Simultaneous binding of mouse monoclonal antibody and streptavidin to heterobifunctional dendritic L-lysine core bearing T-antigen tumor marker and biotin

Article abstract of DOI:10.1016/S0968-0896(01)00205-X

Thiolate T-antigen Galβ-(1-3)-GalNAcα, T-Ag] (6 ), derive in situ from thioacetate 5 was couple to N-chloroacetylate glycylglycyl L-lysine dendritic cores (7 9) using high yielding substitution reactions to affor di- (10), tetra- (11), and octa-valent (12) glycodendrimers in good yields (76 86%). Heterobifunctional conjugate 14 was prepare as a biosensor from tetravalent conjugate 11 an biotin hydrazide 13 using TBTU strategy. In a solid-phase double sandwich enzyme linked immuno-sorbent assays (ELISA), biotinylate conjugate 14 was shown to bin to streptavidin used as a coating material. Mouse monoclonal anti T-Ag antibody (IgG3) an horseradish peroxydase-labele goat anti mouse IgG, used for quantification, were found to bind T-Ag tetramer 14 immobilize on the surface of the streptavin layer. A typical saturation curve was observed for 14 while non-biotinylated tetramer 11 showed no binding in the entire concentration range. These results demonstrate the availability of both haptens toward the T-Ag antibody and streptavidin receptors. Copyright

Full text of DOI:10.1016/S0968-0896(01)00205-X

Bioorganic & Medicinal Chemistry 9 (2001) 3005–3011
Simultaneous Binding of Mouse Monoclonal Antibody and
Streptavidin to Heterobifunctional Dendritic L-Lysine Core
Bearing T-Antigen Tumor Marker and Biotin
Myung-Gi Baek and Rene Roy*
Department of Chemistry, Centre for Research in Biopharmaceuticals, University of Ottawa, Ottawa, Ontario, Canada, K1N 6N5
Received 13 March 2001; accepted 29 May 2001
Abstract—Thiolated T-antigen [Galb-(1-3)-GalNAca, T-Ag] (6), derived in situ from thioacetate 5 was coupled to N-chlo-
roacetylated glycylglycyl l-lysine dendritic cores (7–9) using high yielding substitution reactions to afford di- (10), tetra- (11), and
octa-valent (12) glycodendrimers in good yields (76–86%). Heterobifunctional conjugate 14 was prepared as a biosensor from
tetravalent conjugate 11 and biotin hydrazide 13 using TBTU strategy. In a solid-phase double sandwich enzyme linked immuno-
sorbent assays (ELISA), biotinylated conjugate 14 was shown to bind to streptavidin used as a coating material. Mouse monoclonal
anti T-Ag antibody (IgG3) and horseradish peroxydase-labeled goat anti mouse IgG, used for quantification, were found to bind T-
Ag tetramer 14 immobilized on the surface of the streptavin layer. A typical saturation curve was observed for 14 while non-bio-
tinylated tetramer 11 showed no binding in the entire concentration range. These results demonstrate the availability of both hap-
tens toward the T-Ag antibody and streptavidin receptors. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
chemical applications, synthetic T-Ag containing
glycoproteins have been used to develop mouse mono-
clonal antibodies (MAb), JAA-F11 (IgG3) and C5
(IgM) for immunohistochemical staining of breast ade-
nocarcinomas.9a These antibodies were applied as
receptors for several T-Ag conjugates including
glycodendrimers.10aꢀc Glycopolymers bearing T-Ag
have also been employed in high-throughput screening
toward the development of solid-phase glycosyl-
transferase assays for drug discovery research.9b
Cell-surface carbohydrates derived from glycoconju-
gates mediate the associative interactions of enzymes,
hormones, toxins, viruses, bacteria, lectins, and anti-
bodies.1ꢀ6 Carbohydrate–protein interactions are neces-
sary for stimulation of immunological surveillances,
protection, or clearance of senescent glycoproteins from
circulation, inflammation, and microbial infections.1ꢀ6
For a fundamental understanding of these interactions,
their intrinsically low affinity has to be overcome.7 In
fact, nature utilizes multiple expression of carbohy-
drates on the cell surface that act cooperatively to
increase the binding affinity toward their receptors.6,8
To this end, glycopolymers9 and glycodendrimers10ꢀ12
have been successfully employed as potent multivalent
antagonists.
