Synthesis of N,N′-bis(acrylamido)acetic acid-based T-antigen glycodendrimers and their mouse monoclonal IgG antibody binding properties

Article abstract of DOI:10.1021/ja002596w

Novel glycodendrimers based on N,N′-bis(acrylamido)acetic acid core with valencies between two and six were synthesized. The breast cancer-associated T-antigen carbohydrate marker, (β-Gal-(1-3)-α-GalNAc-OR), was then conjugated by (i) 1,4-conjugate additi

Full text of DOI:10.1021/ja002596w

J. Am. Chem. Soc. 2001, 123, 1809-1816
1809
Synthesis of N,N′-bis(Acrylamido)acetic Acid-Based T-Antigen
Glycodendrimers and Their Mouse Monoclonal IgG Antibody
Binding Properties
Rene´ Roy,*,† Myung-Gi Baek,§,† and Kate Rittenhouse-Olson‡
Contribution from the Department of Chemistry, UniVersity of Ottawa,
Ottawa, Ontario K1N 6N5, Canada, and the Department of Microbiology and Immunology,
Roswell Park DiVision, State UniVersity of New York at Buffalo, Buffalo, New York 14214
ReceiVed July 14, 2000
Abstract: Novel glycodendrimers based on N,N’-bis(acrylamido)acetic acid core with valencies between two
and six were synthesized. The breast cancer-associated T-antigen carbohydrate marker, (â-Gal-(1-3)-R-GalNAc-
OR), was then conjugated by (i) 1,4-conjugate addition of thiolated T-antigen to the N-acrylamido dendritic
cores and by (ii) amide bond formation between an acid derivative of the T-antigen and the polyamino
dendrimers. The protein-binding ability of these new glycodendrimers was fully demonstrated by turbidimetric
analysis and by enzyme-linked immunosorbent assay (ELISA) using peanut lectin from Arachis hypogaea
and a mouse monoclonal antibody (MAb) FAA-J11 (IgG3). When tested as inhibitors of binding between
MAb and a polymeric form of the T-antigen (T-antigen-co-polyacrylamide) used as a coating antigen, di-
(17), tetra- (20), hexa- (21), and tetravalent (22) dendrimers showed IC50 values of 174, 19, 48, and 18 nM,
respectively. Two tetramers showed 120- to ∼128-fold increased inhibitory properties over the monovalent
antigen 6 used as a standard (IC50 2.3 mM). Heterobifunctional glycodendrimer bearing a biotin probe was
also prepared for cancer cell labeling.
Introduction
The Thomsen-Friedenreich (T or TF) carbohydrate antigen
(T-Ag), â-Gal-(1-3)-R-GalNAc, has been well-documented as
a cancer-related marker1 and as an important antigen for the
detection and immunotherapy of carcinomas, particularly rel-
evant to breast cancer. In related pharmaceutical applications,
T-Ag-containing glycopolymers (Figure 1) have been employed
in high-throughput screening toward the development of a solid-
phase glycosyltransferase assay for drug discovery research.2
Mouse monoclonal antibodies, JAA-F11 (IgG3) and C5 (IgM)
were also recently developed for successful immunohistochemi-
cal staining of breast adenocarcinomas.3 These antibodies were
obtained from T-antigen-BSA conjugates (Figure 1) that did
not contain any peptide segment of the natural T-antigen present
on mucins, thus demonstrating the thorough carbohydrate nature
of the cancer marker. The expression of T-Ag has been proposed
Figure 1. Structure of T-antigen bearing neoglycoconjugates.
† University of Ottawa.
as a tool for the detection of tumors and as a criterion for
prognosis.4 Moreover, there is an increased expression of T-Ag
in metastatic tumors, and a plant lectin (Arachis hypogaea) that
recognizes T-Ag has been shown to bind to common sites of
metastatic tumor growth.5 Receptors for T-Ag adhesion have
been proposed to be in the liver, the bone marrow, and the lymph
nodes.
With the necessary antibodies in hand, it therefore became
of interest to synthesize nonimmunogenic, high-affinity T-Ag-
containing glycodendrimers that could be used to block the
potential metastatic sites of invasion tumors cells. These
molecules might be particularly useful after surgery to protect
‡ State University of New York at Buffalo.
§ Current address: National Research Council, Steacie Institute for
Molecular Sciences, 100 Sussex Dr., Ottawa, Ontario K1A 0R6 Canada.
(1) (a) Livingston, P. O.; Koganty, R.; Longenecker, B. M.; Lloyd, K.
O.; Calves, M. Vaccine Res. 1992, 1, 99. (b) Springer, G. F.; Murthy, M.
S.; Desai, P. R.; Scanlon, E. F. Cancer 1980, 45, 2949. (c) Itzkowitz, S.
H.; Yuan, M.; Montgomery, C. K.; Kjeldsen, T.; Takahashi, H. K.; Bigbee,
W. L.; Kim, Y. S. Cancer Res. 1989, 49, 197. (d) Bray, J.; Leumieux, R.
U.; McPherson, T. A. J. Immunol. 1981, 126, 1966. (e) Kichler, A.; Schuber,
F. Glycoconjugate J. 1995, 12, 275. (f) Ho¨ppner, W.; Fischer, K.;
Poschmann, A.; Paulsen, H. Mol. Immunol. 1985, 22, 1341. (g) Hung, P.
Y. S.; Madej, M.; Koganty, R. R.; Longenecker, B. M. Cancer Res. 1990,
50, 4308. (h) Chen, Y.; Jain, R. K.; Chandrasekaran, E. V.; Matta, K. L.
Glycoconjugate J. 1995, 12, 55.
(2) (a) Donovan, R. S.; Datti, A.; Baek, M. G.; Wu, Q. Q.; Sas, I. J.;
Korczak, B.; Berger, E. G.; Roy, R.; Dennis, J. W. Glycoconjugate J. 1999,
16, 607. (b) Baek, M.-G.; Roy, R. Biomacromolecules 2000, 1, 768.
(3) Rittenhouse-Diakun, K.; Xia, Z.; Pickhardt, D.; Morey, P. S.; Baek,
M. G.; Roy, R. Hybridoma 1998, 17, 165.
(4) Maclean, G. D.; Longenecker, B. M. Semin. Cancer Biol. 1991, 2,
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Histochem. Cytochem. 1990, 38, 1625.
10.1021/ja002596w CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/13/2001

1810 J. Am. Chem. Soc., Vol. 123, No. 9, 2001
Roy et al.
Scheme 1a
a Hg(CN)2, PhH/CH3NO2, room temp, 98%; (b) (i) CH3O-Na+/MeOH, (ii) aq AcOH, 60 °C, quant; (c) AcSH/AIBN, MeOH, reflux, 71%, then
CH3O-Na+/MeOH, quant; (d) HSCH2CH2NH2 (13) or (e) HSCH2CH2CO2H, hV (254 nm), water, 63 or 83%; (f) AcSH/AIBN, MeOH, heat, 71%;
(g) CH3O-Na+/MeOH, quant.
against the spreading of cancer cells that have evaded the sites
of intervention. Toward this goal, we synthesized and evaluated
the relative binding properties of a new type of T-antigen
dendrimers (17, 20, 21, and 22) with valencies varying between
two and six on the basis of N,N′-bis(acrylamido)acetic acid (11)
used as the seed molecule. The cluster effect of the T-antigen
dendrimers was also evaluated toward the highly specific protein
from the plant lectin of A. hypogaea and to our mouse MAb
JAA-F11 in comparison to monomeric T-antigen (6).
Multivalent neoglycoconjugates are excellent tools for the
understanding of multiple carbohydrate-protein binding interac-
tions via cooperative association energies.6 Several glyco-
polymers,7-9 including our recently synthesized T-Ag copoly-
acrylamides,2 showed strong inhibitory properties against a wide
range of carbohydrate receptors. However, glyco-co-polymers
have ill-defined chemical structures with random distribution
of carbohydrates, and some condensation polymers are too
dense.
