A self-assembled multivalent pseudopolyrotaxane for binding galectin-1

Article abstract of DOI:10.1021/ja0491073

A self-assembled pseudopolyrotaxane consisting of lactoside-displaying cyclodextrin (CD) "beads" threaded onto a linear polyviologen "string" was investigated for its ability to inhibit galectin-1-mediated T-cell agglutination. The CDs of the pseudopolyro

Full text of DOI:10.1021/ja0491073

Published on Web 09/04/2004
A Self-Assembled Multivalent Pseudopolyrotaxane for Binding
Galectin-1
Alshakim Nelson,† Jason M. Belitsky,† Se´bastien Vidal,§ C. Steven Joiner,†
Linda G. Baum,*,‡ and J. Fraser Stoddart*,†
Contribution from the California NanoSystems Institute (CNSI), the Department of Chemistry
and Biochemistry, and the Department of Pathology, UniVersity of California,
Los Angeles, California 90095
Received February 17, 2004; E-mail: lbaum@mednet.ucla.edu; stoddart@chem.ucla.edu
Abstract: A self-assembled pseudopolyrotaxane consisting of lactoside-displaying cyclodextrin (CD) “beads”
threaded onto a linear polyviologen “string” was investigated for its ability to inhibit galectin-1-mediated
T-cell agglutination. The CDs of the pseudopolyrotaxane are able to spin around the axis of the polymer
chain as well as to move back and forth along its backbone to alter the presentation of its ligand. This
supramolecular superstructure incorporates all the advantages of polymeric structures, such as the ability
to span large distances, along with a distinctively dynamic presentation of its lactoside ligands to afford a
neoglycoconjugate that can adjust to the relative stereochemistries of the lectin’s binding sites. The
pseudopolyrotaxane exhibited a valency-corrected 10-fold enhancement over native lactose in the
agglutination assay, which was greater than the enhancements observed for lactoside-bearing trivalent
glycoclusters and a lactoside-bearing chitosan polymer tested using the same assay. The experimental
results indicate that supramolecular architectures, such as the pseudopolyrotaxane, provide tools for
investigating protein-carbohydrate interactions.
Phenomena that control the form and function of living
systems, such as self-assembly,1 molecular recognition,2 and
multivalency,3 have served to hasten the development of
supramolecular chemistry4 and template-directed synthesis,5
particularly of mechanically interlocked molecular compounds.6
Once considered chemical curiosities, complexes such as
pseudorotaxanes7 and compounds such as catenanes8 and
rotaxanes9 are now contributing to advances in molecular
electronics10 and the construction of artificial molecular ma-
chines.11 In addition, polymeric pseudorotaxanes12 and mechani-
cally interlocked polyrotaxanes13 are enriching polymer chem-
istry and materials science in terms of both structure14 and
function.15 While biology has provided much of the inspiration
† California NanoSystems Institute and Department of Chemistry and
Biochemistry, University of California, Los Angeles, CA.
‡ Department of Pathology, University of California, Los Angeles, CA.
§ Current address: Laboratoire de Chimie Organique 2, Universite Claude
Bernard Lyon 1, France.
(1) (a) Lindsey, J. S. New J. Chem. 1991, 15, 153-180. (b) Whitesides, G.
M.; Mathias, J. P.; Seto, C. T. Science 1991, 254, 1312-1319. (c) Philp,
D.; Stoddart J. F. Synlett 1991, 445-458. (d) Philp, D.; Stoddart, J. F.
Angew. Chem., Int. Ed. Engl. 1996, 35, 1154-1196. (e) Fujita, M. Acc.
Chem. Res. 1999, 32, 53-61. (f) Rebek, J., Jr. Acc. Chem. Res. 1999, 32,
278-286. (g) Bong, D. T.; Clark, T. D.; Granja, J. R.; Ghadiri, M. R.
Angew. Chem., Int. Ed. 2001, 40, 988-1011. (h) Prins, L. J.; Reinhoudt,
D. N.; Timmerman, P. Angew. Chem., Int. Ed. 2001, 40, 2382-2426. (i)
Seidel, S. R.; Stang, P. J. Acc. Chem. Res. 2002, 35, 972-983. (j) Elemans,
J. A. A. W.; Rowan, A. E.; Nolte, R. J. M. J. Mater. Chem. 2003, 13,
2661-2670.
(6) (a) Amabilino, D. B.; Stoddart, J. F. Chem. ReV. 1995, 95, 2725-2828.
(b) Vo¨gtle, F.; Du¨nwald, T.; Schmidt, T. Acc. Chem. Res. 1996, 29, 451-
460. (c) Breault, G. A.; Hunter, C. A.; Mayers, P. C. Tetrahedron 1999,
55, 5265-5293. (d) Cantrill, S. J.; Pease, A. R.; Stoddart, J. F. J. Chem.
Soc., Dalton Trans. 2000, 3715-3734.
(7) Ashton, P. R.; Belohradsky, M.; Philp, D.; Spencer, N.; Stoddart, J. F. J.
Chem. Soc., Chem. Commun. 1993, 1274-1277.
(8) (a) Leigh, D. A.; Wong, J. K. Y.; Dehez, F.; Zerbetto, F. Nature 2003,
424, 174-179. (b) Dietrich-Buchecker, C.; Colasson, B.; Fujita, M.; Hori,
A.; Geum, N.; Sakamoto, S.; Yamaguchi, K.; Sauvage, J.-P. J. Am. Chem.
Soc. 2003, 125, 5717-5725.
(2) See special issue on molecular recognition in Chem. ReV. 1997, 97, 1231-
1734.
(3) (a) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int. Ed.
1998, 37, 2754-2794. (b) Lee, R. T.; Lee, Y. C. Glycoconjugate J. 2000,
17, 543-551. (c) Lundquist, J. J.; Toone, E. J. Chem. ReV. 2002, 102,
555-578. (d) Ercolani, G. J. Am. Chem. Soc. 2003, 125, 16097-16103.
(e) Kitov, P. I.; Bundle, D. R. J. Am. Chem. Soc. 2003, 125, 16271-16284.
(4) (a) Lehn, J. M. Supramolecular Chemistry; VCH: Weinheim, Germany,
1995. (b) Reinhoudt, D. N.; Crego-Calama, M. Science 2002, 295, 2403-
2407. (c) Whitesides, G. M.; Grzybowski, B. Science 2002, 295, 2418-
2421.
(9) (a) Pomeranc, D.; Jouvenot, D.; Chambron, J.-C.; Collin, J.-P.; Heitz, V.;
Sauvage, J. P. Chem.-Eur. J. 2003, 9, 4247-4254. (b) Kilbinger, A. F.
M.; Cantrill, S. J.; Waltman, A. W.; Day, M. W.; Grubbs, R. H. Angew.
Chem., Int. Ed. 2003, 42, 3281-3285.