The expression of T-Ag has been proposed as a tool for
the detection of tumors and as a criteria for prognosis.14
Moreover, there is an increased expression of T-Ag in
metastatic tumors and a plant lectin (Arachis hypogaea)
recognizing T-Ag has been shown to bind to common
sites of metastatic tumor growth.15 To date, only a few
hypervalent T-Ag clusters have been prepared for bio-
logical and pharmaceutical applications.9 These con-
jugates have architectures varying in size and
carbohydrate orientations. Changes in their structure
shapes for precise quantitative analysis of bioassays are
necessary. In this respect, glycoclusters offer distinct
advantages over random coil glycopolymers and glyco-
proteins. Moreover, they are unlikely to be immuno-
genic. The preparation of l-lysine-based dendritic
polypeptides has attracted considerable attention due to
The Thomson–Friedenrich carbohydrate antigen [T-Ag,
Galb-(1-3)-GalNAca], has been known as a cancer
related marker and is therefore an important antigen for
the detection and immunotherapy of carcinomas, parti-
cularly relevant to breast cancer.13 In related immuno-
*Corresponding author. Tel.: +1-613-562-5800 x 6055; fax: +1-613-
562-5170; e-mail: rroy@science.uottawa.ca
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00205-X

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M.-G. Baek, R. Roy / Bioorg. Med. Chem. 9 (2001) 3005–3011
potential vaccine applications,16ꢀ18 and few recent syn-
theses of glycodendrimers scaffolded onto hyperbranched
l-lysine core have been described.19,20
13C NMR, and MS spectral data. The 1H NMR spectra
systematically showed two anomeric protons of the b-
and a-galactosides at d 4.55 and 4.96 ppm and l-lysyl
and b-alanyl methylene residues at 1.3–3.3 ppm. At
room temperature (500 MHz), no distinctive signals
could be observed for the l-lysyl a- or e-branches in
The efficient synthesis of T-Ag containing l-lysine di-
(10), tetra- (11), and octa-meric (12) dendrons is descri-
bed herein. A biotin labeled heterobifunctional T-Ag
tetrameric conjugate has been prepared using con-
venient peptide coupling methods. Its binding proper-
ties toward mouse MAb IgG39a is described using
streptavidin as a coating protein. It is anticipated that
such biomacromolecules would constitute chemically
well-defined, monodispersed, heterobifunctional ligands
that can be used to detect T-Ag receptors (metastatic
sites), thus providing improvement over ill-defined
artificial polymers and glycoproteins.
1
either the H and 13C NMR.
For the purpose of broadening the potential application
of these conjugates, biotin-labeled heterobifunctional
T-Ag conjugate 14 was prepared using tetravalent 11 and
biotin hydrazide 13 by a TBTU strategy in 92% yield after
gel permeation chromatography (Biogel P-4, H2O)
(Scheme 3). High resolution 1H and COSY-NMR spectra
(500 MHz, D2O) showed the key signals, which represent
the glycoside (4.55 and 4.96 ppm for H-1s), l-lysyl dendritic
core (1.65–1.51 ppm for lysyl d-CH2) and biotin hydrazide
(4.68 and 4.50 ppm for protons in the ring system).
Results and Discussion
In previous work,9a,b,10aꢀc T-Ag containing dendritic
and polymeric conjugates demonstrated strong binding
interactions as inhibitors in biological tests which
expanded their contribution as potential immuno-
diagnostic and therapeutic materials as well as for drug
delivery systems. To evaluate the relative affinity of the
above glycodendrimers toward mouse monoclonal anti-
body (mAb IgG), a competitive solid-phase inhibition
experiment was set using a T-Ag-containing polymer,
obtained by copolymerization of 4 with acrylamide,10c as
a coating antigen. The relative inhibitory properties of
10–12 was evaluated as previously described using per-
oxidase-labeled goat anti-mouse antibody.10a The results
indicated relative inhibitory potencies of ꢁ50 (dimer 10),
300 (tetramer 11), and 200 (octamer 12) when measured
on a per saccharide basis compared to monomer 4 (IC50
2.3 mM). These results indicate once more that in some
cases, smaller glycoclusters are better inhibitors than
larger one, presumably because of the increasing steric
inaccessibility of the more crowded species.