Due to the potential applications in medicinal engineering,
drug delivery, gene- and chemo-therapy, sensory devices, and
as bioactive function carriers with immunochemical and phar-
maceutical applications, monodisperse high-molecular-weight
dendrimers have stimulated wide interest in the field of
chemistry and biology.10 Recently, novel classes of nonimmu-
nogenic glycodendrimers have provided a better understanding
of multiple-carbohydrate-protein interactions.11-13
As cell surface carbohydrates are involved in a wide variety
of important biological phenomena, such as recognition of
bacteria, viruses, hormones, and other cells; cell growth;
regulation and differentiation; cancer metastasis; and cellular
trafficking;14 it can be stated that chemically well-defined
glycoconjugates (glycodendrimers) provide a better understand-
ing of biological recognition processes by virtue of well-
organized carbohydrate valencies, shapes, and orientations in
comparison to random copolymers. Many such glycodendrimers
containing diverse carbohydrate ligands have been reported.11-13
Results and Discussion
Although several syntheses of the T-Ag with various agly-
cones exist,15 we chose to synthesize the required allyl T-antigen
(â-D-Gal-(1-3)-R-D-GalNAc-O-allyl) in large scale from 1 and
2 under classical Helferich glycosylation method in excellent
yields (PhH/MeNO2, Hg(CN)2, 78 to ∼98%, respectively)
(Scheme 1). The T-antigen derivatives 3 and 4 were deprotected
under Zemple´n conditions (NaOMe, MeOH), followed by
aqueous acetic acid hydrolysis of the benzylidene acetal to afford
6 quantitatively. The thiol functionality was introduced onto
the alkenyl moiety of 4 and 6 using photochemical activation
with thioacetic acid. The resulting thioacetates were successively
treated under Zemple´n conditions to provide 5 and 8 (degassed
MeOH, reflux, 82 and 71%, respectively). Other functional
groups such as acid (9) and amine (10) were also introduced
via sulfide linker formation using thiol derivatives such as
3-mercaptopropionic acid and cysteamine (13) (254 nm, de-
gassed water, 63 and 83% yields, respectively).
(6) (a) Bundle, D. R.; Young, N. M. Curr. Opin. Struct. Biol. 1995, 2,
666. (b) Vyas, N. K. Curr. Opin. Struct. Biol. 1996, 6, 732. (c) Toone, E.
J. Curr. Opin. Struct. Biol. 1991, 4, 719.
(7) (a) Roy, R. In Carbohydrate Chemistry; Boon, G.-J., Ed.; Blackie
Academic & Professional: London, U.K. 1998, p 243. (b) Roy, R. Curr.
Opin. Struct. Biol. 1996, 6, 692. (c) Roy, R. Top. Curr. Chem. 1997, 187,
241. (d) Roy, R. Trends Glycosci. Glycotechnol. 1996, 8, 79.
(8) Mammen, M.; Choi, S. K.; Whitesides, G. M. Angew. Chem., Int.
Ed. Engl. 1998, 37, 2754.
Synthesis of Dendritic Cores Based on N,N′-Bis(acryl-
amido)acetic Acid. Due to its bifunctionality and its commercial
accessibility, N,N′-bis(acrylamido)acetic acid (11) was chosen
(13) Ashton, P. R.; Hounsell, E. F.; Jayaraman, N.; Nilsen, T. M.;
Spencer, N.; Stoddart, J. F.; Young, M. J. Org. Chem. 1998, 63, 3429.
(14) (a) Varki, A. Glycobiology 1993, 3, 97.
(9) (a) Bovin, N. V.; Gabius, H.-J. Chem. Soc. ReV. 1995, 24, 413. (b)
Kiessling, L. L.; Pohl, N. L. Chem. Biol. 1996, 3, 71.
(10) (a) Ardoin, N.; Astruc, D. Bull. Soc. Chim. Fr. 1995, 132, 875. (b)
Fre´chet, J. M. J. Science 1994, 263, 1710. (c) Gensch, T.; Hofkens, J.;
Heirmann, A.; Tsuda, K.; Verheijen, W.; Vosch, T.; Christ, T.; Basche´, T.;
Mu¨ller, K.; De Schryver, T. C. Angew. Chem., Int. Ed. Engl. 1999, 38,
3752. (d) Issberner, J.; Moors, R.; Vo¨gtle, F. Angew. Chem., Int. Ed. Engl.
1994, 33, 2413.
(11) (a) Roy, R. Polymer News 1996, 21, 226. (b) Roy, R. Carbohydr.
Eur. 1999, 27, 34. (b) Zanini, D.; Roy, R. J. Am. Chem. Soc. 1997, 119,
2088. (c) Zanini, D.; Roy, R. J. Org. Chem. 1996, 61, 7348.
(12) Lindhorst, T. K.; Kieburg, C. Angew. Chem., Int. Ed. Engl. 1996,
35, 1953.
(15) (a) Danishefsky, S. J.; Allen, J. R. Angew. Chem., Int. Ed. Engl.
2000, 39, 837. (b) Paulsen, H.; Peters, S.; Bielfeldt, T.; Meldal, M.; Bock,
K. Carbohydr. Res. 1995, 268, 17. (c) Kunz, H.; Birnbach, S.; Wernig, P.
Carbohydr. Res. 1990, 202, 207. (d) Kinzy, W.; Schmidt, R. R. Carbohydr.
Res. 1987, 166, 265. (e) Qiu, D.; Gandhi, S. S.; Koganty, R. R. Tetrahedron
Lett. 1996, 37, 595. (f) Lubineau, A.; Bienayme´, H. Carbohydr. Res. 1991,
212, 267. (g) Gambert, U.; Thiem, J. Carbohydr. Res. 1997, 299, 85. (h)
Toyokuni, T.; Singhal, A. K. Chem. Soc. ReV. 1995, 231. (i) Bay, S.;
Berthier-Vergnes, O.; Cantacuzene, D. Carbohydr. Res. 1997, 298, 153.
(j) Roy, R.; Tropper, F. D.; Romanowska, A.; Jain, R. K.; Piskorz, C. F.;
Matta, K. L. Bioorg. Med. Chem. Lett. 1992, 2, 911.

T-Antigen Glycodendrimers
J. Am. Chem. Soc., Vol. 123, No. 9, 2001 1811
Scheme 2a
Scheme 3
Scheme 4
a SOCl2/MeOH, -15 f 25 °C, 80%; (b) for 16: (i) HSCH2CH2NH2
(13), MeOH, DIPEA, overnight, 25 °C, (ii) OH- resin, 93%; for 17:
(i) 8, Et3N, MeOH, 25 °C, N2, quant; (c) H2N(CH)6NH2 (14), HOBt/
EDC/DIPEA, DMSO, overnight, 60%; (d) N(CH2CH2NH2)3 (15).
as a building block. Compound 11 was esterified to give methyl
ester 12 (SOCl2, MeOH, 80%). Subsequently, 12 was coupled
with cysteamine 13 to provide a molecule with extended spacer
length under mild condition to give bis-amino methyl ester 16
in an excellent yield [(i) 13, DIPEA, MeOH; (ii) HO-, Amberlite
IRA-400, 93%] (Scheme 2).
Acid 11 was also successfully coupled to thiolated T-antigen
10 by a 1,4-conjugate addition to give divalent cluster 17 (Et3N,
degassed MeOH, quantitative). Construction of tetra- and hexa-
valent dendritic cores 18 and 19 were efficiently accomplished
using a carbodiimide-hydroxybenzotriazole (EDC/HOBt) method
(Scheme 2). Briefly, 11 was coupled with 1,6-hexamethylene-
diamine (14) or tris(2-ethylamino)amine (15) to afford the
corresponding glycodendrimer precursors 18 and 19, which were
separated from the diisopropylcarbodiimide urea by precipitation
(60 and 62%, respectively).
The synthesis of the small glycoclusters was based on the
coupling of dendritic cores and the T-antigen derivatives by
Michael-type addition of thiolated carbohydrates or by peptide
coupling, depending on the functionality of each component,
to give the corresponding glycodendrimers with valencies
between four and six. Thus, tetravalent T-antigen dendrimer 20
was prepared as follows: Pre-built tetravalent acrylamido
derivative 18 was treated with a slight excess of thiolated
T-antigen 8 (Et3N, degassed DMSO/MeOH, N2). Alternatively,
1,6-hexamethylenediamine 14 was coupled to divalent T-antigen
17 (TBTU, DIPEA, DMSO) to give tethered T-Ag dendrimer
20 in 50% yield. Dendrimer 20 was isolated by gel permeation
chromatography (GPC, Bio-gel P-2) using water as eluent. The
hexavalent cluster 21 was obtained from either thiolated 8 and
hexavalent core 19 (1,4-conjugate addition) or, alternatively,
from trisamine 15 and acid 17 (amide coupling), as described
above, in 45 and 46% yields, respectively (Scheme 4).
