
CD conjugate 32:DTX complex as compared to the free host
32. This result supports our hypothesis of a docetaxel-promoted
clusterization of the glycodendritic system in the case of 32, a
mechanism that is inhibited in the case of 39 due to the
sandwich-type supramolecular organization. Moreover, these
data indicate that both host-glycodendrimer architectures, not
only as such but also as the corresponding docetaxel complexes,
are efficiently recognized by the lectin and that the sugar ligand-
lectin affinity can be allosterically modulated, to some extent,
by acting at the host end.
Targeting of âCD-Polymannoside Dendrimers to
Macrophages. The above results illustrate the concept that the
CD-glycodendrimer architecture, in combination with a ver-
satile diversity-oriented synthetic methodology, allows the
systematic study of the different factors influencing sugar-lectin
and host-guest interactions. From these results, information can
thus be gained for the design of new systems for saccharide-
directed molecular delivery of specific guests to a biological
receptor. Because lectins having identical sugar binding motifs
may differ significantly in their binding-site, quaternary struc-
ture, and preferred binding mechanisms, the conclusions ob-
tained for a particular receptor, for example, the plant lectin
Con A in the present study, must be taken with care when
building block 1 by the methyl R-D-glucopyranosyl-derived core
element 7 in the synthetic scheme, the model conjugate 40,
lacking the dual CD cavity system, was prepared as a reference
compound for control experiments.
The choice of nitrogen-centered TREN-derived branching
elements in the above synthetic design responds to two important
considerations: (i) according to molecular modeling calcula-
tions, the length of the corresponding bis(thiourea) linker
between the CD units in 39 matches the distance between the
aromatic rings in DTX, allowing the appropriate orientation of
the two cavities for an optimal cooperative complexation; (ii)
the use of nitrogen-centered dendrons prevents the occurrence
of diasterotopism in the NMR spectra of the final conjugates,
thus facilitating NMR analysis.
Comparison of Docetaxel Carrier Capabilities and Lectin-
Binding Properties for CD Mono and Dimer Conjugates.
Both the monomeric and the dimeric hosts 32 and 39 formed
supramolecular complexes with docetaxel and dramatically
increased its solubility in water. Association constants (Kc) of
4000 and 1.5 × 105 M-1, respectively, were estimated from
the slope of the corresponding phase solubility diagrams,
assuming a 1:1 host:guest stoichiometry.34 The 37.5-fold
increase in association constant for the dimeric with respect to
the monomeric host is in agreement with the simultaneous
interaction of the drug with both CD cavities in a sandwich-
type complex (see Figure 6).35 Both conjugates exhibited strong
binding affinities toward Con A as determined by ELLA, with
similar IC50 values (10 and 13 µM for 32 and 39, respectively).
(34) The corresponding solubility diagrams for the systems DTX:32 and DTX:
39, in the range of host concentrations investigated (10-25 mM; see
Supporting Information), provided quasi-linear plots with slopes of less
than unity. Although this situation doest not necessarily mean that only a
1:1 complex is formed (actually, the lectin-binding experiments pointed to
the presence of a 1:2 complex in the case of 32), it is consistent with these
species being the predominant ones in the solutions. See: Connors, K. A.
Binding Constants, The Measurement of Molecular Complex Stability; John
Wiley & Sons: New York, 1987; pp 261-281.
(35) For the generation of the three-dimensional model of the DTX:39 complex,
the thiourea groups in the host were set in the Z,Z-conformation, the dihedral
angle about the C-5I-C-6I bond was set in the gauche-trans conformation
(i.e., the C-4I and the thiourea substituents in the anti disposition), the carbon
segments were set in the all-trans conformation, and the carbamate bond
in the guest was set in the Z-conformation. The host and the guest structures
were extensively minimized using the GSB continuum solvent model for
water, and the minimized structures were used in the docking experiments.
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Glycodendrimer−Cyclodextrin Conjugates
A R T I C L E S
changing the protein target. To test the potential of these systems
in drug targeting, assaying a mammalian lectin was, therefore,
of interest.
the macrophage surface as compared to uncomplexed 32. No
inhibition effect was observed, however, for the corresponding
complex of docetaxel with â-cyclodextrin, discarding a contri-
bution of the included drug to macrophage surface binding.
These results parallel those discussed above for the association
of multivalent âCD conjugates and the corresponding docetaxel
complexes to Con A, supporting a guest-promoted clusterization
mechanism that results in higher-valent species with increased
mannose receptor affinity. In the case of 39, the competition
experiment did not show any difference between the uncom-
plexed host and the corresponding docetaxel complex, in
agreement with the proposed sandwich-type structure of the
later.