Allyl T-antigen (3) was synthesized from per-benzo-
ylated galactosyl bromide (1) and 4,6-O-benzylidene
protected allyl GalNAc (2) which was prepared in 98%
yield under classical Helferich glycosylation method
according to
a known procedure [PhH/MeNO2,
Hg(CN)2]10a (Scheme 1). The required disaccharide 3
was successfully deprotected under Zemplen conditions
(MeONa/MeOH) followed by aqueous acetic acid
hydrolysis for deacetalation. Fully deprotected T-Ag 4
was obtained in quantitative yield after aqueous work up
and lyophilization. The thiol functional group was intro-
duced onto the alkenyl group of 4 by radical activation
(AIBN) with thioacetic acid to give thiacetate 5 in 71%
yield. Compound 5 was then deprotected under Zemplen
conditions (NaOMe, MeOH) to afford thiol 6 (Scheme 2).
l-Lysine based T-Ag dendrons with valencies of two
(10), four (11), and eight (12) were synthesized in 86, 76,
and 79% yields, respectively.11a,19 Each N-chloroacetyl
group in the known lysine cores, 7, 8, and 9 was sub-
stituted by thiolated T-Ag 6 prepared in situ from
thioacatate 5 under basic conditions. The T-Ag con-
taining conjugates (10, 11, and 12) were then purified by
gel permeation chromatography (Biogel P-2 or P-4,
H2O). Completion of conjugation was confirmed by 1H,
Scheme 1. Synthesis of b-d-Gal-(1-3)-a-d-GalNAc (T-antigen): (i)
Hg(CN)2, PhH/MeNO2, rt, N2; (ii) cat. MeONa/MeOH (1 M), 30 min;
(iii) 60% aq AcOH, 60 ꢂC, 1.5 h; (iv) AcSH, AIBN, MeOH, N2, reflux,
12 h; (v) NaOMe, MeOH, rt, N2.

M.-G. Baek, R. Roy / Bioorg. Med. Chem. 9 (2001) 3005–3011
3007
Of all T-Ag dendrimers prepared so far,10a,b tetrameric
structures were shown to bind with the strongest affinity
when expressed on a per saccharide basis. Based on
these results, heterobifunctional tetramer 14, containing
both T-Ag and biotin ligands, was prepared for the
purpose of detecting cancer cell receptors, known to
localize in the lymph nodes, lung, and liver where
metastatic cells are often captured. Quantitative labeling
measurements were performed using streptavidin as a
general biosensor. The cooperative binding interactions
between biotin and streptavidin were demonstrated via
antigen (T-Ag)–antibody (mouse MAb–IgG3) interac-
tions (Fig. 1). The assays were performed with con-
Figure 1. Solid-phase double sandwich enzyme linked immunosorbent
assay (ELISA) illustrating the binding of biotin-labeled tetravalent T-
Ag dendrimer 14 (&) to streptavidin used as coating material. The
detection was based on mouse monoclonal antibody (IgG3) and
horseradish peroxidase-labeled goat anti-mouse antibodies. T-antigen
dendrimer 11 (*), deprived of biotin, was used as a negative control.
Scheme 3. Synthesis of biotin-labeled heterobifunctional tetravalent T-antigen dendron 14.

3008
M.-G. Baek, R. Roy / Bioorg. Med. Chem. 9 (2001) 3005–3011
jugate 11 as a reference negative control. Thus, coating
microtiter plates with streptavidin, binding to 14
through the intermediacy of biotin, followed by detec-
tion of bound T-Ag with the mAb IgG indicated
bifunctional ligand behavior.
chromatography was performed using Biogel P-2 and
P-4 columns using water as an eluent. Optical density
values (ODs) for the ELISA were measured on a
Dynatech MR600 microplate reader. Mouse MAb IgG3
was obtained from T-Ag–BSA glycoprotein by mice
immunization.9a
The optical density (OD) values were dramatically
increased from 0.2 to 1.2 in the concentration range
between 1.5ꢃ10ꢀ10 and 1.5ꢃ10ꢀ12 mol/well. There was
no further increase of OD even at higher concentration
of conjugate 14. Meanwhile, conjugate 11 showed flat
OD values over the entire range of concentrations.
Allyl 3-O-(2,3,4,6-tetra-O-benzoyl-ꢀ-D-galactopyrano-
syl)-2-acetamido-4,6-O-benzylidene-2-deoxy-ꢁ-D-galacto-
pyranoside (3). Allyl 2-acetamido-4,6-O-benzylidene-2-
deoxy-a-d-galactopyranose (2, 1.67 g, 4.78 mmol) was
dissolved in a mixture of anhydrous MeNO2/PhH (60
mL, 1/1, v/v). To ensure dryness, the solution was con-
centrated under reduced pressure. The same volume of
solvent mixture was added and then concentrated until
approximately half the volume remained. The tempera-
ture was adjusted to 25 ꢂC, then 1-bromo-2,3,4,6-tetra-
benzoyl-a-d-galactopyranoside (1, 4.70 g, 7.13 mmol)
and Hg(CN)2 (1.80 g, 7.13 mmol) were added. The
solution was stirred for 18 h under the same conditions.