Complete couplings between the dendritic cores and the
T-antigen were verified by 1H NMR spectra for compounds 17,
20, and 21. Disappearance of the acryloyl group at δ 6.35, 6.12,
and 5.62 ppm (DMSO-d6) and the emergence of new signals
corresponding to the anomeric protons of the disaccharide (δ
4.96 and 4.55 ppm, D2O) and the integration of these signals
relative to methylene signals of the cores fully established the
complete extent of T-antigen incorporation.
An additional tetravalent T-antigen cluster 22 was analogously
obtained from diamine methyl ester 16 and divalent T-antigen
17 using carbodiimide coupling as above (55%) (Scheme 5).
The strategy described for the efficient syntheses of the
glycoclusters follows a convergent approach that was shown
to be efficient to provide all the desired products in good yields.
The chemical characterization of the dendritic structures was
straightforward. Structural analysis of each dendrimer was
performed by high-field NMR spectroscopy using the well-
resolved doublets at δ 4.96 and 4.55 ppm, which correspond to
the two anomeric protons relative to other signals, especially
from the methylene or methine signals of the core.
For the purpose of broadening the potential application of
these conjugates, a heterobifunctional glycocluster 24 was also
prepared using divalent acid 17 and biotin derivative 2316
(TBTU, DIPEA, DMF, 66%) (Scheme 6). The resulting probe
24 was purified by GPC using Biogel P-4 in water. Such a
labeled glycocluster can be used to screen T-Ag receptors in
cancer cells using well-established streptavidin assays. Further
investigation is under way to broaden the application and
efficiency of the multi-functional glycoconjugates, and the
results will be reported elsewhere.
Turbidimetric Analysis of T-Antigen Dendrimers. A.
hypogaea, having four noncovalently associated subunits (Mr
(16) Zanini, D. Ph.D. Dissertation, University of Ottawa, 1997.

1812 J. Am. Chem. Soc., Vol. 123, No. 9, 2001
Roy et al.
Scheme 5
Scheme 6
of 0.13, and 0.12, respectively. Dimer 17 showed the lowest
value (OD ) 0.1). The relative turbidities of di- and tetravalent
dendrimers were up to only 50% when compared to the value
of hexamer. These preliminary results clearly confirmed the
multivalency effect for the binding to the receptor protein and
also convinced us of its further applications in the fields of
biology and immunology as a potential inhibitor for biological
applications.
Enzyme-Linked Immunosorbent Assays (ELISA). The
relative inhibitory potency of each dendrimer toward the mouse
monoclonal antibody (MAb) JAA-F113 bound to the coated
antigen (Gal-â-(1-3)-GalNAc-R-O-)-co-polyacrylamide (T-
antigen/acrylamide, 1/10)2 was determined by a competitive
enzyme-linked immunosorbent assay (ELISA). Horseradish
peroxidase-labeled goat-antimouse IgG was used for quantita-
tive detection. The results for the inhibition of binding of JAA-
F11 to the coated antigen are shown in Figure 3. In general, an
increase in multivalency resulted in an increase in inhibitory
potency. At low dendrimer concentrations, no inhibition was
detected without the multivalency effect, regardless of dendrimer
valencies. As the concentration of inhibitors increased, these
effects were more significant.
) 120 000), is a plant having lectin with strong T-antigen (â-
Gal-(1-3)-R-GalNAc)-binding specificity. It does not ag-
glutinate normal human erythrocytes but does strongly agglu-
tinate neuraminidase-treated erythrocytes and has potent anti-T
activity similar to that of the anti-T antibody in human sera.17
The relative binding ability of T-antigen dendrimers to peanut
lectin was determined by a microturbidimetric analysis, which
served to determine the cross-linking ability of the glycoclusters
to form insoluble complexes with the tetrameric lectin.
Further addition resulted in the saturation of inhibition for
all dendrimers. Among these series, two tetravalent dendrimers,
The time course formation of precipitin complexes between
peanut lectin and di- (17), tetra- (20), hexa-(21), and tetravalent
(22) dendrimers is compared in Figure 2. In most cases, the
maximum turbidity was obtained within 1 h. These micro-
quantitative experiments demonstrated that the hexamer 21
showed the strongest/fastest ability to precipitate the lectin (OD
) 0.43). Two tetramers, 20 and 22, showed moderate OD values
Figure 2. Time-course turbidimetric analysis of peanut lectin with
T-antigen dendrimers 17 (9), 20 (2), 21 (b), and 22 ([).
(17) (a) Lotan, R J. Biol. Chem. 1975, 250, 8518. (b) London, J. J.
Immunol. 1978, 121, 438.

T-Antigen Glycodendrimers
J. Am. Chem. Soc., Vol. 123, No. 9, 2001 1813
22) against a mouse monoclonal IgG antibody binding using a
polymeric form of the T-Ag used as coating material (1 µg/
well). These results further substantiate the enhanced carbohy-
drate affinity toward the antibody by means of a multivalency
effect.
However, due care should be taken in the interpretation of
the origin of the observed enhanced binding. Indeed, we have
recently demonstrated that the increases in Ka of multivalent
glycoforms are due to more positive entropy (T∆S) contributions
that critically depended on the extent of both the valency of
the glycoclusters and the protein receptors.18,22 These results
are further corroborated by several other studies on polyman-
nosides binding toward concanavalin A.23,24 Clearly, glycoforms
have the added beneficial potential to form cross-linked
complexes. The nature and stability of these are dependent on
whether the proteins are themselves divalent (soluble complexes)
or tetravalent or higher (insoluble).25 So far, however, polymeric
carbohydrates have almost always surpassed glycodendrimers,
with the exception of hybrids containing dendronized poly-
mers.26
Theses results open various medicinal and pharmaceutical
applications for these conjugates to interrupt and modulate
biological pathways of cancer cell metastasis.19 In this manner,
the applications of well-organized hyperbranched glycoconju-
gates can be expanded toward biological contributions such as
immuno diagnostic and therapeutic purposes and in drug
delivery systems.
Figure 3. Inhibition of the binding of mouse monoclonal antibody
(IgG3) to T-antigen-copolyacrylamide by dendrimers 17 (9), 20 (2),
21 (b), and 22 ([).
Table 1. Inhibitory Potencies (IC50’s) of T-antigen Dendrimers to
Mouse Monoclonal Antibody JAA-F11 (IgG3)
compd
IC50 (nM)a
rel potencya
6 (monomer)
17 (dimer)
20 (tetramer)
21 (hexamer)
22 (tetramer)
2300
1
174 (347)
19 (76)
48 (288)
18 (72)
13.3 (6.6)
120.5 (30.1)
47.8 (8.0)
128.1 (32.2)
a Values in parentheses are based on per T-antigen residue.
20 and 22, showed the best results. The IC50 values were 19
and 18 nM, respectively. These values represent 120 to ∼130-
fold increased inhibition over that of the T-antigen monomer 6
(IC50 2.3 mM), which was used as a reference (Table 1). On a
per-T-antigen basis, each residue was ∼30 times more potent
than the corresponding monomer. Divalent cluster 17 had an
intermediate inhibitory potency (IC50 174 nM) 13 times more
potent than 6. Interestingly, hexavalent dendrimer 21 showed
less inhibition (IC50 48 nM) than the tetravalent ones, with a
potency only 48-fold higher than that of 6 and 8 times more
potent on a T-antigen basis. These results imply that not all of
the carbohydrate residues are properly participating in the
inhibition event. It may due to inappropriate conformational
environments, such as insufficient length of linkers and lack of
spatial freedom of each the carbohydrate components, resulting
in insufficient participation in binding. As a result, unbound
T-antigen residues reduce the overall inhibitory ability on a per-
saccharide basis. This phenomenon has been previously ob-
served with divalent IgG antibodies.18 However, all dendrimers
provided multivalent effect in terms of the inhibition of antibody
(JAA-F11) binding to the coated antigen.