(10) (a) Pease, A. R.; Jeppesen, J. O.; Stoddart, J. F.; Luo, Y.; Collier, C. P.;
Heath, J. R. Acc. Chem. Res. 2001, 34, 433-444. (b) Raehm, L.; Kern,
J.-M.; Sauvage, J. P.; Hamann, C.; Palacin, S.; Bourgoin, J.-P. Chem.-
Eur. J. 2002, 8, 2153-2162.
(11) (a) Balzani, V.; Credi, A.; Raymo, F. M.; Stoddart, J. F. Angew. Chem.,
Int. Ed. 2000, 39, 3348-3391. (b) Harada, A. Acc. Chem. Res. 2001, 34,
456-464. (c) Balzani, V.; Credi, A.; Venturi, M. Molecular DeVices and
Machines: A Journey into the Nano World; Wiley-VCH: Weinheim,
Germany, 2003. (d) Flood, A. H.; Ramirez, R. J. A.; Deng, W.-Q.; Muller,
R. P.; Goddard, W. A., III; Stoddart, J. F. Aust. J. Chem. 2004, 57, 301-
322.
(5) (a) Busch, D. H.; Stephensen, N. A. Coord. Chem. ReV. 1990, 100, 119-
154. (b) Anderson, S.; Anderson, H. L.; Sanders, J. K. M. Acc. Chem. Res.
1993, 26, 469-475. (c) Cacciapaglia, R.; Mandolini, L. Chem. Soc. ReV.
1993, 22, 221-231. (d) Hoss, R.; Vo¨gtle, F. Angew. Chem., Int. Ed. 1994,
33, 375-384. (e) Hubin, T. J.; Kolchinski, A. G.; Vance, A. L.; Busch, D.
L. AdV. Supramol. Chem. 1999, 5, 237-357. (f) Templated Organic
Synthesis; Diederich, F., Stang, P. J., Eds.; Wiley-VCH: Weinheim,
Germany, 1999. (g) Stoddart, J. F.; Tseng, H. R. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 4797-4800.
(12) Amabilino, D. B.; Parsons, I. W.; Stoddart, J. F. Trends Polym. Chem.
1994, 2, 146-152.
(13) Raymo, F. M.; Stoddart, J. F. Chem. ReV. 1999, 99, 1643-1663.
9
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J. AM. CHEM. SOC. 2004, 126, 11914-11922
10.1021/ja0491073 CCC: $27.50 © 2004 American Chemical Society

Multivalent Pseudopolyrotaxane for Binding Gal-1
A R T I C L E S
Scheme 1. Formation of Pseudopolyrotaxane 3
and many of the conceptual tools for the growth of supra-
molecular chemistry4 and template-directed synthesis,5 the
biochemistry that interfaces with wholly synthetic complexes
and mechanically interlocked molecules is still in its infancy.16
The concept of multivalency3 is especially important in
glycobiology.17 Nature uses multivalent interactions between
multimeric and/or membrane-bound carbohydrate-binding pro-
teins (lectins) and their matching carbohydrate ligands (epitopes)
to mediate both physiological and pathological processes.
Although a typical monovalent interaction between a lectin and
its epitope is in the millimolar regime, Nature attains much
higher avidities through multivalency.3 Affinity enhancements
over and beyond those expected on the basis of valency have
been defined by Lee18 as the glycoside cluster effect. Chemists
have synthesized a variety of neoglycoconjugates using den-
dritic,19 polymeric,20 and self-assembled21 architectures in
attempts to mimic the complex multivalent scaffolds found in
biological systems. There is considerable interest22 in how the
architectural features including rigidity, spacing, topology, and
density of saccharidesscharacteristic of these synthetic multi-
valent structuressinfluence their avidity for their respective
lectins. Glycodendrimers19 generally fill some kind of three-
dimensional space and usually display saccharide residues on
their peripheries, while glycopolymers20 are composed of a
polymeric backbone with pendant saccharide residues. In
general, the extended lengths and high valencies of glycopoly-
mers offer particular advantages20e that have led to their
extensive use20 in binding lectins. Self-assembled dynamic
systems, such as those provided by gold nanoparticles,21e
micelles,21f,g and liposomes21a represent yet other avenues by
which multivalency can be investigated. These systems support
(20) (a) Spaltenstein, A.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 686-
687. (b) Choi, S. K.; Mammen, M.; Whitesides, G. M. J. Am. Chem. Soc.
1997, 119, 4103-4111. (c) Bovin, N. V. Glycoconjugate J. 1998, 15, 431-
446. (d) Mortell, K. H.; Weatherman, R. V.; Kiessling, L. L. J. Am. Chem.
Soc. 1996, 118, 2297-2298. (e) Cairo, C. W.; Gestwicki, J. E.; Kanai, M.;
Kiessling, L. L. J. Am. Chem. Soc. 2002, 124, 1615-1619. (f) Arranz-
Plaza, E.; Tracy, A. S.; Siriwardena, A.; Pierce, J. M.; Boons, G.-J. J. Am.
Chem. Soc. 2002, 124, 13035-13046. (g) Thobhani, S.; Ember, B.;
Siriwardena, A. Boons, G.-J. J. Am. Chem. Soc. 2003, 125, 7154-7155.
(21) (a) Kingery-Wood, J. E.; Williams, K. W.; Sigal, G. B.; Whitesides, G. M.
J. Am. Chem. Soc. 1992, 114, 7303-7305. (b) Spevak, W.; Nagy, J. O.;
Charych, D. H.; Schaefer, M. E.; Gilgert, J. H.; Bednarski, M. D. J. Am.
Chem. Soc. 1993, 115, 1146-1147. (c) Kamitakahara, H.; Suzuki, T.;
Nishigori, N.; Suzuki, Y.; Kanie, O.; Wong, C.-H. Angew. Chem., Int. Ed.
1998, 37, 1524-1528. (d) Bruehl, R. E.; Dasgupta, F.; Katsumoto, T. R.;
Tan, J. H.; Bertozzi, C. R.; Spevak, W.; Ahn, D. J.; Rosen, S. D.; Nagy,
J. O. Biochemistry 2001, 40, 5964-5974. (e) Rojas, T. C.; de la Fuente, J.
M.; Barrientos, A. G.; Penade´s, S.; Ponsonnet, L.; Ferna´ndez, A. AdV. Mater.
2002, 14, 585-588. (f) Thoma, G.; Katopodis, A. G.; Voelcker, N.;
Duthaler, R. O.; Streiff, M. B. Angew. Chem., Int. Ed. 2002, 41, 3195-
3198. (g) Jule, E.; Nagasaki, Y.; Kataoka, K. Bioconjugate Chem. 2003,
14, 177-186.
(14) (a) Taylor, P. N.; O’Connell, M. J.; McNeill, L. A.; Hall, M. J.; Aplin, R.