The solvent was removed under reduced pressure, then
the residue was dissolved in CHCl3 (40 mL) and filtered
through a Celite pad. The filtrate was washed succes-
sively with 10% aq KI, saturated NaHCO3 and water,
and then dried over MgSO4. After the solvent was
evaporated, the residue was purified by silica gel column
chromatography (PhH/EtOAc, 15:1) to give compound
3 in 98% yield (4.31 g, 4.67 mmol). Mp 109.7–111.0 ꢂC,
Rf 0.59 (PhH/EtOAc, 1/2), [a]D+116.0ꢂ (c 1, CHCl3),
(+) FAB-MS (glycerol): 928.3 (M+1), 1856 [2(M+1)].
1H NMR (CDCl3) d 8.06–7.19 (multi, 25H, Ar), 5.98
Conclusions
Chemically well-defined T-Ag-l-lysine dendrons having
valencies of 2, 4, and 8 (10, 11, and 12) were synthesized
by nucleophilic substitution of thiolated T-Ag derivative
(6) to l-lysyl-containing dendritic cores. Although the
chemistry represents yet another example of small
glycoclusters,19,20 using l-lysine core, it further demon-
strates the usefulness of these ‘glycotope mimetics’ in
evaluating factors that dictate multiple protein–carbo-
hydrate interactions. Besides numerous multivalent lec-
tin–carbohydrate interactions studies,10 there is very
scarce examples wherein these multiple interactions are
studied in the context of antibodies, particularly of the
case of divalent IgGs. The first example of glycoden-
drimer synthesis having biotin label to provide a het-
erobifunctional tetramer 14 was also demonstrated from
the corresponding precursor 11 and biotin hydrazide
(13) via efficient peptide bond formation. The doubly
associative binding interactions between conjugate 14 and
the coating streptavidin together with mouse monoclonal
T-Ag–antibody recognition were fully demonstrated
herein using conventional sandwich solid-phase immuno-
assays. The virtue of the T-Ag glycopolymer used as
coating antigen in the present case and elsewhere10aꢀc
should not be minimized since, not only it represents
very effective, high avidity anchoring motif (nanomolar
coating), as shown implicitly by its binding to the anti-
body in the ELISA format, but it also constitutes an
efficient cell surface model.
(dd, 1H, J34 =3.3 Hz, J45 ꢄ 1 Hz, H-40), 5.85–5.78
0
0
(multi, 2H, CH,0 H-20), 5.60 (dd, 1H, J23 =10.2 Hz,
PhCH), 5.23 (dd, 1H, Jgem=1.5 Hz, Jtrans=17.2 Hz,
0
0
J34 =3.4 Hz, H-3 ), 5.48 (broad s, 1H, NH), 5.38 (s, 1H,
CH), 5.16 (multi, 2H, Jcis=8.0 Hz, CH, H-10), 5.10 (d,
1H, J12=3.4 Hz, H-1), 4.68 (dd, 1H, J560=6.9 Hz,
0
0
J6ab =11.4 Hz, H-6a ), 4.63–4.58 (multi, 1H, H-2), 4.46–
4.36 (multi, 3H, H-4, H-30, H-6b0), 4.15–4.07 (multi, 3H,
1H6a, CH, H-3), 3.96 (dd, 1H, Jgem=6.1 Hz, Jab=13.0
Hz, CH), 3.75 (dd, 1H, J56=1.4 Hz, Jgem=12.4 Hz,
H6b), 3.51 (multi, 1H, H-3), 1.40 (s, 3H, Ac); 13C NMR
(CDCl3) d 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 (2 C), 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 (2M+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.
Experimental
Materials and methods
a-d-Galactosamine, biotin hydrazide, streptavidin, and
goat anti mouse monoclonal IgG antibody–horseradish
peroxydase conjugates were purchased from Sigma.
N-Chloroacetylated dendrimers were prepared as pre-
viously described.17 The 1H and 13C NMR spectral data
were obtained using a Bruker 500 MHz AMX NMR
spectrometer. Proton chemical shifts (d) were assigned
relative to internal HOD (4.76 ppm) for D2O solutions.