Experimental Section
General Methods. The 1H and 13C NMR spectra were recorded on
a Bru¨ker 500 MHz NMR spectrometer. Proton chemical shifts (δ) are
given relative to internal CHCl3 (7.24 ppm) for CDCl3 solutions, to
internal dimethyl sulfoxide (2.49 ppm) for DMSO-d6 solutions, and to
internal HOD (4.76 ppm) for D2O solutions. Carbon chemical shifts
are given relative to CDCl3 (77.0 ppm) and DMSO-d6 (39.4 ppm).
Assignments were based on COSY, DEPT, and HMQC experiments.
Mass spectra were obtained either on a VG 7070-E spectrometer (CI,
ether) or on a Kratos Concept IIH spectrometer (FAB-MS, glycerol
matrix). Optical rotation values were measured on a Perkin-Elmer 241
polarimeter and were run at 25 °C. Absorbance for the turbidimetric
and ELISA assays were performed on a Dynatech MR 600 microplate
reader. Peanut lectin from A. hypogaea and its peroxidase-labeled form
were purchased from Sigma. Mouse monoclonal IgG antibodies were
generated using bovine serum albumin (BSA)-T-antigen conjugates.3
Thin-layer chromatography was performed on silica gel 60 F-254, and
column chromatography was carried out on silica gel 60. Gel permeation
chromatography (GPC) was performed using Biogel P-2, and P-4 using
water as eluent.
Synthesis of Allyl (2,3,4,6-Tetra-O-benzoyl-â-D-galactopyranosyl)-
(1-3)-2-acetamido-4,6-O-benzylidene-2-deoxy-r-D-galactopyrano-
side (3). A solution of allyl 2-acetamido-4,6-O-benzylidene-2-deoxy-
R-D-galactopyranoside (2)20 (1.67 g, 4.78 mmol) in a mixture of
nitromethane/benzene (60 mL; 1:1, v:v) was stirred for a couple of
hours at room temperature. To ensure dryness, the solution was
concentrated under reduced pressure. This process was repeated three
Conclusions
A new family of small glycodendrons (17, 22) and glyco-
dendrimers (20, 21) based on a N,N′-bis(acrylamido)acetic acid
core were synthesized in an efficient, convergent strategy. An
important cancer marker, the T-antigen, was conjugated to the
dendritic cores by either 1,4-conjugate addition of thiolated
disaccharide derivatives to the acrylamido group or by amide
bond formation between a T-antigen acid derivative (9) and
multiamino dendritic cores to give the desired glycodendrimers.
Turbidimetric analysis confirmed the binding abilities of gly-
codendrimers 17, 20, 21, and 22 toward the plant lectin from
A. hypogaea, forming insoluble cross-linked lattice complexes.
Solid-phase competitive ELISA demonstrated strong inhibitory
properties of e30-fold enhancement on a per-T-Ag basis (20,
(19) De´prez, B.; Gras-Masse, H.; Martinon, F.; Gomard, E.; Le´vy, J.-
P.; Tartar, A. J. Med. Chem. 1995, 38, 459.
(20) Lee, R. T.; Wong, T. C.; Lee, Y. C. J. Carbohydr. Chem. 1986, 5,
343.
(21) Kartha, K. P. R.; Jennings, H. J. J. Carbohydr. Chem. 1990, 9, 777.
(22) Dam, T. K.; Roy, R.; Das, S. K.; Oscoarson, S.; Brewer, C. F. J.
Biol. Chem. 2000, 275, 14223.
(23) Page´, D.; Zanini, D.; Roy, R. Bioorg. Med. Chem. 1996, 4, 1949.
(24) Dimick, S. M.; Powell, S. C.; McMahon, S. A.; Moothoo, D. N.;
Naismith, J. H.; Toone, E. J. J. Am. Chem. Soc. 1999, 121, 10286.
(25) Brewer, C. F. Chemtracts Biochem. Mol. Biol. 1996, 6, 165.
(26) Reuter, J. D.; Myc, A.; Hayes, M. M.; Gan, Z.; Roy, R.; Qin, D.;
Yin, R.; Piehler, L. T.; Esfand, R.; Tomalia, D. A.; Baker, J. R., Jr.
Bioconjugate Chem. 1999, 10, 271.
(18) Andre´, S.; Cejas Ortega, P. J.; Alamino Perez, M.; Roy, R.; Gabius,
H.-J. Glycobiology 1999, 9, 1253.

1814 J. Am. Chem. Soc., Vol. 123, No. 9, 2001
Roy et al.
times. The same volume of solvent was added and then concentrated
until one-half of the volume remained. The temperature was adjusted
to 25 °C and 2,3,4,6-tetra-O-benzoyl-R-D-galactopyranosyl bromide
(1)21 (4.70 g, 7.13 mmol) and Hg(CN)2 (1.80 g, 7.13 mmol) were added
successively at N2 atmosphere. The resulting solution was stirred at
room temperature for 18 h. The solvent was removed under vacuum.
The residue was dissolved in CHCl3 (40 mL) and then filtered through
a Celite pad. The filtrate was successively washed with 10% aqueous
KI, saturated NaHCO3, and distilled water, and then was dried over
Na2SO4. After concentration, the residue was purified by silica gel
column chromatography (benzene:ethyl acetate, 15:1) to give a white
foamy solid in 98% yield (4.31 g, 4.67 mmol): mp 109.7 to ∼111.0
°C; Rf 0.59 (benzene:EtOAc, 1:2); [R]D + 116.0 (c 1, CHCl3); 1H NMR
(CDCl3) δ 8.06 to ∼7.19 (m, 25H, Ar), 5.98 (dd, 1H, J34′ ) 3.3, J45′
e 1 Hz, H-4′), 5.85 to ∼5.78 (m, 2H, CH, H-2′), 5.60 (dd, 1H, J23′ )
10.2, J34′ ) 3.4 Hz, H-3′), 5.48 (br s, 1H, NH), 5.38 (s, 1H, CH), 5.23
(dd, 1H, Jgem ) 1.5, Jtrans ) 17.2 Hz, CH), 5.16 (m, 2H, Jcis ) 8.0 Hz,
CH, H-1′), 5.10 (d, 1H, J12 ) 3.4 Hz, H-1), 4.68 (dd, 1H, J56′ ) 6.9,
Hz, H-3), 4.06 to ∼4.03 (m, 1H, H-5), 3.98 (d, 1H, J34′ ) 3.4 Hz,
H-4′), 3.86 to ∼3.79 (m, 5H, H-6, H-6′, 1H of CH2), 3.74 to ∼3.72
(m, 1H, H-5′), 3.70 (dd, 1H, J23′ ) 10.0 Hz, J34′ ) 3.3 Hz, H-3′), 3.59
(dd, 1H, J12′ ) 7.8 Hz, J23′ ) 9.9 Hz, H-2′), 3.60 to ∼3.56 (m, 1H, 1H
of CH2), 3.13 to ∼3.04 (m, 2H, CH2), 2.46 (s, 3H, Ac), 2.10 (s, 3H,
Ac), 2.00 to ∼1.94 (m, 2H, CH2); 13C NMR (D2O) δ 201.4, 174.1,
104.2, 96.7, 76.8, 74.5, 72.0, 70.1, 70.1, 68.3, 68.1, 65.7, 60.7, 60.5,
48.2, 29.5, 27.8, 25.2, 21.5; Calcd. for C19H33O12NS (499.4). (+) FAB-
MS (glycerol) m/z: 500.2 (M + 1).