T.; Anderson, H. L. Angew. Chem., Int. Ed. 2000, 39, 3456-3460. (b)
Oku, T.; Furusho, Y.; Takata, T. Angew. Chem., Int. Ed. 2004, 43, 966-
969.
(15) (a) Yui, N.; Ooya, T.; Kumeno, T. Bioconjugate Chem. 1998, 9, 118-
125. (b) Tamura, M.; Gao, D.; Ueno, A. Chem.-Eur. J. 2001, 7, 1390-
1397. (b) Fustin, C.-A.; Bailly, C.; Clarkson, G. J.; De Groote, P.; Galow,
T. H.; Leigh, D. A.; Robertson, D.; Slawin, A. M. Z.; Wong, J. K. Y. J.
Am. Chem. Soc. 2003, 125, 2200-2207.
(16) (a) Niemeyer, C. M. Trends Biotechnol. 2002, 20, 395-401. (b) Fiam-
mengo, R.; Crego-Calama, M.; Reinhoudt, D. N. Curr. Opin. Chem. Biol.
2002, 5, 660-673. (c) Dvornikovs, V.; House, B. E.; Kaetzel, M.; Dedman,
J. R.; Smithrud, D. B. J. Am. Chem. Soc. 2003, 125, 8290-8301.
(17) (a) Varki, A. Glycobiology 1993, 3, 97-130. (b) Essentials of Glycobiology;
Varki, A., Cummings, R., Esko´, J., Freeze, H., Hart, G., Marth, J., Eds.;
Cold Spring Harbor Laboratory Press: Plainview, NY, 1999.
(18) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321-327.
(19) (a) Roy, R. Polym. News 1996, 21, 226-232. (b) Rockendorf, N.; Lindhorst,
T. Top. Curr. Chem. 2001, 217, 201-238. (c) Turnbull, W. B.; Stoddart,
J. F. ReV. Mol. Biotechnol. 2002, 90, 231-255. (d) Cloninger, M. J. Curr.
Opin. Chem. Biol. 2002, 6, 742-748. (e) Woller, E. K.; Walter, E. D.;
Morgon, J. R.; Singel, D. J.; Cloninger, M. J. J. Am. Chem. Soc. 2003,
125, 8820-8826.
(22) (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) Kalovidouris, S.
A.; Blixt, O.; Nelson, A.; Vidal, S.; Turnbull, W. B.; Paulson, J. C.; Stoddart,
J. F. J. Org. Chem. 2003, 68, 8485-8493. (c) Kitov, P. I.; Shimizu, H.;
Homans, S. W.; Bundle, D. R. J. Am. Chem. Soc. 2003, 125, 3284-3294.
9
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A R T I C L E S
Nelson et al.
fluid surfaces, reminiscent of natural cellular membranes,23
which allow for the arrangement of saccharide ligands coated
on their surfaces to find the appropriate positions and orienta-
tions to maximize their interactions with the particular lectin
binding site.
and to shed light on the supramolecular interactions of Gal-1
and its natural and oncogenic carbohydrate ligands. Chemists
targeting Gal-1 have focused primarily on glycodendrimers and
smaller glycoclusters with mixed results.33 Topologically, Gal-1
is a particularly interesting challenge34 since it is a rigid dimer
with two binding sites oriented in opposite directions such that
the entrance to each of the binding sites is located 6 nm apart.
Herein, we describe the formation of a lactoside-displaying CD-
based polyviologen pseudopolyrotaxane and the supramolecular
inhibition of Gal-1-mediated T-cell agglutination in comparison
with a series of trivalent glycoclusters 8, 9, and 10 and a rigid
covalent neoglycopolymer 11 as controls.
Recently, we have become interested24 in carbohydrate-
displaying pseudopolyrotaxanes and polyrotaxanes of cyclo-
dextrins (CDs) threaded onto linear polymer chains as dynamic
multivalent neoglycoconjugates. Although the supramolecular
chemistry of CD-based pseudopolyrotaxanes and polyrotaxanes,
which was pioneered by Harada,25 is well-established,26 these
“beads-on-a-string” have only recently been adapted27 for the
multivalent display of biological ligands. While polyrotaxanes
displaying maltosides have been synthesized by Yui and co-
workers,28 CD-based pseudopolyrotaxanes displaying lactosides
have been self-assembled recently in our own laboratory.24 The
CDs are able to spin around the axes of the polymer chains as
well as move back and forth along the polymers’ backbones
and thus alter the presentations of their saccharide ligands. In
addition to the convenience of the synthesis, which relies on a
self-assembly protocol, these “beads-on-a-string” offer the
advantages of polymeric features such as the ability to span
nanometer lengths as well as adaptable presentation of epitopes
on account of the dynamic flexibility of the pseudopolyrotaxane
architecture.
In this article, we describe the preparation (Scheme 1) of a
stable pseudopolyrotaxane 3 using the lactoside-bearing R-CD
1 and polyviologen 2 for targeting galectin-1 (Gal-1).29 Gal-1
is a soluble 14 kDa dimeric galactoside-binding lectin that can
organize cell surface glycoproteins through binding and cross-
linking of terminal or polymeric lactose or N-acetyllactosamine
residues. Gal-1 is the prototypical member of the galectins, a
ubiquitous family of galactoside-binding lectins that mediate
cell adhesion, signaling, and death. Certain galectins, particularly
Gal-1 and galectin-3, are overexpressed in many types of
cancer.30 Gal-1 plays multiple roles in cancer, from defense
against the immune system by exploiting its natural pro-
apoptotic function31 to promoting motility and homotypic cell
agglutination, which is believed32 to be important for aggregating
cancer cells into tumor emboli, thus increasing malignant cell
survival in circulation. Synthetic multivalent ligands for Gal-1
have the potential to act as cancer diagnostics and therapeutics
Results
Synthesis of Trivalent Lactoside Glycoclusters. The three
trifunctional core compounds, 4, 5, and 6, which present methyl-
amino residues for the attachment of dendrons by reductive
amination, were prepared35 for the synthesis (Scheme 2) of the
trivalent glycoclusters 8, 9, and 10. The arms of these cores
differ by (a) the number of atoms that separate the methylamino
groups and (b) the overall rigidity of the structures, which may
alter the interaction of the resulting trivalent structure with its
receptor. Both cores 4 and 6 possess a nitrogen atom at their
center, and while 4 is the smaller in size, 6 contains para-phenyl-
ene spacers within its arms to yield a more extended conforma-
tion. Core 5, instead of having a central nitrogen atom, uses
phloroglucinol as a more rigid central core. The trisaccharide22b
7 was coupled (Scheme 2) with trifunctional trismethylamino
core compounds 4, 5, and 6 to afford the lactose clusters 8, 9,
(23) (a) Menger, F. M.; Gabrielson, K. D. Angew. Chem., Int. Ed. 1995, 34,
2091-2106. (b) Kataoka, K.; Harada, A.; Nagasaki, Y. AdV. Drug DeliVery
ReV. 2001, 47, 113-131.
