Synthesis and antibody binding properties of glycodendrimers bearing the tumor related T-antigen

Article abstract of DOI:10.1039/b008664i

Breast cancer marker T-antigen (Gal(β1-3)αGalNAc) was prepared as an allyl glycoside that was transformed into an active ester and coupled to a series of poly(amidoamine) dendrimers showing strong binding to mouse monoclonal IgG antibodies and serving as

Full text of DOI:10.1039/b008664i

Synthesis and antibody binding properties of glycodendrimers bearing the
tumor related T-antigen
Myung-Gi Baek,a Kate Rittenhouse-Olson,b and René Roy*a
a Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa, Ontario K1N 6N5 Canada
b Department of Microbiology and Immunology, Roswell Park Division, State University of New York at Buffalo,
Buffalo, NY 14214. USA. E-Mail: rroy@science.uottawa.ca; Fax: +1-613-562-5170; Tel: +1-613-562-5800
ext 6055
Received (in Corvallis, OR, USA) 23rd October 2000, Accepted 8th December 2000
First published as an Advance Article on the web 23rd January 2001
Breast cancer marker T-antigen (Gal(b1-3)aGalNAc) was
prepared as an allyl glycoside that was transformed into an
active ester and coupled to a series of poly(amidoamine)
dendrimers showing strong binding to mouse monoclonal
IgG antibodies and serving as useful coating antigens in
microtiter plates.
deoxygenated aqueous conditions gave acid functionalized 9 in
83% yield.
PAMAM cores 10–13 were conjugated via amide bond
formation with a slight excess acid 9 (1.1 eq.) using TBTU‡
reagent (Scheme 2). The reaction mixtures were lyophilized to
dryness and the residues were purified by either gel permeation
chromatography (P-2 or P-4, H2O) or dialysed against water
(2000 molecular weight cut-off). After purification, T-antigen-
glycoPAMAMs, 14–17 (Fig. 1) with 4, 8, 16, and 32 T-antigen
residues were obtained in excellent yields (73, 81, 99, and 79%,
respectively).
On the basis of negative ninhydrin tests, mass spectrometry
(FAB-MS), and high field 1H NMR spectral data, the extent of
conjugations were shown to be complete. The ratios of the NMR
signals, fully assigned using COSY and HMQC experiments
(D2O, sensisitivity @2 ~ 3%; ± 1 residue/30), clearly confirmed
the total incorporation of the T-antigen residues by comparing
the relative integration of the two methylene signals of the
dendritic cores (d 2.80 for 14 and 15, 2.77 for 16 and 3.05 ppm
for 17, respectively) to those of the two anomeric protons of the
T-antigen (d 4.96 for H-1 and 4.56 ppm for H-1A re-
spectively).
Poly(amidoamine) (PAMAM) dendrimers1 are actually scruti-
nized as core structures bearing various pharmacophores for
potential applications in biomedical fields such as DNA
transfection,2 inhibition of pathogenic infections,3 and toxicity,
immunogenicity and biodistribution to organs.4 PAMAM have
further broadened their potential usefulness to biological
systems by combination with different carbohydrate moie-
ties.5–7 Chemically well defined monodisperse neoglycoconju-