3-(2-Carboxyethylthio)propyl
â-D-galactopyranosyl-(1-3)-2-
acetamido-2-deoxy-r-D-galactopyranoside (9). To a solution of allyl
â-D-Gal-(1-3)-R-D-GalNAc (6) (100 mg, 0.24 mmol) in deoxygenated
distilled water (2.5 mL) was added 3-mercaptopropionic acid (21 µL,
1 equiv), and the resulting solution was irradiated (254 nm) for 7 h
under N2 atmosphere. The reaction solution was then loaded onto an
anion-exchange resin column (Amberite IRA 400 OH-) and washed
with water. The eluent was then changed gradually to aq acetic acid
with an increasing acetic acid gradient. The fractions containing 9 were
collected and evaporated under reduced pressure, followed by multiple
coevaporation with ethanol. A small amount of water was added and
the fraction was lyophilized to afford a white spongy solid in 83%
yield (105.9 mg, 2.0 mmol): mp 90.0 to ∼92.5 °C; [R]D + 76.0 (c 1,
H2O); Rf 0.33 (CHCl3:MeOH:H2O, 10:9:1); 1H NMR (D2O) δ 4.96 (d,
1H, J12 ) 3.7 Hz, H-1), 4.54 (d, 1H, J12′ ) 7.7 Hz, H-1′), 4.39 (dd,
1H, J12 ) 3.7 Hz, J23 ) 11.0 Hz, H-2), 4.31 (br d, 1H, H-4), 4.10 (dd,
1H, J23 ) 11.1 Hz, J34 ) 3.1 Hz, H-3), 4.07 (br s, 1H, H-5), 3.98 (br
d, 1H, J34′ ) 3.5 Hz, H-4′), 3.893.79 (m, 5H, H-6, H-6′, 1H of CH2),
3.74 to ∼3.72 (m, 1H, H-5′), 3.69 (dd, 1H, J23′ ) 10.0 Hz, J34′ ) 3.4
Hz, H-3′), 3.65 to ∼3.57 (m, 2H, H-2′, 1H of CH2), 2.89 (t, 2H, J )
7.5 Hz, CH2), 2.80 to ∼2.73 (m, 4H, 2 CH2), 2.10 (s, 3H, Ac), 2.01 to
∼1.96 (m, 2H, CH2); 13C NMR (D2O) δ 174.0, 104.2, 96.7, 76.8, 74.5,
72.0, 70.1, 70.1, 68.2, 68.1, 66.0, 60.7, 60.5, 48.2, 34.2, 27.9, 27.6,
26.0, 21.5; Calcd. for C20H35O13NS (529.4); (+) FAB-MS (glycerol)
m/z: 530.3 (M + 1).
J
6ab′ ) 11.4 Hz, H-6′), 4.63 to ∼4.58 (m, 1H, H-2), 4.46 to ∼4.36 (m,
3H, H-4, H-5′, H-6′), 4.15 to ∼4.07 (m, 3H, 1H-6, CH, H-3), 3.96
(dd, 1H, Jgem ) 6.1, Jab ) 13.0 Hz, CH), 3.75 (dd, 1H, J56 ) 1.4, Jgem
) 12.4 Hz, H-6), 3.51 (m, 1H, H-5), 1.40 (s, 3H, Ac); 13C NMR
(CDCl3)_δ 170.0, 166.0, 165.6, 165.5, 165.0, 137.7, 133.7, 133.5, 133.4,
133.1, 130.0, 129.9, 129.8, 129.7, 129.4, 129.2, 128.9, 128.7, 128.6,
128.4, 128.3, 128.2, 128.1, 126.2, 126.0 (31 C between 137.7 and
126.0), 117.7, 102.0, 100.9, 97.4, 75.8, 75.4, 71.8 (2C), 70.2, 69.2,
68.7, 68.1, 63.0, 62.7, 48.4, 22.4; (+) FAB-MS (glycerol) m/z 928.3
(M + 1), 1856 (2 M + 1); Anal. Calcd. for C52H49O15N (927.5): C,
67.20; H, 5.31; N, 1.53. Found: C, 66.84; H, 5.27; N, 1.48.
Preparation of Thiol Functionalized T-antigen Derivatives 5 and
8. These compounds were prepared in situ from their mother compounds
4 and 7 due to their readily oxidation into disulfide byproducts.
Allyl O-(â-D-galactopyranosyl)-(1-3)-2-acetamido-2-deoxy-r-D-
galactopyranoside (6). Compound 3 (1 g, 2.0 mmol) was dissolved
in MeOH (10 mL) and MeONa/MeOH (cat.) was added to adjust the
pH to 9. The resulting solution was stirred for 30 min at room
temperature. Solvent was eliminated by rotary evaporator. The residue
was dissolved in water (10 mL), and the aqueous layer was separated
from the organic layer then was lyophilized to give de-benzoylated
intermediate. Subsequently, this intermediate was dissolved in 60% aq
acetic acid (15 mL), and the resulting solution was stirred for 1.5 h at
60 °C. The solvent was evaporated under reduced pressure, and the
crude product was purified by silica gel column chromatography
(CHCl3:MeOH:H2O, 11:6:1) to give a white solid in 93% yield (0.79
g, 1.86 mmol): mp 230 to ∼232 °C; Rf 0.53 (CHCl3/MeOH/H2O);
3-(2-Aminoethylthio)propyl â-D-galactopyranosyl-(1-3)-2-acet-
amido-2-deoxy-r-D-galactopyranoside (10). A solution of allyl â-D-
Gal-(1-3)-R-D-GalNAc (6) (50 mg, 0.12 mmol) and cysteamine
hydrochloride (13) (12.5 mg, 0.11 mmol) in deoxygenated water (1
mL) was irradiated (254 nm) for 4.5 h under nitrogen atmosphere. The
product was loaded onto a cation-exchange resin column (Dowex 50W,
+
100 mesh, NH4 form) and washed with water. The eluent was then
changed gradually to NH4OH with an increasing concentration from 0
to 1.5 M with a linear gradient. The fractions containing the product
were collected and evaporated under reduced pressure. A small amount
of water was added and the fraction was lyophilized to afford a white
spongy solid in 63% yield (36.9 mg, 74 µmol): mp 108.5 to ∼109.6
1
[R]D + 120.0 (c 1, H2O); H NMR (D2O) δ 6.08 to ∼6.01 (m, 1H,
1
CH), 5.42 (dd, 1H, Jgem ) 1.6 Hz, Jtrans ) 17.3 Hz, CH), 5.33 (dd, 1H,
Jgem ) 1.7 Hz, Jcis ) 10.4 Hz, CH), 5.01 (d, 1H, J12 ) 3.8 Hz, H-1),
°C; [R]D + 74.0 (c 1, H2O); H NMR (D2Ã) δ 4.97 (d, 1H, J12 ) 3.8
Hz, H-1), 4.54 (d, 1H, J12′ ) 7.8 Hz, H-1′), 4.40 (dd, 1H, J12 ) 3.8
Hz, J23 ) 11.1 Hz, H-2), 4.31 (d, 1H, J34 ) 3.0 Hz, H-4), 4.10 (dd,
1H, J23 ) 11.1 Hz, J34 ) 3.0 Hz, H-3), 4.06 (m, 1H, H-5), 3.98 (d, 1H,
J34′ ) 3.4 Hz, H-4′), 3.90 to ∼3.79 (m, 5H, H-6, H-6′, 1H of CH2),
3.73 (m, 1H, H-5′), 3.69 (dd, 1H, J23′ ) 9.9 Hz, J34′ ) 3.4 Hz, H-3′),
3.64 (m, 1H, 1H of CH2), 3.59 (dd, 1H, J12′ ) 7.8 Hz, J23′ ) 9.9 Hz,
H-2′), 3.05 (m, 2H, CH2), 2.82 (t, 2H, J ) 6.6 Hz, CH2), 2.76 (t, 2H,
J ) 7.2 Hz, CH2), 2.10 (s, 3H, Ac), 1.98 (m, 2H, CH2); 13C NMR
(D2O) δ 174.0, 104.2, 96.7, 76.7, 74.5, 72.0, 70.1 (2C), 68.2, 68.1,
66.0, 60.7, 60.5, 48.2, 38.6, 30.8, 27.9, 27.2, 21.5; Anal. Calcd. for
C19H36O11N2S (500.2): C, 45.63; H, 7.31; N, 5.64. Found: C, 45.09;
H, 6.95; N, 5.07. (+) FAB-MS (glycerol) m/z: 501.2 (M + 1).