(24) Nelson, A.; Stoddart, J. F. Org. Lett. 2003, 5, 3783-3786.
(25) (a) Harada, A.; Kamachi, M. Macromolecules 1990, 23, 2821-2823. (b)
Harada, A.; Li, J.; Kamachi, M. Macromolecules 1993, 26, 5698-5703.
(26) (a) Wenz, G. Angew. Chem., Int. Ed. Engl. 1994, 33, 803-822. (b) Harada,
A. Coord. Chem. ReV. 1996, 148, 115-133. (c) Harada, A. Carbohydr.
Polym. 1997, 34, 183-188. (d) Nepogodiev, S. A.; Stoddart, J. F. Chem.
ReV. 1998, 98, 1959-1976.
(27) (a) Yui, N.; Ooya, T.; Kawashima, T.; Saito, Y.; Tamai, I.; Sai, Y.; Tsuji,
A. Bioconjugate Chem. 2002, 13, 582-587. (b) Ooya, T.; Yui, N. J.
Controlled Release 2002, 80, 219-228.
(28) Ooya, T.; Eguchi, M.; Yui, N. J. Am. Chem. Soc. 2003, 125, 13016-13017.
(29) (a) Pace, K. E.; Lee, C.; Stewart, P. L.; Baum, L. G. J. Immunol. 1999,
163, 3801-3811. (b) Hernandez, J. D.; Baum, L. G. Glycobiology 2002,
12, 127R-136R. (c) Nagia-Makker, P.; Conklin, J.; Hogan, V.; Raz, A.
Trends Mol. Med. 2002, 8, 187-192. (d) Brewer, C. F.; Miceli, M. C.;
Baum, L. G. Curr. Opin. Struct. Biol. 2002, 12, 616-623. (e) Amano, M.;
Galvan, M.; He, J.; Baum, L. G. J. Biol. Chem. 2003, 278, 7469-7475.
(30) Danguy, A.; Camby, I.; Kiss, R. Biochim. Biophys. Acta 2002, 1572, 285-
293.
(33) (a) Andre´, S.; Cejas Ortega, P. J.; Alamino Perez, M.; Roy, R.; Gabius,
H.-J. Glycobiology 1999, 9, 1253-1261. (b) Andre´, S.; Pieters, R. J.;
Vrasidas, I.; Kaltner, H.; Nishimura, S.-I.; Liu, F.-U.; Liskamp, R. M. J.;
Gabius, H.-J. ChemBioChem 2001, 2, 822-830. (c) Vrasidas, I.; Andre´,
S.; Valentini, P.; Bock, C.; Lensch, M.; Kaltner, H.; Liskamp, R. M. J.;
Gabius, H.-J.; Pieters, R. J. Org. Biomol. Chem. 2003, 1, 803-810. (d)
Andre´, S.; Liu, B.; Gabius, H.-J.; Roy, R. Org. Biomol. Chem. 2003, 1,
3909-3916. (e) Andre´, S.; Kaltner, H.; Furuiki, T.; Nishimura, S.-I.; Gabius,
H.-J. Bioconjugate Chem. 2004, 15, 87-98.
(31) Rubinstein, N.; Alvarez, M.; Zwirner, N. W.; Toscano, M. A.; Ilarregui, J.
M.; Bravo, A.; Mordoh, J.; Fainboim, L.; Podhajhcer, O. L.; Rabinovich,
G. A. Cancer Cell 2004, 5, 241-251.
(32) (a) Perillo, N. L.; Marcus, M. E.; Baum, L. G. J. Mol. Med. 1998, 76,
402-412. (b) He, J.; Baum, L. G. J. Biol. Chem. 2004, 279, 4705-4712.
(34) Bourne, Y.; Bolgiano, B.; Liao, D.-I.; Strecker, G.; Cantau, P.; Herzberg,
O.; Feizi, T.; Cambillau, C. Nat. Struct. Biol. 1994, 1, 863-870.
(35) The synthesis of these cores is provided in the Supporting Information.
9
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Multivalent Pseudopolyrotaxane for Binding Gal-1
A R T I C L E S
Scheme 2. Synthesis of Trivalent Glycoclusters by Reductive Amination
and 10, respectively, by reductive amination. All three glyco-
cluster compounds were fully characterized using high-resolution
mass spectrometry and 1D and 2D NMR spectroscopies.
Synthesis of Chitosan-Based Polymer. Chitosan is a linear
polysaccharide of repeating â-1,4-linked glucosamine and
N-acetylglucosamine, obtained by the partial hydrolysis of the
N-acetyl groups of chitin.36 Like cellulose, chitosan possesses
a rigid linear conformation due to the presence of intramolecular
hydrogen bonds in its secondary structure. Functionalized
chitosan has been investigated by a number of groups for
applications such as gene37 and drug38 delivery, as well as
antimicrobial agents.39 Roy and co-workers40 have demonstrated
that functionalization of the amino groups in chitosan can be
achieved by reductive amination. Commercially available chi-
tosan was reacted with the trisaccharide 7 under reductive
amination conditions using NaCNBH3. Chitosan provides a rigid
rod scaffold for the multivalent display of lactose. On the basis
nitrogen atoms on the bipyridinium segments of the polymer,
should act42,43 as electronic “speed bumps” over which the CD
rings must pass. Thus, the R-CDs should thread onto the polymer
chain in aqueous solution, resting predominantly on its deca-
methylene segments, stabilized by the hydrophobic interactions
inside the cavities of the CD beads. The polyviologen 2 was
synthesized as reported in the literature.41a The number average
molecular weight (Mn ) 7760) of 2, which was determined (end
1
group analysis) by H NMR spectroscopy, corresponds to its
having an average of 17 repeating AB units.
Formation of the Pseudopolyrotaxane. The lactoside-
bearing CD derivative 1 was synthesized as reported in the
literature.24 The self-assembly (Scheme 1) of the pseudopoly-
rotaxane 3 from 1 and 2 was monitored (Figure 1) by 1H NMR
spectroscopy. When 2 was added to a 17-fold excess of 1 (20
mM) in D2O, changes in the proton resonances of both the
polymer and CD were observed. In addition to the broadening
of the CD signals, upfield shifts were observed for the H-3
protons (which are pointing into the center of the CD cavity),
an observation that indicates that the CDs have threaded onto
the polymer chains. We estimate44 that >90% of the CDs are
threaded after 4 days. Moreover, the signals (δ ) 1.3-1.5 and
2.12 ppm) arising from the protons on the decamethylene
segments of free 2 (HE-G(uc)) decrease in their intensities with
time, while new signals, arising from decamethylene segments
onto which the CDs have threaded in 3 (HE-G(c)), appear at δ
) 1.5-1.7 and 2.22 ppm. Additional signals for the complexed
HD and HE-G overlap with the original uncomplexed signals.