Carbon chemical shifts were assigned relative to
DMSO-d6 (39.4 ppm). These assignments were based on
COSY, HMQC and DEPT experiments. Mass spectral
data were obtained on a Kratos Concept IIH spectro-
meter (FAB-MS, glycerol matrix). Gel permeation
Allyl 3-O-(ꢀ-D-galactopyranosyl)-2-acetamido-2-deoxy-
ꢁ-D-galactopyranoside (4). To compound 3 (1.9 g, 2
mmol) in MeOH (20 mL) was added a catalytic amount
of MeONa/MeOH (1 M) to adjust the pH to 9. The
reaction mixture was stirred for 30 min at room tem-
perature. The solution was neutralized with H+ resin
(Amberite IR-120) then, condensed under reduced
pressure. The de-benzoylated intermediate was dis-
solved in 60% aqueous acetic acid (15 mL) and the
resulting solution was stirred for 1.5 h at 60 ꢂC. After
evaporation, the residue was dissolved in water (10 mL)

M.-G. Baek, R. Roy / Bioorg. Med. Chem. 9 (2001) 3005–3011
3009
and washed with CHCl3 (20 mL). The solution was
condensed and the residue was purified by silica gel
column chromatography (CHCl3/MeOH/H2O, 11:6:1)
followed by evaporation of CHCl3 and MeOH, and
lyophilization to give T-antigen 4 in quantitative yield
(0.9 g 2 mmol). Mp 230–232 ꢂC, Rf 0.53 (CHCl3/MeOH/
H2O), [a]D+120.0ꢂ (c 1, H2O), (+) FAB-MS (glycerol):
pleted, H+ resin (IRA 120) was added to neutralize
excess CH3OꢀNa+ and the mixture was then filtered.
The filtrate was directly transferred to the solution of
lysine dendritic cores in DMSO and Et3N was added to
adjust the pH to 9. The solution was stirred overnight
under the same conditions. After the solvent was eva-
porated, the residue was purified by gel permeation
chromatography (P-2 or P-4, H2O) to afford the desired
T-Ag conjugates, 10, 11, and 12.
1
424.2 (M+1), H NMR (D2O) d 6.08–6.01 (multi, 1H,
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), 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–
4.27 (multi, 2H, H-4, CH), 4.13–4.07 (multi, 3H, H-3,
H-5, CH), 3.98 (dd, 1H, J34=3.4 Hz, J45=0.8 Hz, H-4),
3.86–3.78 (multi, 4H, 2H-6, 2H-6), 3.74–3.71 (multi, 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) d 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; (+) FAB-MS (glycerol) m/z 424.2
(M+1). 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.
ꢀ-D-Gal-(1-3)-ꢁ-D-GalNAc-OCH2CH2CH2SCH2C(O)-
Gly-Gly2-Lys-ꢀ-Ala-OH (10). T-Ag 6 (35 mg, 1.2 equiv
per chloroacetyl group) and divalent dendritic lysine
core 7 (20 mg, 32 mmol) were used. After purification by
gel permeation chromatography (P-2), conjugate 10 was
1
obtained in 86% yield (42.5 mg, 27.6 mmol). H NMR
(D2O) d 4.95 (d, 2H, J12=3.7 Hz, H-1), 4.54 (d, 2H,
J12=7.8 Hz, H-10), 4.39 (dd, 2H, J12=3.7 Hz, J23=11.0
Hz, H-2), 4.42–4.31 (multi, 4H, H-4, lysyl a-CH2), 4.14–
3.98 (multi, 14H, H-3, H-40, H-5, glycyl CH2), 3.89–3.79
(multi, 10H, H-6, H-60, CH), 3.74–3.70 (multi, 4H, H-30,
H-50), 3.69–3.60 (multi, 4H, H-20, CH), 3.45 (broad s,
2H, b-alanyl b-CH2), 3.07 (t, 2H, Jde=7.3 Hz, lysyl e-
CH2), 2.81 (t, 4H, J=7.2 Hz, CH2S), 2.49 (broad t, 2H,
b-alanyl a-CH2), 2.09 (s, 6H, CH3), 2.00–1.96 (multi,
4H, CH2), 1.92–1.80 (multi, 2H, lysyl b-CH2), 1.78–1.71
(multi, 2H, lysyl d-CH2), 1.51–1.42 (multi, 2H, lysyl g-
CH2); 13C NMR (D2O) d 174.0, 170.8, 104.2, 96.71,
76.9, 74.5, 72.1, 70.2, 68.3, 68.1, 65.9, 60.7, 60.5, 53.2,
48.2, 42.2, 41.9, 38.7, 36.4, 34.3, 30.0, 28.4, 27.6, 25.7,
21.6; (+) FAB-MS (glycerol) m/z; cacld for
C55H93O31N9S2, 1439.5, found, 1482 (M+Na+H2O).