Methyl N,N′-Bis(acrylamido)acetate (12). A solution of N,N′-bis-
(acrylamido)acetic acid, 11 (2 g, 10.1 mmol) in MeOH was cooled to
-15 °C. Thionyl chloride (884 µL, 12.1 mmol, 1.2 equivs) was added
dropwise over a 1-h period. The resulting mixture was stirred another
1 h at the same temperature and 3 h at room temperature. The solution
was condensed to one-half of the volume, then purified by flash silica
gel column chromatography (CH3Cl:MeOH, 10:2) to give a white solid,
11, in 80% yield (1.7 g, 8.1 mmol): mp 229 to ∼231 °C (soften temp),
>350 °C; Rf 0.73 (EtOAc:MeOH, 24:1); 1H NMR (DMSO-d6) δ 9.08
(d, 2H, J ) 7.5 Hz, 2 NH), 6.33 (dd, 2H, Jcis ) 10.2, Jtrans ) 17.1 Hz),
6.14 (dd, 2H, Jgem ) 2.0, Jtrans ) 17.1 Hz), 5.76 (t, 1H, J ) 7.6 Hz),
5.66 (dd, 2H, Jgem ) 2.0, Jcis ) 10.2 Hz), 3.64 (s, 3H, CH3); 13C NMR
4.53 (d, 1H, J12′ ) 7.8 Hz, H-1′), 4.41 (dd, 1H, J12 ) 3.7 Hz, J23
)
11.2 Hz, H-2), 4.31 to ∼4.27 (m, 2H, H-4, 1H of CH2), 4.13 to ∼4.07
(m, 3H, H-3, H-5, 1H of CH2), 3.98 (dd, 1H, J34′ ) 3.4 Hz, J45′ ) 0.8
Hz, H-4′), 3.86 to ∼3.78 (m, 4H, H-6, H-6′), 3.7 to ∼3.71 (m, 1H,
H-5′), 3.69 (dd, 1H, J23′ ) 10.0 Hz, J34′ ) 3.4 Hz, H-3′), 3.59 (dd, 1H,
J12′ ) 7.7 Hz, J23′ ) 10.0 Hz, H-2′), 2.09 (s, 3H, Ac); 13C NMR
(D2O)_δ 174.1, 133.2, 117.4, 104.2, 95.9, 76.8, 74.5, 72.1, 70.2, 70.1,
68.3, 68.1, 68.0, 60.7, 60.5, 48.1, 21.5; Anal. Calcd. for C17H29O11N
(423.2): C, 45.30; H, 6.92; N, 3.30. Found: C, 45.30; H, 6.93; N,
3.07. (+) FAB-MS (glycerol) m/z: 424.2 (M + 1).
(3-Thioacetyl)propyl (â-D-galactopyranosyl)-(1-3)-2-acetamido-
2-deoxy-r-D-galactopyranoside (7). To a solution of allyl â-D-Gal-
(1-3)-R-D-GalNAc (6) (60 mg, 0.14 mmol) and thioacetic acid (63 µL,
0.85 mmol) in deoxygenated MeOH (3 mL) was added a catalytic
amount of AIBN, and the resulting solution was refluxed for 1 day
under an N2 atmosphere. The solution was then evaporated under
reduced pressure, and the residue was purified by silica gel column
chromatography (CHCl3:MeOH:H2O, 7:4:0.8) to afford an ivory-colored
powder in 71% yield (50 mg, 0.1 mmol): mp 119.6 to ∼122.5 °C;
[R]D + 83.0 (c 1, H2O); Rf 0.69 (CHCl3:MeOH:H2O, 10:5:1); 1H NMR
(D2O) δ 4.95 (d, 1H, J12 ) 3.8 Hz, H-1), 4.55 (d, 1H, J12′ ) 7.8 Hz,
H-1′), 4.39 (dd, 1H, J12 ) 3.8 Hz, J23 ) 11.1 Hz, H-2), 4.31 (d, 1H,
J34 ) 3.1 Hz, J45 e 1 Hz, H-4), 4.10 (dd, 1H, J23 ) 11.1 Hz, J34 ) 3.1

T-Antigen Glycodendrimers
J. Am. Chem. Soc., Vol. 123, No. 9, 2001 1815
(DMSO-d6) δ 168.7 (2C), 164.6, 130.7 (2C), 126.9 (2C), 56.1, 52.5;
Calcd. for C9H12O4N2 (212.1), CI-MS (ether) m/z 213.1 (M + 1).
Preparation of Methyl Bis[N,N′-(2-(2-aminoethyl)thioethyl-
amido)]acetate (16). To a solution of 12 (0.4 g, 1.89 mmol) in MeOH
(30 mL) was added a solution of cysteamine, 13 (0.44 g, 3.87 mmol)
and DIPEA (680 µL, 3.87 mmol) in MeOH (3 mL). The solution was
stirred for 6 h at room temperature. After solvent evaporation, the
residue was purified by silica gel column chromatography (CHCl3:
MeOH:H2O, 10:6:1 and EtOAc:AcOH:H2O, 3:2:2). The fractions
containing 15 were collected and evaporated under reduced pressure.
The residue was dissolved in 50 mL of distilled water and neutralized
with OH- resin (amberite IRA 400, OH-). The solution was filtered,
and the filtrate was lyophilized to give an amorphous hygroscopic solid,
15, in 93% yield (0.64 g, 1.76 mmol): 1H NMR (D2O) δ 6.00 (br s,
1H, CH), 3.95 (s, 3H, CH3), 3.37 (t, 4H, J ) 6.7 Hz, 2CH2), 3.01 (t,
4H, J ) 6.8 Hz, 2CH2), 2.99 (t, 4H, J ) 6.8 Hz, 2CH2), 2.77 (t, 4H,
J ) 6.8 Hz, 2CH2); 13C NMR (D2O) δ 173.8 (2C), 169.3, 56.1, 53.2,
37.9 (2C), 34.4 (2C), 27.9 (2C), 25.8 (2C); Calcd. for C13H26O4N4S2
(366.4), CI-MS (ether) m/z: 367.2 (M + 1).
Preparation of Divalent T-Antigen Dendrimer (17). A solution
of 11 (23.8 mg, 0.12 mmol) and triethylamine (33.5 µL, 0.24 mmol)
in degassed MeOH (5 mL) was saturated with N2. Thiol 8 (150 mg,
0.3 mmol) which was prepared in-situ from 7 (NaOMe, MeOH) in
degassed MeOH (1 mL) was added to the reaction flask using a syringe.
The reaction was continued for 5 h under N2 atmosphere. After
evaporation of the solvent, the crude product was purified by silica gel
column chromatography (CHCl3:MeOH:H2O, 10:8:2) to give 17 in
quantitative yield: mp 162.8 to ∼164.7 °C; [R]D + 80.7 (c 1, H2O); Rf
0.26 (CHCl3:MeOH:H2O, 10:8:2); 1H NMR (D2O) δ 5.56 (s, 1H, CH),
4.96 (d, 2H, J12 ) 3.8 Hz, 2H-1), 4.55 (d, 2H, J12′ ) 7.8 Hz, 2H-1′),
4.40 (dd, 2H, J12 ) 3.8, J23 ) 11.1 Hz, 2H-2), 4.32 (d, 2H, J34 ) 2.9
Hz, J45 e 1, 2H-4), 4.10 (dd, 2H, J23 ) 11.1, J34 ) 3.1 Hz, 2H-3),
4.09 to ∼4.06 (br t, 2H, J45 e 1, J56 ) 6.4 Hz, 2H-5), 3.99 (d, 2H, J34′
) 3.4, J45′ e 1 Hz, 2H-4′), 3.89 to ∼3.79 (m, 10H, 4H-6, 4H-6′, 2H-
a), 3.75 to ∼3.72 (m, 2H, 2H-5′), 3.71 (dd, 2H, J23′ ) 10.0, J34′ ) 3.4
Hz, 2H-3), 3.65 to ∼3.60 (m, 2H, 2H-a), 3.59 (dd, 2H, J12′ ) 7.8, J23′
) 10.0 Hz, 2H-2′), 2.90 (t, 4H, J ) 7.1 Hz, 2CH2), 2.77 (t, 4H, J )
7.4 Hz, 2CH2), 2.67 (t, 4H, J ) 6.9 Hz, 2CH2), 2.04 to ∼1.95 (m, 4H,
2CH2); 13C NMR (D2O) δ 174.3, 173.4 (2C), 172.6 (2C), 104.2 (2C),
96.7 (2C), 76.8 (2C), 74.5 (2C), 72.1 (2C), 70.2 (2C), 70.1 (2C), 68.3
(2C), 68.1 (2C), 66.1 (2C), 60.7 (2C), 60.5 (2C), 57.7, 48.2 (2C), 35.0
(2C), 28.0 (2C), 27.6 (2C), 26.4 (2C), 21.6 (2C); Calcd. for C42H72O26N4S2
(1112.4), (+) FAB-MS (glycerol) m/z: 1135.6 (M + Na).