The presence of two sets of resonances for HD and HE-G at
equilibrium is attributed to the asymmetry of the two rims of
the CDs, a property that renders each half of the decamethylene
chain chemically nonequivalent.43b
1
of integrations of the H NMR spectra, 25% of the monosac-
charide residues of the chitosan polymer 11 were found to be
substituted with a lactoside residue.
Synthesis of the Polyviologen Polymer. Previously, we had
focused24 our attention on dynamic systems using R- and â-CD
derivatives threaded onto poly(tetrahydrofuran) and poly-
(propylene glycol). We had also observed that when 2,6-di-O-
methlyated CD derivatives were used, the threading/dethreading
1
of the CDs were fast on the H NMR time scale to the extent
that isolating the pseudopolyrotaxanes was not possible. In an
effort to lower the rate of translational motion of the CD beads
along the polymer chain, we decided to investigate polycationic
polymers. Polyviologen AB-copolymers, comprising alternating
decamethylene (A) and positively charged bipyridinium (B)
segments, are known41 to form stable, water-soluble complexes
with R-CD. The positive charges, associated formally with the
(41) (a) Harada, A.; Adachi, H.; Kawaguchi, Y.; Okada, M.; Kamachi, M. Polym.
J. (Tokyo) 1996, 28, 159-163. (b) Herrmann, W.; Keller, B.; Wenz, G.
Macromolecules 1997, 30, 4966-4972.
(36) Dutta, P. K.; Ravikumar, M. N. V.; Dutta, J. J. Macromol. Sci., Polym.
ReV. 2002, C42, 307-354.
(37) Liu, W. G.; Zhang, X.; Sun, S. J.; Sun, G. J.; Yao, K. D. Bioconjugate
Chem. 2003, 14, 782-789.
(42) (a) Hermann, W.; Keller, B.; Wenz, G. Macromolecules 1997, 30, 4966-
4972. (b) Kelch, S.; Caseri, W.; Shelden, R. A.; Suter, U. W.; Wenz, G.;
Keller, B. Langmuir 2000, 16, 5311-5316.
(38) Li, F.; Liu, W. G.; Yao, K. D. Biomaterials 2002, 23, 343-347.
(39) Rabea, E. I.; Badawy, M. E.-T.; Stevens, C. V.; Smagghe, G.; Steurbaut,
W. Biomacromolecules 2003, 4, 1457-1465.
(40) (a) Sashiwa, H.; Makimura, Y.; Shigemasa, Y.; Roy, R. Chem. Commun.
2000, 909-910. (b) Sashiwa, H.; Thompson, J.; Das, S. K.; Shigemasa, Y.;
Tripathy, S.; Roy, R. Biomacromolecules 2001, 1, 303-305. (c) Sashiwa,
H.; Shigemasa, Y.; Roy, R. Bull. Chem. Soc. Jpn. 2001, 74, 937-943.
(43) (a) Lyon, A. P.; Banton, N. J.; Macartney, D. H. Can. J. Chem. 1998, 76,
843-850. (b) Avram, L.; Cohen, Y. J. Org. Chem. 2002, 62, 2639-2644.
(44) The formation of the pseudopolyrotaxane was also monitored by TLC (4:
3:3:2 EtOAc/MeOH/H2O/AcOH) using 5% H2SO4 in EtOH as the develop-
ing solution. The free CD 1 (RF ) 0.5) disappeared over time as a new
spot representing complex 3 appeared at the baseline.
9
J. AM. CHEM. SOC. VOL. 126, NO. 38, 2004 11917

A R T I C L E S
Nelson et al.
Figure 1. 1H NMR spectra (500 MHz, D2O, 298 K) of (a) CD 1, and the mixture of CD 1 and polymer 2 at (b) 0.5 h, (c) 1 d, (d) 2 d, and (e) 4 d after
mixing.
Complexation of the decamethylene segments also influences
the chemical shifts of the protons on the bipyridinium segments
as evidenced by the presence of new signals, HA(c) and HB(c),
in the aromatic region of the 1H NMR spectrum. The presence
of these signals, arising from the CD-occupied segments,
demonstrates that the equilibrium is displaced toward the
formation of the pseudopolyrotaxane. Once equilibrium is
attained (4 days), the spectrum does not change, and even
after a 50-fold dilution of the D2O solution, the spectrum
still stays the same over the course of a week, demonstrating
that the CDs and polymer chains form a stable pseudopoly-
rotaxane.
2D NMR spectroscopy was particularly useful in determining
that the CDs were resting on the decamethylene (and not the
bipyridinium) segments of the polymer. The H-3 and H-5
protons of the CD torus point into the center of its cavity and
therefore serve as probes for the complexation of a guest
molecule. The presence of cross-peaks in the 2D TROESY
NMR of pseudopolyrotaxane 3 between the decamethylene
signals of the polymer and the H-3 and H-5 resonances is
consistent with the close proximity of the decamethylene protons
to the H-3 and H-5 protons as a result of formation of the
complex.
Gal-1-Induced T-cell Agglutination. The ability of Gal-1
to aggregate cells, used to malignant advantage in cancer, formed
the basis of a straightforward assay. Treatment of CEM cells
(cultured human T-leukemia cells) with 10 µM recombinant
human Gal-1 for 5 min at 37 °C resulted in large aggregates
that resemble tumor emboli (Figure 2i,ii). As determined by
light microscopy, pretreating Gal-1 with 2 mM lactose (final
concentration) for 10 min at room temperature allows the cells
to remain largely dispersed (Figure 2iii), whereas a mixed state
of aggregated and dispersed cells (Figure 2iv) is observed down
to 1.5 mM lactose. A value of 1.5 mM for the minimum
inhibitory concentration (MIC) of lactose with Gal-1 matches
well with the literature values in other agglutination assays.33b,45
The trivalent glycoclusters, the chitosan-derived polymer, the
pseudopolyrotaxane, and its components were evaluated quali-
tatively using this T-cell agglutination assay.
Evaluation of the Trivalent Glycoclusters with Gal-1. The
trivalent lactosides 8 and 9 completely inhibited Gal-1-induced
aggregation (Table 1) at a concentration of 0.7 mM of the
glycocluster in solution, with the trivalent lactoside 10 perform-
ing nominally better (0.6 mM). On a valency-corrected basis,
taking into account three lactose residues per glycocluster, the
performance of trivalent glycoclusters 8 and 9 was equivalent
to native lactose, while the tris-lactoside 10 exhibited a slight
enhancement to attain the dispersed state (Figure 3iv-vi). The
lowest concentration of the mixed state (0.5 mM) was the same
for all three trivalent lactosides, and this valency-corrected MIC
(1.5 mM) is identical to lactose. Thus, the trivalent compounds,
at best, demonstrated only a small improvement in inhibiting
Gal-1-induced cell agglutination.