gates with several covalently attached carbohydrate residues,
can efficiently compensate for the usually weak affinities of
individual haptens via cooperative binding interactions.8 How-
ever, in spite of these potential biological applications,
systematic preparation and biological evaluation of PAMAM-
based T-antigen, Gal(b13)aGalNAc, glycodendrimers have not
been reported to date.
T-antigen (Thomsen–Friedenrich antigen) has been reported
as a cancer-related epitope and as an important antigen for the
detection and immunotherapy of carcinomas, particularly
relevant in breast cancer.9 For pharmaceutical applications, T-
antigen containing linear glycopolymers have been employed in
solid-phase glycosyl transferase assays for high-throughput
screening in cancer drug discovery research.10 Recently, we
have reported that mouse monoclonal antibodies raised against
the T-antigen recognised breast cancer tissues selectively.11
These biological and immunochemical results motivated the
synthesis of well defined T-antigen-glycoPAMAMs that can be
used as vaccines or as antibody targeting species. Their binding
properties against mouse monoclonal IgG antibody has also
been evaluated. The synthetic strategy for the construction of T-
antigen-containing glycoPAMAMs was based on amide bond
formation between amine terminated PAMAM cores 10–13 and
acid functionalized T-antigen derivative 9.
a- -GalNAc derivative 2† was prepared in 3 steps from N-
D
acetylglucosamine (1) by a C-4 epimerization strategy using an
adaptation of documented procedure (Scheme 1).12 Compound
2 was debenzoylated under Zemplén conditions (NaOMe,
MeOH) and subsequently exposed to either benzaldehyde–zinc
chloride or benzaldehyde dimethyl acetal and a catalytic amount
of toluene-p-sulfonic acid monohydrate to give 4,6-benzylidene
protected glycosyl acceptor 3 in 87% yield after deprotection.
Compound 3 was coupled with peracylated galactosyl bromide
(4 or 5) using mercuric(II) cyanide as a promoter at rt to provide
disaccharides 6 and 7 in 98 and 78% yields, respectively.
Successive de-O-acylation (MeONa–MeOH) and de-acetala-
tion (aq. AcOH, 60 °C) afforded the desired T-antigen 8 in 95%
overall yield. Finally, radical initiated 3-mercaptopropionic
acid addition to 8 in the presence of UV light (254 nm) under
Scheme 1 (a) allyl alcohol, BF3OEt2, reflux for 3 h, 65 %; (b) BzCl (2.2 eq.),
Py–CHCl3, 260 °C, 92%; (c) (i)Tf2O (1.5 equiv), Py (3.3 eq.)/CH2Cl2,
215 °C, (ii) NaNO2 (10 eq.), DMF, 25 °C, 85% (2 steps); (d) MeONa,
MeOH, quantitative; (e) PhCHO, ZnCl2 (2 eq.) or PhCH(OMe)2 (5 eq.), 25
°C, 87%; (f) Gal-Br (4 (Bz) or 5 (Ac)) (1.5 eq), Hg(CN)2 (1.5 eq) , PhH–
MeNO2, 25 °C 78–98 %; (g) i) MeONa, MeOH, (ii) 60% aq. AcOH, 60°C,
93%; (h) HSCH2CH2CO2H (1.1 eq), UV (254 nm), 83%. BzCl = benzoyl
chloride, Py = pyridine, Tf2O = trifluoromethanesulfonic anhydride,
AcOH = acetic acid.
DOI: 10.1039/b008664i
Chem. Commun., 2001, 257–258
This journal is © The Royal Society of Chemistry 2001
257