δ 167.6 (3C), 164.5 (6C), 131.1 (6C), 126.3 (6C), 57.0 (3C), 52.8 (3C),
37.1 (3C); Calcd. for C30H42O9N10 (686.4), (+) FAB-MS (glycerol)
m/z: 687.4 (M + 1).
Synthesis of Tetra- and Hexavalent T-Antigen Dendrimers (20
and 21). Typical Procedure 1. To a solution of 18 (5 mg, 10.5 µmol)
and triethylamine (7 µL, 5 equivs) in degassed DMSO (1 mL) was
added 8 (25 mg, 50 µmol) in degassed MeOH (1 mL), which was
prepared in situ from 7 under N2 atmosphere (NaOMe, MeOH). The
resulting solution was stirred overnight at room temperature. After
evaporation of the solvent under vacuum, the residue was purified by
gel permeation chromatography (P-2) in water. The fractions containing
pure 20 were collected and lyophilized to give a white solid in 50%
yield (12 mg, 5.3 mmol). Typical Procedure 2. To a solution of 14
and 17 in DMSO was added DIPEA (pH ) 9) and TBTU (1.2 equivs).
The resulting solution was stirred overnight at room temperature. After
solvent evaporation, the residue was purified as described above. The
fraction containing the desired product was collected and lyophilized
to give a white spongy solid in 50% yield. Hexavalent T-antigen
dendrimer 21 was obtained as described above from 8/19 or 15/17 in
45 to ∼46% yields, respectively. Data for 20: mp 149.2 to ∼153.5
°C; [R]D + 69.2 (c 0.65, H2O); 1H NMR, (D2O) 4.97 (d, 4H, J12 ) 3.8
Hz, H-1), 4.55 (d, 4H, J12′ ) 7.8 Hz, H-1′), 3.89 to ∼3.79 (m, 20H,
8H-6, 8H-6′, 2CH2), 3.65 to ∼3.61 (m, 4H, 2CH2), 3.30 (t, 4H, J )
6.8 Hz, 2CH2), 2.91 (t, 8H, J ) 6.9 Hz, 4CH2), 2.77 (t, 8H, J ) 7.2
Hz, 4CH2), 2.69 (t, 8H, J ) 6.9 Hz, 4CH2), 2.10 (s, 12H, Ac), 2.01 to
∼1.95 (m, 8H, 4CH2), 1.58 to ∼1.56 (m, 4H, 2CH2), 1.40 to ∼1.37
(m, 4H, 2CH2); 13C NMR (D2O) δ 174.0, 173.9 (6C between 174.0
and 173.9), 168.1 (4C), 104.2 (4C), 96.7 (4C), 76.8 (4C), 74.5 (4C),
72.1 (4C), 70.2 (4C), 70.1 (4C), 68.2 (4C), 68.1 (4C), 66.0 (4C), 60.7
(4C), 60.5 (4C), 56.9 (2C), 48.2 (4C), 39.1 (2C), 34.8 (4C), 28.0 (4C),
27.7 (2C), 27.6 (4C), 26.4 (4C), 25.1 (2C), 21.6 (4C); Calcd. for
C90H156O50N10S4 (2304.9), (+) FAB-MS (glycerol) m/z 2328.5 (M +
Na). Data for 21: mp 194 to ∼195 °C; [R]D + 81.2 (c 0.85, H2O); 1H
NMR (D2O) δ 5.54 (s, 3H, 3CH), 4.96 (d, 6H, J12 ) 3.7 Hz, H-1),
4.54 (d, 6H, J12′ ) 7.7 Hz, H-1′), 3.86 to ∼3.78 (m, 30H, 12H-6, 12H-
6′, 3CH2), 3.63 to ∼3.56 (m, 12H, 6H-2′, 3CH2), 2.88 (t, 12H, J ) 6.9
Hz, 6CH2), 2.85 (t, 6H, J ) 7.3 Hz, 3CH2), 2.75 (t, 12H, J ) 7.1 Hz,
6CH2), 2.65 (t, 12H, J ) 6.8 Hz, 6CH2), 2.56 (t, 6H, J ) 7.2 Hz,
3CH2), 2.09 (s, 18H, Ac); 13C NMR (D2O) δ 174.1 (3C), 173.9 (6C),
173.6 (6C), 104.2 (6C), 96.7 (6C), 76.8 (6C), 74.5 (6C), 72.0 (6C),
70.2 (6C), 70.1 (6C), 68.3 (6C), 68.01 (6C), 66.0 (6C), 60.7 (6C), 60.5
(6C), 57.7 (3C), 48.2 (6C), 34.9 (6C), 33.8 (6C), 28.1 (6C), 27.6 (6C),
27.5 (3C), 26.4 (6C), 21.7 (6C); Calcd. for C132H228O75N16S6 (3429.3),
ES-MS (10 V) m/z 1144.4 (1/3 M + 1).
Synthesis of N,N′-Bis(acrylamido)acetic Acid-Based Dendritic
Cores 18 and 19. Typical Procedure. To compound 11 (0.44 g, 2.22
mmol) in DMSO (40 mL) successively was added 1-hydroxybenzo-
triazole hydrate (HOBt) (0.45 g, 3.33 mmol), hexamethylenediamine,
14 (0.12 g, 1 mmol), and DIPEA (87.1 µL, 0.5 mmol) at 0 °C. The
heterogeneous solution was stirred for 10 min, after which was added
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)
(0.98 g, 3.3 mmol). The suspension was allowed to reach room
temperature and then was stirred for another 6 h. The solvent was
removed by lyophilization, and the yellow residue was dissolved in a
mixture of CH2Cl2:H2O (1:1, v:v; 100 mL) and left in the refrigerator
for 1 day. The precipitate was filtered, washed successively with MeOH
and water, and dried under vacuum to give a solid in 60% yield (0.29
g, 1.33 mmol). Hexavalent dendritic core, 19, was isolated as described
above from trisamine, 15 and a seed molecule, 11, as a light brown
solid in 62% yield. Data for 18: mp >350 °C; 1H NMR (DMSO-d6) δ
8.64 (d, 4H, J ) 6.5 Hz, 4 NH), 8.02 (br s, 2H, 2 NH), 6.38 to ∼6.33
(m, 4H, 4CH), 6.10 (d, 4H, Jtrans ) 17.1, Jgem ) 2.0 Hz, 4CH), 5.84 (br
s, 2H, 2CH), 5.61 (d, 4H, Jcis ) 9.9, Jgem ) 1.9 Hz, 4CH), 3.04 (br s,
4H, 2CH2), 1.37 (br s, 4H, 2CH2), 1.21 (br s, 4H, 2CH2); 13C NMR
(DMSO-d6) δ 167.4 (2C), 164.3 (4C), 131.2 (4C), 126.2 (4C), 57.1
(2C), 39.2 (2C), 38.8 (2C), 28.8 (2C); Calcd. for C22H32O6N6 (476.3),
(+) FAB-MS (glycerol) m/z 951.4 (2 M). Data for 19: 1H NMR
(DMSO-d6) δ 8.71 (d, 6H, J ) 7.6 Hz, 6 NH), 7.97 (m, 3H, 3 NH),
Preparation of Tetravalent T-Antigen Dendrimer (22). To a
solution of 16 (2.1 mg, 5.8 µmol) and 17 (13 mg, 11.7 µmol) in DMSO
(1 mL) was added HOBt (4 mg, 29.6 µmol), EDC (8.6 mg, 29.0 µmol),
and DIPEA (100 µL) at 0 °C. The resulting solution was stirred for 40
min, then the temperature was allowed to rise to room temperature.