3-(Thioacetyl)propyl 3-O-(ꢀ-D-galactopyranosyl)-2-acet-
amido-2-deoxy-ꢁ-D-galactopyranoside (5). To com-
pound 4 (60 mg, 0.14 mmol) and AcSH (63 mL, 0.85
mmol) in deoxygenated MeOH (3 mL) was added a
catalytic amount of AIBN. The resulting solution was
refluxed for 1 day under nitrogen atmosphere. The
solution was then condensed under reduced pressure
and the residue was dissolved in CHCl3. The organic
layer was washed with saturated NaHCO3, water and
then dried over Na2SO4. The solution was condensed
and the crude product was purified by silica gel column
chromatography (CHCl3/MeOH/H2O, 7:4:0.8) to afford
thioacetate 5 in 71% yield (50 mg, 0.1 mmol). Mp
119.6–122.5 ꢂC, aD+83.0ꢂ (c 1, H2O), RIf 0.69 (CHCl3/
MeOH/H2O, 10/5/1); (+) FAB-MS (glycerol): calcd for
C19H33O12N1S1, 499.17, found, 500.2 (M+1). 1H NMR
(D2O) d 4.95 (d,0 1H, J12=3.8 Hz, H-1), 4.55 (d, 1H,
ꢀ-D-Gal-(1-3)-ꢁ-D-GalNAc-OCH2CH2CH2SCH2C(O)-
Gly-Gly4-Lys2-Lys-ꢀ-Ala-OH (11). T-Ag 6 (34 mg, 1.2
equiv per chloroacetyl group) and tetravalent dendritic
lysine core 8 (16.7 mg, 13.5 mmol) were used. After
purification by gel permeation chromatography (P-4),
conjugate 11 was obtained in 76% yield (30.1 mg, 10.3
1
mmol). H NMR (D2O) d 4.96 (d, 4H, J12=3.8 Hz, H-
0
0
0
1), 4.55 (d, 4H, J12 =7.8 Hz, H-1 ), 4.40 (dd, 4H,
J12 =7.8 Hz, H-1 ), 4.39 (dd, 1H, J12=3.8 Hz, J23=11.9
Hz, H-2), 4.31 (d, 1H, J34=3.1 Hz, J45 ꢄ 1 Hz, H-4),
J12=3.8 Hz, J23=11.1 Hz, H-2), 4.32 (d, 4H, J34=3.0
Hz, J45ꢄ1 Hz, H-4), 4.37–4.35, 4.31–4.28 (multi, 3H,
lysyl a-CH2), 4.11–4.05 (multi, 20H, H-3, H-5, glycyl
CH2), 3.99 (multi, 8H, H-40, glycyl CH2), 3.89–3.79
(multi, 20H, H-6, H-60, OCH2), 3.75–3.69 (multi, 8H,
H-30, H-50), 3.65–3.58 (multi, 8H, H-20, OCH2), 3.46–
3.43 (multi, 2H, b-alanyl b-CH2), 3.29–3.23 (multi, 6H,
lysyl e-CH2), 2.83–2.77 (multi, 8H, SCH2), 2.47–2.45 (t,
2H, J=7.0 Hz, b-alanyl a-CH2), 2.01 (s, 12H, CH3),
2.02–1.95 (multi, 8H, CH2), 1.93–1.71 (multi, 6H, lysyl
b-CH2), 1.60–1.57 (multi, 6H, lysyl d-CH2), 1.44–1.37
(multi, 6H, lysyl g-CH2); 13C NMR (D2O) d 174.02,
170.54, 104.21, 96.7, 76.8, 74.5, 72.1, 70.2, 70.2, 68.3,
68.2, 65.9, 60.7, 60.6, 53.6, 53.3, 48.2, 42.4, 41.9, 38.6,
38.3, 36.2, 34.3, 30.1, 28.5, 27.7, 27.3, 21.9, 21.6; (+)
FAB-MS (glycerol) m/z; cacld for C111H191O61N19S4,
2895.1, found, 1448 (1/2M+1), 724 (1/4M+1).
4.10 (dd, 1H, J23=11.1 Hz, J34=3.1 Hz, H-3), 4.06–
0
0
4.03 (multi, 1H, H-5), 3.98 (d, 1H, J34 =3.4 Hz, H-4 ),
3.86–3.79 (multi, 5H, H-6, H-60, CH), 3.74–3.72 (multi,
1H, H-50), 3.70 (dd,1H, J23 =10.0 Hz, J34 =3.30 Hz, H-
0
0
30), 3.59 (dd, 1H, J12 =7.8 Hz, J23 =9.9 Hz, H-2 ), 3.60–
3.56 (multi, 1H, CH), 3.13–3.04 (multi, 2H, CH), 2.46
(s, 3H,-SAc), 2.10 (s, 3H, AcNH–), 2.00–1.94 (multi,
2H, CH): 13C NMR (D2O) d 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).