Table 1. Gal-1 Inhibition with Glycoclusters 8, 9, and 10a
glycocluster
concn [ M]
lactose- and valency-
corrected concn [mM]
cluster
cluster
cluster
µ
lactose
8
9
10
800
700
600
500
400
300
200
100
2.4
2.1
1.8
1.5
1.2
0.9
0.6
0.3
D
D
M
M
A
A
A
A
D
D
M
M
A
A
A
A
D
D
M
M
A
A
A
A
D
D
D
M
A
A
A
A
a Series of concentrations tested for lactose and each glycocluster at which
an aggregated (A), mixed (M), or dispersed (D) state of T-cells was observed
in the presence of 10 µM galectin-1 in the agglutination assay. The
compounds were premixed with galectin-1 for 10 min at ambient temper-
ature before being incubated with the CEM cells for 5 min at 37 °C. Each
compound was tested at least three times at each concentration.
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11918 J. AM. CHEM. SOC. VOL. 126, NO. 38, 2004

Multivalent Pseudopolyrotaxane for Binding Gal-1
A R T I C L E S
Figure 2. Light microscopy images (10×) of CEM cells in the presence or absence of Gal-1 and inhibitors. (i) In the absence of Gal-1, cells are dispersed
(D). (ii) With 10 µM Gal-1 cells are aggregated (A). (iii) With 10 µM Gal-1 and 2 mM lactose, cells are dispersed (D). (iv) With 10 µM Gal-1 and 1.8 mM
lactose, cells are in a mixed state (M). (v) With 10 µM Gal-1 and 1.8 mM (valency-corrected) 8, cells are in a mixed state (D). (vi) With 10 µM Gal-1 and
1.8 mM (valency-corrected) 10, cells are dispersed (M).
Evaluation of Chitosan-Based Polymer with Gal-1. The
HCl salt of the commercially available chitosan polymer was
not inhibitory at over 9 mM of the repeating unit. When 25%
of the monosaccharides of this polymer were substituted with
the galactityl-linked lactoside to yield neoglycopolymer 11, an
inhibitor was obtained. Cells were dispersed (Table 2) with 1.8
mM 11 and exhibited a mixed state down to 900 µM 11
(concentrations based on lactoside). This valency-corrected MIC
of 900 µM compares favorably to monovalent lactose (1.5 mM),
but only by 1.7-fold.
valency-corrected MICs of 1 mM and 0.9 mM. In fact, CD 1
attains the dispersed state at a lower concentration (1.5 vs 1.8
mM, based on lactoside concentration) than does the polymer
11. The monovalent CD 1 also outperforms the trivalent
lactosides on a valency-corrected basis. Considerably more
potent inhibition was uncovered, however, when the CD 1 was
presented multivalently in the pseudopolyrotaxane 3.
Table 3. Gal-1 Inhibition with CD 1 and Pseudopolyrotaxane 3a
concn (valency-corrected)[mM]
lactose
CD 1
complex 3
2.0
1.5
1.0
0.75
0.5
0.25
0.15
0.1
D
M
A
A
A
A
A
A
A
D
D
M
A
A
A
A
A
A
D
D
D
D
M
M
M
A
A
Table 2. Gal-1 Inhibition with the Chitosan 11a
concn (valency-corrected)[mM]
lactose
chitosan 11
1.8
1.7
1.5
1.3
0.9
0.75
0.6
M
M
M
A
A
A
A
D
M
M
M
M
A
0.05
a Series of valency-corrected concentrations tested for lactose, CD 1,
and pseudopolyrotaxane 3 at which an aggregated (A), mixed (M), or
dispersed (D) state of T-cells was observed in the presence of 10 µM
galectin-1 in the agglutination assay. The compounds were premixed with
galectin-1 for 10 min at ambient temperature before being incubated with
the CEM cells for 5 min at 37 °C. Each compound was tested at least three
times at each concentration.
A
a Series of valency-corrected concentrations tested for lactose and chitosan
11 at which an aggregated (A), mixed (M), or dispersed (D) state of T-cells
was observed in the presence of 10 µM galectin-1 in the agglutination assay.
The compounds were premixed with galectin-1 for 10 min at ambient
temperature before being incubated with the CEM cells for 5 min at 37 °C.
Each compound was tested at least three times at each concentration.
The assays revealed a dramatic effect of the self-assembled
multivalent pseudopolyrotaxane upon the inhibition of Gal-1-
induced cell agglutination. (It should be noted that the concen-
trations recorded in Figure 3 are “valency-corrected” based on
lactose residues.) Controls of the polymer 2 alone and the
pseudopolyrotaxane formed with native R-CD (without lactose)
were not inhibitory at concentrations equivalent to the highest
tested for 3. At identical “lactose” concentrations (horizontal
comparisons in Figure 3), large differences are observed in the
response of the cells to Gal-1; for example, at a 1 mM “lactose”
concentration, the cells (i) remain aggregated when they are
treated with native lactose, (ii) assume a mixed state of
Evaluation of Pseudopolyrotaxanes with Gal-1. Interest-
ingly, the lactoside-bearing R-CD had a noticeable shift in the
onset of aggregation compared (Table 3) to native lactose.
Whereas a mixed state was not observed below 1.5 mM of lac-
tose or 500 µM trivalent lactoside, the cells were still dispersed
at 1.5 mM of the CD 1 and the mixed state was observed at 1
mM of the CD 1 (Figure 3ii). Thus, the monovalent CD 1
performed similarly to the rigid chitosan-based polymer 11 with
(45) Ahmad, N.; Gabius, H.-J.; Kaltner, H.; Andre´, S.; Kuwabara, I.; Liu, F.-
T.; Oscarson, S.; Norberg, T.; Brewer, C. F. Can. J. Chem. 2002, 80, 1096-
1104.
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A R T I C L E S
Nelson et al.