Among the receptors present at the surface of macrophages,
the well-characterized mannosyl/fucosyl receptor is attractive
for targeting purposes.6e,36 Previous results using mannosylated
liposomes evidenced a parallelism between binding affinity
toward Con A and binding affinity toward the macrophage
surface.37 In an in vitro experiment, the interaction of the
hexavalent â-cyclodextrin-dendrimer conjugates 32 and 39 with
resident peritoneal macrophages (mice), which present such a
lectin at their surface, was studied. Binding of the targeted
conjugates and the control (native â-cyclodextrin) to cells was
quantified by a fluorimetric method adapted from that reported
by Muller and Schuber37 for mannosylated liposomes, using 6-p-
toluidino-2-naphthalenesulfonic acid (TNS) as the fluorescent
dye. TNS forms a strong 1:1 inclusion complex with âCD,38
and it was also strongly complexed by 32 and 39.39
To define the specificity of the host interaction with the
surface of the macrophage, we have performed competition
experiments at 4 °C, a temperature that precludes phagocytosis.
The cells were incubated at this temperature with three
concentrations of the 1:1 TNS:32 or TNS:39 complexes (0.1-
80 µmol/2 mL Dulbecco’s modified Eagle’s medium), and the
amount of cell-associated complex was determined fluorimetri-
cally. As compared to the nontargeted TNS:âCD complex, an
above 20-fold preferential association of 32 and 39 was observed
at a concentration of 650 nM. An inhibition of this binding was
observed in the presence of increasing concentrations of the
uncomplexed hosts. IC50 values of 45 and 48 ( 5 µM,
respectively, were obtained from classical displacement curves
for the mono and dimeric hosts (concentration of TNS:host )
40 µM). These results are consistent with a reversible binding
of the mannosylated âCD derivatives 32 and 39 to the cell
surface under these experimental conditions, both the uncom-
plexed conjugates and the corresponding TNS inclusion com-
plexes competing for the same binding site. In contrast, when
â-cyclodextrin was used, no direct competition was observed
for the binding of the TNS:32 or TNS:39 complexes.
Conclusions
Targeting involving sugar binding to biological receptors has
generally relied on the generation of large multivalent displays
of the putative epitopes on a macromolecular scaffold. Coupling
such high molecular weight ligands to single-molecule molecular
containers (e.g., cyclodextrins) would be, however, unpractical.
Our results show that it is possible to attain biologically useful
affinities by using relatively small glycoclusters of appropriate
geometry, even though cross-linking of different carbohydrate-
binding domains is unlikely to occur for short intersaccharide
bridges. Synthetic methods that allow not only the efficient
coupling of the glycocluster and the host moieties but also tailor
the ligand-lectin and host-guest interactions should facilitate
exploration of the potential of the microcluster effect in
vectorized molecular delivery. In this context, the significance
of the present study is three-fold. First, it demonstrates an
efficient and very flexible strategy for the preparation of tailor-
made CD dendrimers bearing biorecognizable markers or
reporting groups. Second, the present system suggests a potential
utility in sugar-directed delivery of particular drugs or probes
to specific saccharide-recepting biological surfaces. Third, the
possibility of exploiting guest-promoted clusterization of mul-
tivalent carriers may actually represent a new approach in active
drug targeting.
Acknowledgment. This paper is dedicated to the memory of
Professor Christian Pedersen. We thank the Ministerio de
Ciencia y Tecnolog´ıa for financial support (grant nos. BQU2003-
00937 and BMC2001-2366-CO3-03) and for a doctoral fellow-
ship (M.G.-G.).
Interestingly, when the DTX:32 complex (lyophilizate con-
taining 1:5 guest:host molar ratio) was used as competing agent,
a value of IC50 ) 30 ( 5 µM (referred to 32) was obtained
under the same conditions, indicating a higher affinity toward
(36) Du¨ffels, A.; Green, L. G.; Ley, S. V.; Miller, A. D. Chem.-Eur. J. 2000, 6,
1416-1430.
Supporting Information Available: Experimental details,
purification and characterization data for the prepared com-
pounds, and experimental procedures for the determination of
Con A and macrophage binding efficiency. This material is
available free of charge via the Internet at http://pubs.acs.org.
(37) Muller, C. D.; Schuber, F. Biochim. Biophys. Acta 1989, 986, 97-105.
(38) Rekharsky, M. V.; Inoue, Y. Chem. ReV. 1998, 98, 1875-1917.
(39) Association constants (K11) of 1.8 × 104, 1.2 × 104, and 9.5 × 105 M-1
for the corresponding 1:1 TNS:âCD, TNS:32, and TNS:39 complexes were
determined by fluorescence titration experiments. Note that this implies
that less than 1% of the complex is dissociated in solution, discarding any
significant influence of the dissociation equilibrium in the lectin-binding
evaluation experiments.
JA047864V
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J. AM. CHEM. SOC. VOL. 126, NO. 33, 2004 10363