The reaction was continued overnight. After solvent elimination, the
residue was dissolved in a minimum amount of water and purified by
gel permeation chromatography (P-4) in water. The fractions containing
22 were collected and lyophilized to give a white solid in 65% yield
(9.2 mg, 7.61 µmol): mp 127.0 to ∼131.0 °C; [R]D + 57.5 (c 0.8,
H2O); 1H NMR (D2O) δ 5.90 (s, 3H, 3CH), 4.95 (d, 4H, J12 ) 3.7 Hz,
H-1), 4.54 (d, 4H, J12′ ) 7.8 Hz, H-1′), 3.87 (s, 3H, CH3), 3.85 to
∼3.78 (m, 20H, 4H-6, 4H-6′, 2CH2), 3.64 to ∼3.60 (m, 4H, 2CH2),
3.51 (t, 4H, J ) 6.6 Hz, 2CH2), 2.91 to ∼2.85 (m, 12H, 6CH2), 2.79
to ∼2.74 (m, 12H, 6CH2), 2.73 to ∼2.65 (m, 12H, 6CH2), 2.09 (s,
12H, AC), 2.00 to ∼1.94 (m, 8H, 4CH2); 13C NMR (D2O) δ 174.0,
173.9 (9C between 174.0 and 173.9), 168.4 (4C), 104.2 (4C), 96.7 (4C),
76.8 (4C), 74.5 (4C), 72.2 (4C), 70.1 (8C), 68.2 (4C), 68.1 (4C), 66.0
(4C), 60.7 (4C), 60.5 (4C), 56.8 (3C), 53.2, 48.2 (4C), 38.5 (2C), 34.8
(4C), 34.7 (2C), 29.9 (2C), 28.0 (4C), 27.6 (4C), 26.4 (4C), 26.1 (2C),
21.6 (4C); Calcd. for C97H166O54N12S6 (2556.0), ES-MS (10 V) m/z
852.7 (1/3 M + 1).
Preparation of Biotin-Labeled Divalent T-Antigen Dendrimer
(24). To a solution of divalent T-antigen 17 (16 mg, 14.4 µmol) and
4-aminobutanamide biotin derivative 2316 (4.53 mg, 1 equiv) in DMF
(3 mL) was added TBTU (5.6 mg, 1.2 equivs) and DIPEA (pH ) 8 to
∼9). The reaction mixture was stirred for 5 h at room temperature.
6.36 (dd, 6H, Jcis ) 10.2, Jtrans ) 17.1 Hz, 6CH), 6.12 (dd, 6H, Jgem
)
2.0, Jtrans ) 17.1 Hz, 6H of allylic CH2), 5.88 (d, 3H, J ) 7.7 Hz,
3CH), 5.62 (dd, 6H, Jgem ) 1.9, Jcis ) 10.3 Hz, 6H of allylic CH2),
3.11 (br s, 6H, 3CH2), 2.53 (hidden, 6H, 3CH2); 13C NMR (DMSO-d6)

1816 J. Am. Chem. Soc., Vol. 123, No. 9, 2001
Roy et al.
Completion of the reaction was confirmed by a negative Kaiser test.
The solvent was removed under reduced pressure, and the residue was
purified by gel permeation chromatography (P-4) in water. The fractions
containing 24 were collected and lyophilized to give a white solid in
79% yield (16.1 mg, 11.4 µmol): mp 170 to ∼172 °C; [R]D + 22.2 (c
were coated overnight with T-antigen containing co-polyacrylamide2
at 100 µL of a polymer stock solution, (10 µg/mL in 0.01 M phosphate
buffer (pH ) 7.3) at room temperature. Each well contained 1 µg/well
of polymer, which corresponded to 0.36 µg (0.85 nmol) T-antigen. The
wells were then washed 3 times with 400 µL of PBST (0.01 M
phosphate buffer (pH ) 7.3) containing 0.05% (v/v) Tween 20. BSA
solution (1% in PBS, 150 µL/well) was added to each well and
incubated for 1 h at 37 °C. At the same time, 50 µL of mouse
monoclonal IgG antibodies solution in PBST (10 times dilution of
ascitic fluid, 0.25 µmol/50 µL) and 50 µL of inhibitor solution in PBST
with varying concentration from 138 to 2.15 nmol/well of T-antigen
by 2-fold serial dilution were mixed in Nunclon (Delta) microtiter plates
and preincubated for 1 h at 37 °C. After excess BSA was washed out
with with PBST, each well was filled with 100 µL/well of preincubated
mouse IgG MAb/inhibitor solution and incubated again for 1 h at 37
°C. The wells were washed with PBST as described above and then
filled with 100 µL of goat anti-mouse IgG in PBST (1000 times dilution
of ascitic fluid), followed by incubation for 1 h at 37 °C. The wells
were washed with PBST, and 50 µL of 2,2′-azinobis(3-ethylbenzothia-
zoline-6-sulfonic acid) diammonium salt (ABTS) [1 mg/4 mL of citrate-
phosphate buffer (0.2 M, pH ) 4.0 with 0.015% H2O2)] was added.
The reaction was stopped after 20 min by 50 µL/well of 1 M aqueous
sulfuric acid solution. Optical density was measured at 410 nm relative
to 570 nm. Percent inhibitions were calculated as follows:
1
1.4, MeOH); H NMR (D2O) δ 4.95 (d, 2H, J12 ) 3.7 Hz, H-1), 4.67
to ∼4.65 (m, 1H, CH), 4.54 (d, 2H, J12′ ) 7.8 Hz, H-1′), 4.48 to ∼4.45
(m, 1H, CH), 3.87 to ∼3.78 (m, 10H, H-6, H-6′, CH2), 3.63 to ∼3.57
(m, 3H, H-2′, CH2), 3.39 to ∼3.35 (m, 1H, CH), 3.31 (br t, 2H, J )
6.3 Hz, CH2), 3.24 (br t, 2H, J ) 6.5 Hz, CH2), 3.05 (dd, 1H, J ) 5.0,
J ) 13.1 Hz, CH), 2.93 to ∼2.81 (m, 5H, 2CH2, CH), 2.75 (t, 4H, J )
7.1 Hz, 2CH2), 2.68 (t, 4H, J ) 6.8 Hz, 2CH2), 2.30 (t, 2H, J ) 7.3
Hz, CH2), 2.10 (s, 6H, 2CH3), 1.99 to ∼1.93 (m, 4H, 2CH2), 1.81 to
∼1.64 (m, 4H, 2CH2), 1.63 to ∼1.50 (br s, 4H, 2CH2), 1.49 to ∼1.42
(m, 2H, CH2); 13C NMR (D2O) δ 176.1, 174.0, 173.9 (3C between
174.0 and 173.9), 168.2 (2C), 165.3, 104.2 (2C), 96.8 (2C), 76.9 (2C),
74.5 (2C), 72.1 (2C), 70.2 (4C), 68.3 (2C), 68.2 (2C), 66.0 (2C), 61.6,
60.7 (2C), 60.5 (2C), 59.8, 57.0, 54.9, 48.3 (2C), 39.3, 38.9, 38.4, 35.1,
34.8 (2C), 28.0 (2C), 27.6 (2C), 27.4, 26.4 (2C), 25.3 (2C), 24.7 (2C),
21.6 (2C); Calcd. for C56H96O27N8S3 (1408.6), ES-MS (10 V) m/z 705.3
(1/2 M + 1).
Turbidimetric Analysis between Peanut Lectin and Di- (17),
Tetra- (20, 22), and Hexavalent (21) T-Antigen Dendrimers.
Turbidimetric experiments were performed in Linbro (Titertek) mi-
crotitration plates where 50 µL/well of stock lectin solutions prepared
from peanut lectin (2 mg/mL in PBS) were mixed with 50 µL of a
stock solution of glycodendrimers, 17, 20, 21, and 22 (11 nmol/well
of T-antigen for all dendrimers) and incubated at room temperature
for 3 h. The turbidity of the solutions was monitored by reading the
optical density (OD) at 490 nm at regular time intervals until no
noticeable changes were observed. Each test was performed in triplicate.
Competitive Double Sandwich Inhibition ELISA Using Mouse
Monoclonal Antibody JAA-F11 IgG3 and T-Antigen Dendrimers,
17, 20, 21, and 22 as Inhibitors. Linbro (Titertek) microtiter plates
% inhibition ) [A(no inhibitor) - A(with inhibitor)/A(no inhibitor)] × 100
IC50’s were calculated as the concentration required for 50% inhibition
of the coating antigen. All test were performed in triplicate.
JA002596W