14–17 showed IC50 values of 5.0, 2.4, 1.4 and 0.6 nmol,
respectively where monomeric T-antigen 8 needed 2.3 mmol.
The inhibitory abilities of these conjugates were thus 460, 960,
1700 and 3800 times higher than that of monomer 8.
Interestingly, when expressed on a per saccharide basis, all
dendrimers were equivalent to one another (115 fold better than
8), thus supporting previous observations that low density
glycoPAMAMs are efficient inhibitors.5
In conclusion, geometrically well designed starburst T-
antigen-glycoPAMAMs were efficiently synthesized with va-
lencies between 4 to 32 using efficient peptide coupling
strategy. A series of bioassays with mouse MAb IgG demon-
strated that these conjugates strongly bound to the antibody. All
conjugates were antigenetically active as previously demon-
strated with analogous structures.5–7 Moreover, some carci-
noma cells have been reported to express T-antigen binding
sites,14 it is therefore possible that the compounds described
herein may also find applications in specific cancer cell
targeting.
Scheme 2 (a) 1.1 eq 9 per amine, TBTU, DIPEA, DMSO, 25 °C, (b) gel
permeation chromatography (P-2, or P-4, water) or dialysis (2000 molecular
weight cut-off) 73–99%. DIPEA
= diisopropylethylamine, DMSO =
dimethylsulfoxide.
Notes and references
† Physical data for representative compounds, NMR assignments are based
on COSY and HMQC.
6: mp 109.7–111.0 °C; Rf = 0.59 (benzene–ethyl acetate, 1+2); [a]D
=
+116.0° (c = 1.00, CHCl3); (+) FAB-MS (glycerol) (m/z): 928.3 (M + 1);
dH (500 MHz, CDCl3): 5.16 (m, Jcis = 8.0 Hz, 2 H, CH, H1A), 5.10 (d, J12
= 3.4 Hz, 1 H, H1); dC (500 MHz, CDCl3): 102.0 (C1A), 100.9 (CH), 97.4
(C1); Anal. Calcd. for C52H49O15N (927.49): C, 67.20; H, 5.31; N, 1.53.
Found: C, 66.84; H, 5.27; N, 1.48%. 9: mp 90.0–92.5 °C; [a]D = +76.0° (c
Fig. 1 ELISA of T-antigen-glycoPAMAMs, 14(5), 15 (3), 16 (/) and 17
(:) with mouse monoclonal IgG antibody, goat anti-mouse IgG-horse-
radish peroxidase conjugate and ABTS-H2O2 as enzyme substrate. O.D.
(410 nm) = optical density, ABTS = 2,2A-azinobis(3-ethylbenzothiazo-
line-6-sulfonic acid). C: Relative concentration.
=
1, H2O); Rf = 0.33 (CHCl3–MeOH–H2O, 10+9+1); (+) FAB-MS
(glycerol): 530.3 (M + 1); dH (ppm, D2O): 4.96 (d, J12 = 3.7 Hz, 1 H, H1),
4.54 (d, J1A2A = 7.7 Hz, 1 H, H1A); dH (ppm, D2O): 104.2 (C1A), 96.7 (C1).
17: dH (500 MHz, D2O): 4.96 (d, J12 = 3.7 Hz, 32 H, H1), 4.56 (d, J1A2A
=
7.7 Hz, 32 H, H1A); dC (D2O): 104.2 (C1A), 96.8 (C1). Anal. Calcd. for
C942H1664O444N154S32(23,278.7): C, 48.60; H, 7.20; N, 9.27. Found: C,
48.85; H, 7.21; N, 8.79%.
The binding properties of glycoPAMAMs to mouse mono-
clonal IgG antibody (MAb)11 were initially evaluated by
enzyme linked immunosorbent assay (ELISA) (Fig. 1). Glyco-
PAMAMs were used as coating antigens to determine their
ability to capture antibodies on the surface of the microtiter
plates. Goat anti-mouse MAb IgG-horseradish peroxidase
(HRP) conjugate was used for quantitative detection. The
ability of the mouse MAb to recognise all glycodendrimers was
clearly demonstrated (Fig. 1). Compound 17, having 32 T-
antigen residues, was shown to be the best ligand. To obtain an
optical density value of 0.6, 90 mmol of coating glycoden-
drimers was needed. This value represents 2- to 4-fold increased
binding potentials over those of 15 (16-mer) and 16 (8-mer)
(330 and 150 mmol, respectively) which where > 25-fold more
potent than tetramer 14 ( > 2 mmol).
The efficiency of these glycoPAMAMs for protein-binding
interactions were further substantiated by competitive double
sandwich inhibition where conjugates 14–17 were employed as
inhibitors. T-antigen-co-polyacrylamide (T-antigen–acryla-
mide, 1+10)13 was used as a coating antigen. To this end, the T-
antigen polymer (1mg well21, 0.85 nmol of T-antigen in PBS)
was deposited in the microtiter plates. In separated plates
(Nunclon, Delta), mouse-MAb IgG (50 mL well21, 0.25 mmol in
PBS-tween) and glycoPAMAM inhibitors (50 mL well21 in
PBS-tween with varying concentrations from 275 to 1.07 nmol
well21) were preincubated. After blocking the microtiter plates
with bovine serum albumin (BSA), the wells were filled with
the mixture of antibody–glycoPAMAM inhibitors (100
mL well21) and the mixtures were then incubated for 1 h at
37 °C. For the quantitative detection of antibodies, goat anti-
mouse MAb-HRP was then added, as above. Based on bulk
conjugates, the glycoPAMAMs with highest carbohydrate
density (17) exhibited the strongest inhibitions. Conjugates
‡ TBTU
=
O-benzotriazol-1-yl)-N,N,NA,NA-tetramethyluronium tetra-
fluoroborate.
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