0
0
General procedure for T-antigen dendrons synthesis
l-Lysine dendritic cores (7, 8, and 9),19b prepared by
solid-phase, peptide synthesis, were dissolved in deoxy-
genated DMSO under N2 atmosphere. Thioacetylated
T-antigen 5 (1.2 equiv per chloroacetyl group) was dis-
solved in deoxygenated methanol and CH3ONa/MeOH
(degassed, 0.3 M) was added dropwise until compound
5 was changed to thiol 6. Once the reaction was com-
ꢀ-D-Gal-(1-3)-ꢁ-D-GalNAc-OCH2CH2CH2SCH2C(O)-
Gly-Gly8-Lys4-Lys2-Lys-ꢀ-Ala-OH (12). T-Ag 6 (35
mg, 1.2 equiv per chloroacetyl group) and octavalent
dendritic lysyne core 9 (20 mg, 8 mmol) were used. After

3010
M.-G. Baek, R. Roy / Bioorg. Med. Chem. 9 (2001) 3005–3011
purification by gel permeation chromatography (P-4),
conjugate 12 was obtained in 79% yield (39 mg, 6.3
blocking with BSA, pre-incubated mixtures of the T-Ag
dendrimers and mAb IgG were added 2-fold serial
dilution from 1 to 200 nmol/well. Inhibition of binding
was recorded my measuring residual mAb present using
peroxidase-labeled goat anti-mouse antibody. Values
were compared to monomer 4 (IC50 2.3 mM). The
results indicated relative inhibitory potency of ꢁ50
(dimer 10), 300 (tetramer 11), and 200 (octamer 12)
when measured on a per saccharide basis.
1
mmol). H NMR (D2O) d 4.94 (d, 8H, J12=3.7 Hz, H-
0
0
1), 4.53 (d, 8H, J12 =7.8 Hz, H-1 ), 4.38 (dd, 8H,
J12=3.7 Hz, J23=11.1 Hz, Hꢀ2), 4.30 (d, 8H, J34=3.0
Hz, J45 ꢄ 1 Hz, H-4), 4.36–4.34 and 4.28–4.26 (multi,
7H, lysyl a-CH2), 4.10–4.03 (multi, 32H, H-3, H-5, gly-
cyl CH2), 3.97 (multi, 24H, H-40, glycyl CH2), 3.87–3.80
(multi, 40H, H-6, H-60, OCH2), 3.79–3.69 (multi, 16H,
H-30, H-50), 3.68–3.56 (multi, 16H, H-20, OCH2), 3.43
(broad s, 2H, b-alanyl b-CH2), 3.27–3.25 (multi, 14H,
lysyl e-CH2), 2.80–2.79 (multi, 16H, SCH2), 2.44 (t, 2H,
Jab=7.0 Hz, b-alanyl a-CH2), 2.08 (s, 24H, CH3), 1.98–
1.96 (multi, 16H, CH2), 1.85–1.78 (multi, 14H, lysyl b-
CH2), 1.59–1.57 (multi, 14H, lysyl d-CH2), 1.55–1.36
(multi, 14H, lysyl g-CH2); 13C NMR (D2O) d 174.0,
170.5, 104.2, 96.7, 76.8, 74.5, 72.1, 70.2, 68.2, 68.1, 65.9,
60.7, 60.5, 53.6, 53.3, 48.2, 42.3, 42.0, 38.6, 36.4, 34.3,
30.2, 28.5, 27.7, 27.7, 21.9, 21.6; (+) FAB-MS (glycerol)
m/z; cacld for C232H392O121N40S8, 5930.4, found, 1507
(1/4M+Na).
Solid-phase
double
sandwich
enzyme
linked
immunosorbent assay (ELISA) using streptavidin as
coating material, mouse monoclonal IgG antibodies and
tetrameric L-lysine based dendrons 11 and 14
Linbro (titertek) microtiter plates were coated overnight
with 100 mL/well of a streptavidin stock solution (10 mg/
mL in 0.01 M phosphate buffer, pH 7.3) at room tem-
perature. Each well contained 1 mg/well of streptavidin.
The wells were then washed three times with 400 mL/
well of phosphate buffer (0.01 M, pH 7.3) containing
0.05% (v/v) Tween 20 (PBST). The wells were blocked
with 150 mL/well of 1% BSA in PBS for 1 h at 37 ꢂC.
After washing the excess BSA with PBST, the wells were
filled with 100 mL/well of dendrimer solutions of varying
concentration by serial 10-fold dilutions from 1.5ꢃ10ꢀ7
to 1.5ꢃ10ꢀ21 mol/well and incubated for 1 h at 37 ꢂC.