Figure 3. Pictures (i-ix) taken by light microscopy (10×) of CEM cells with 10 µM Gal-1 and varying concentration of lactose (column 1), CD 1 (column
2), and pseudopolyrotaxane 3 (column 3). Valency-corrected concentrations are shown.
aggregated and dispersed cells when treated with 1, and (iii)
become dispersed when treated with 3. At 750 µM, the lactose
and 1 treated cells are mostly aggregated (iv and v) while the
cells treated with 3 remain dispersed (vi). With 3, a mixed
population (aggregated and dispersed) is observed (vii, viii, and
ix) down to 150 µM of “lactose”. It is of note that, at 150 µM
“lactose”, the concentration of individual polymer chains is 8.8
µM, a value just below the concentration of Gal-1. A comparison
of the lowest concentrations at which the mixed populations
are observed revealed a 6.7-fold improvement of 3 over 1 and
a 10-fold improvement of 3 over lactose itself.
synthesized by Roy and co-workers33a displayed excellent
multivalent enhancements (on a per-lactoside basis) for binding
Gal-1 in solid-phase competition assays. However, these assays
yield highly variable results, depending on the nature of the
competitor and the surface-immobilized species.33 To our
knowledge, multivalent enhancements for the inhibition of Gal-1
in cell-based assays of over 16-fold have not been achieved.33e
For the purpose of comparing the pseudopolyrotaxane with
other multivalent structures, the trivalent lactosides 8, 9, and
10 and chitosan-based polymer 11 were synthesized and
evaluated in the agglutination assay. In our hands, the valency-
corrected MIC for the trivalent lactosides 8, 9, and 10 showed
no improvement over native lactose as inhibitors of Gal-1-
induced T-cell agglutination. These three glycoclusters differ
in their size, shape, and flexibility, yet behave similarly in the
assays. Although all three glycoclusters had identical valency-
corrected MICs compared to that of lactose, the dispersed state
for the largest glycocluster 10 was observed at a lower
concentration than those for the other two glycoclusters.
The rigid chitosan polymer 11 bearing lactose epitopes
showed only a slight enhancement over native lactose in inter-
acting with Gal-1 in the assay. While chitosan-based 11 and
the pseudopolyrotaxane 3 are both polymeric structures, we
observed dramatic differences in their interactions with Gal-1.
The relatively small enhancement in the valency-corrected MIC
(1.7-fold over native lactose) in the binding of Gal-1 by the
Discussion
The topology of Gal-1, where the binding sites are located
on opposite faces of the protein, provides a lectin that is
challenging to target multivalently. A number of glycoclusters
have been investigated,33 with variable results, ranging from
negligible33d to potent33b in vitro assays and up to 14.5-fold
valency-corrected enhancement in cellular assays.33b Glyco-
clusters with selectivities for galectin-3 over Gal-1 have been
reported in the literature.33c,d Recently, Gabius and co-workers33e
have described 2.8-fold and 5.9-fold valency-corrected enhance-
ments for per-substituted lactose and N-acetyllactosamine â-CD
glycoclusters, respectively, in a Gal-1-mediated hemagglutina-
tion assay. These â-CD glycoclusters displayed a higher activity
toward galectin-3 by up to 28-fold. Starburst glycodendrimers
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11920 J. AM. CHEM. SOC. VOL. 126, NO. 38, 2004

Multivalent Pseudopolyrotaxane for Binding Gal-1
A R T I C L E S
polymer 11 may be attributed to the rigidity of the polymer
backbone, which could prevent binding to the lectins in the same
manner as the pseudopolyrotaxane 3 where a 10-fold enhance-
ment was observed. It is of note that Roy and co-workers40b
have successfully targeted plant lectins using R-gal and sialic
acid epitopes appended to chitosan by reductive amination.
While neogylcopolymers have not been widely explored46 for
binding Gal-1, lactoside-functionalized polymers with N-(2-
hydroxypropyl)methacrylamide47 and poly(norbornene-imide)48
backbones, both of which are more flexible than that of chitosan,
have been used to target Gal-3.
protein asialofetuin. This enhancement is higher than was
observed in a hemagglutination assay with â-CD bearing seven
lactosides.33e While monosubstituted R-CD has been employed
in this present study, many research groups are utilizing per-
substituted CDs as glycoclusters for multivalent display.49
Pseudopolyrotaxanes formed with per-substituted CDs will have
a greater local concentration of ligand, which may increase the
potency of the complex.
We emphasize here that the pseudopolyrotaxane 3 is not a
single molecule, but rather a supramolecular species. On
average, it is the confluence of ca. 18 different molecules (the
polyviologen “string” plus ca. 17 CD “beads”) that produce the
multivalent effect. There are many examples50 in biology where
individual proteins self-assemble into superstructures wherein
the complexes gain or enhance functions relative to those of
the individual components. Although the time scale of assembly
(4 days) is slower as a result of the viologen units on the polymer
backbone, the pseudopolyrotaxane 3 is a rudimentary example
of this phenomenon. The ligands, bound by noncovalent
interactions, are able to rotate freely and independently around
the polymer backbone. The CDs can translate within the
confines of the decamethylene units, which have a length that
is twice the depth of a CD cavity. Not only do the lactoside
ligands have the ability to “fine tune” their positions without
enthalpic penalties having to be paid on account of inducing
strain, but the unbound ligands may also reside away from the
bulky protein in order to reduce steric clashes. The polymer
backbone influences directly the dynamics of the translational
movement of the CDs. Whereas the polyviologen contains
electrostatic “speed bumps” that reduce the translational motion
of the CD beads, other polymers, such as poly(ethylene glycol)
and poly(tetrahydrofuran), allow the free movement of the CDs
along the polymer. As the biochemistry involving unnatural
supramolecular species and mechanically interlocked molecules
evolves and matures, the dynamics of ligand display will shed
light on the supramolecular chemistry of natural systems.
Figure 4. Possible binding modes for Gal-1 and pseudopolyrotaxane 3.
The pseudopolyrotaxane 3, the product of numerous self-
assembling components, contains mobile ligands bound by
noncovalent interactions, with the ability to rotate and translate
on the polymer backbone, which may be important factors in
its exhibiting enhanced avidity for Gal-1. In contrast to chitosan,
the polyviologen backbone of the pseudopolyrotaxane 3 pos-
sesses flexible decamethylene units. The flexibility afforded by
the polymer backbone, in conjunction with the dynamic property
of its lactoside ligands, affords a supramolecular structure with
the fluidity and adaptability of cellular membranes, the biologi-
cal target of Gal-1.29a,e Since the lectin is itself bivalent and the
pseudopolyrotaxane 3 is up to 17-valent (on the basis of the
average number of repeating units in the polyviologen), many
binding modes between the protein and pseudopolyrotaxane are
possible (Figure 4). The 10-fold enhancement in the valency-
corrected MIC is on the same order of magnitude as that
observed by Yui and co-workers28 for their CD-based poly-
(ethylene glycol) polyrotaxanes displaying variable amounts of
maltose residues in a concanavalin A hemagglutination assay.
These authors found that the degree of inhibition depended on
both the number of carbohydrate ligands and the dynamics of
the CDs to which they were attached. The 10-fold enhancement
observed in our assay is also on the same order of magnitude
as has been observed in cellular assays for Gal-1 inhibition33b
with wedge-like multivalent lactoside dendrons and the glyco-
Conclusion
A supramolecular species formed from almost 20 self-
assembling components has been used successfully to inhibit
Gal-1 in a T-cell agglutination assay. While other multivalent
ligands tested displayed little to no enhancement in the valency-
corrected MIC, a 6.7-fold improvement over the lactoside-CD
1 and a 10-fold improvement over lactose itself was observed
for the lactoside-displaying CD-based polyviologen pseudopoly-
rotaxane 3. We are pursuing actively a more detailed charac-
terization of the structural and thermodynamic nature of the
multivalent interactions between 3 and Gal-1. Self-assembled
pseudopolyrotaxanes, such as 3, display highly mobile ligands
as a result of the CD rotating about the polymer chain while
exhibiting limited translation along them. This flexible and
dynamic ligand presentation adds a new dimension to the study
of protein-carbohydrate interactions and the exploitation of
multivalency for targeting therapeutically relevant lectins.
(46) Neoglycoproteins derived from bovine serum albumin have been investi-
gated, especially as matrixes in solid-phase assays. See: (a) ref 33. (b)
Stonewell, S. R.; Dias-Baruffi, M.; Penttila, L.; Renkonen, O.; Nyame, A.
K.; Cummings, R. D. Gylcobiology 2004, 14, 157-167. (c) Andre´, S.;
Unverzagt, C.; Kojima, S.; Frank, M.; Seifert, J.; Fink, C.; Kayser, K.;
von der Lieth, C.-W.; Gabius, H.-J. Eur. J. Biochem. 2004, 271, 118-
134.
(47) David, A.; Kopeckova, P.; Minko, T.; Rubinstein, A.; Kopecek, J. Eur. J.
Cancer 2004, 40, 148-157.
(48) Pohl, N.; Kiessling, L. L. Synthesis 1999, 1515-1519.
(49) (a) Ortiz Mellet, C.; Defaye, J.; Garc´ıa Ferna´ndez J. M. Chem.-Eur. J.
2002, 8, 1983-1990. (b) Fulton, D. A.; Stoddart, J. F. Bioconjugate Chem.
2001, 12, 655-672.
(50) (a) Wikoff, W. R.; Liljas, L.; Duda, R. L.; Tsuruta, H.; Hendrix, R. W.;
Johnson, J. E. Science 2000, 289, 2129-2133. (b) Ogata, K.; Sato, K.;
Tahirov, T. Curr. Opin. Struct. Biol. 2003, 13, 40-48. (c) Pillar, S. C.;
Caly, L.; Jans, D. A. Curr. Drug Targets 2003, 4, 409-429.
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A R T I C L E S
Nelson et al.
aqueous solution was allowed to stand 4 days at ambient temperature.
The threading process was monitored by TLC using 4:3:3:2 EtOAc/
MeOH/H2O/AcOH and 5% H2SO4 in EtOH as the developing agent.
This stock solution (20 mM per molar lactose) was diluted accordingly
to provide the appropriate per molar lactose concentrations for the
agglutination assay.
Agglutination Assay. Lactoside (6.26 × final concentration) and
recombinant human51 Gal-1 (62.6 µM) were mixed in phosphate-
buffered saline (8 µL) at room temperature for 10 min. A solution (42
µL) of 7.5 × 106 CEM cells/mL in serum-free RPMI 1640 media was
added. The mixture (10 µM galectin-1, 3.15 × 105 cells) was allowed
to stand for 5 min at 37 °C. The mixture was vortexed briefly, and an
aliquot (10 µL) was taken for imaging by light microscopy. At least
three independent experiments were performed at each lactoside
concentration. CEM cells (subclone of ATCC no. CCL-119) were
grown in RPMI 1640 medium, supplemented with 10% fetal bovine
serum, 10 mM HEPES, and 2 mM L-glutamine at 37 °C and 5% CO2.
Experimental Section
General Methods. Tris-methylamino core compound 4,22b trisac-
charide 7,22b and CD 124 were prepared as described in the literature.
CD 1 was prepared as described in the literature.24 All other chemicals
were purchased from Aldrich and used as received. Solvents were used
as purchased, except for CH2Cl2 (distilled from CaH2) and MeOH
(distilled from Mg turnings). Analytical TLC was performed on silica
gel 60-F254 (Merck) with detection by fluorescence and/or by charring
following immersion in a 5% H2SO4/EtOH. Flash chromatography was
performed with silica gel 60 (Silicycle). Preparative gel permeation
chromatography was performed using (1) a 25 × 900 mm2 column of
Sephadex LH20 resin (Sigma), eluting with MeOH or (2) a 5 × 100
cm2 column of Sephadex G-25 resin (Amersham Pharmacia Biotech),
eluting with 5% nBuOH in water. High-resolution matrix-assisted laser
desorption ionization (HR-MALDI) mass spectra were recorded using
dihydroxybenzoic acid as a matrix and external calibration using
1
substance P on an IonSpec Ultima 7.4 T FTMS instrument. H and
13C NMR spectra were recorded on a Bru¨ker Avance 500 spectrometer
(at 500 and 125 MHz, respectively) or Bru¨ker Avance 600 spectrometer
(at 600 and 150 MHz, respectively) at ambient temperature using D2O
as the solvent.
Acknowledgment. We thank Joseph Hernandez, Mabel Pang,
and Luciana Kohatsu for their advice and guidance. This work
was supported by the National Institutes of Health through the
award of a postdoctoral fellowship to J.M.B. and Grant R01
GM63281, in addition to support from the National Science
Foundation (equipment Grants CHE-9974928).
Polyviologen 2. 1,10-Dibromodecane (2.6 g, 16.7 mmol) and 4,4′-
bipyridine (5 g, 16.7 mmol) were dissolved in 1:1 MeOH/DMF (16
mL), and the reaction mixture was stirred for 20 h at 75 °C. It was
diluted with H2O (50 mL) and then extracted with CHCl3 (50 mL).
The aqueous layer was concentrated under vacuum to afford a dark
Supporting Information Available: Experimental procedures,
data from the agglutination assays, and NMR spectra (PDF).
This material is available free of charge via the Internet at
http://pubs.acs.org.
1
yellow solid (6.5 g). H NMR (500 MHz, D2O) δ ) 1.13-1.5 (m,
HE-G), 2.12 (br s, HD), 4.68 (br t, HC, J ) 7.4 Hz), 7.98 (br d, HB from
end groups), 8.34 (br d, HB′, from end groups), 8.51 (d, HB, J ) 6.4
Hz), 8.78 (br s, HA from end groups), 8.94 (d, HA′, from end groups),
9.08 (d, HA, J ) 6.4 Hz).
JA0491073
Pseudopolyrotaxane 3. The polyviologen 2 (4.5 mg, 0.58 µmol)
was added to 0.5 mL of a 20 mM solution of the CD 1 in H2O. This
(51) Pace, K. E.; Hahn, H. P.; Baum, L. G. Methods Enzymol. 2003, 363, 499-
518.
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11922 J. AM. CHEM. SOC. VOL. 126, NO. 38, 2004