Dendrimers included conjugate 11 and biotin-labeled
conjugate 14. The wells were washed as described above
and filled with 100 mL/well of mouse monoclonal IgG3
antibody solution in PBST (10 times dilution of super-
natant, 0.5 mmol/100 mL). After incubation and wash-
ing, the wells were filled with 100 mL/well of goat-anti-
mouse IgG-MAb horseradish peroxydase conjugate
solution in PBST (1000 times dilution of supernatant)
followed by incubation for 1 h at 37 ꢂC. The wells were
washed and 50 mL/well of 2,2-azinobis (3-ethylben-
zothiazoline-6-sulfonic acid) diammonium salt (ABTS)
(1 mg/4 mL) in citrate–phosphate buffer (0.2 M, pH 4.0
with 0.015% H2O2) was added. The reaction was stop-
ped after 20 min by adding 50 mL/well of aqueous sul-
furic acid (1 M). Optical density was measured at 410 nm
relative to 570 nm. All tests were performed in triplicate.
ꢀ-D-Gal-(1-3)-ꢁ-D-GalNAc-OCH2CH2CH2SCH2C(O)-
Gly-Gly4-Lys2-Lys-ꢀ-Ala-Biotin hydrazide (14). Tetra-
valent conjugate 11 (30 mg, 10.3 mmol) and biotin
hydrazide 13 (3.8 mg, 10.3 mmol) were dissolved in
DMF (3 mL). TBTU (4 mg, 1.2 equiv) and DIPEA (pH
9) were added and the resulting solution was stirred
overnight at room temperature. Solvent was removed
under reduced pressure and the residue was purified by
gel permeation chromatography (P-4) to afford the het-
erobifunctional conjugate 14 in 92% yield (31 mg, 9.5
1
mmol). H NMR (D2O) d 4.96 (d, 4H, J12=3.8 Hz, H-
1), 4.68 (dd, 1H, J=40.5 Hz, J=8.0 Hz, H-8), 4.55 (d,
0
4H, J12 =7.8 Hz, H-1 ), 4.50 (dd, 1H, J=8.0, 4.5 Hz,
CH), 4.40 (dd, 4H, J12=3.7 Hz, J23=11.1 Hz, H-2),
4.31 (d, 4H, J34=2.9 Hz, J45 ꢄ 1 Hz, H-4), 4.37, 4.34–
4.25 (multi, 6H, lysyl a-CH2), 4.11–4.05 (multi, 20H, H-
3, H-5, glycyl CH2), 3.99 (multi, 8H, H-40, glycyl CH2),
3.96–3.78 (multi, 20H, H-6, H-60, OCH2), 3.74–3.67
(multi, 8H, H-30, H-50), 3.65–3.58 (multi, 8H, H-20,
OCH2), 3.57–3.49 (multi, 2H, b-alanyl b-CH2), 3.29–
3.19 (multi, 9H, CH, CH2, lysyl e-CH2), 3.08–3.05 (dd,
1H, J=5.0 Hz, Jgem=13.1 Hz, CH), 2.87–2.79 (multi,
9H, SCH2, CH), 2.62 (t, 2H, J=6.7 Hz, b-alanyl a-
CH2), 2.40 (t, 2H, J=7.4 Hz, CH2), 2.32 (t, 2H, J=7.2
Hz, CH2), 2.10 (s, 12H, CH3), 2.00–1.97 (multi, 8H,
CH2), 1.93–1.66 (multi, 14H, CH2, CH2, CH2, CH2,
lysyl b-CH2), 1.65–1.51 (multi, 8H, lysyl d-CH2), 1.47–
1.36 (multi, 12H, CH2, CH2, lysyl g-CH2); 13C NMR
(D2O) d 174.0, 170.5, 104.2, 96.7, 76.8, 74.5, 72.1, 70.2,
70.2, 68.2, 68.1, 65.9, 61.6, 60.7, 60.5, 59.8, 54.9, 53.6,
53.0, 48.2, 42.4, 41.9, 39.5, 38.6, 32.1, 34.3, 32.8, 30.2,
30.1, 28.5, 27.7, 27.6, 28.5, 27.6, 24.9, 24.7, 22.0, 21.6.
Acknowledgements
The authors thank Dr. D. Zanini for valuable assistance
in the preparation of lysine dendrimers. We also thank
the National Science and Engineering Research Council
of Canada (NSERC) for financial support.
References and Notes
Competitive solid-phase inhibition of binding of mAb
IgG to T-Ag copolyacrylamide by dendrimers 10, 11,
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