Synthesis and protein binding properties of T-antigen containing GlycoPAMAM dendrimers

Article abstract of DOI:10.1016/S0968-0896(01)00248-6

Allyl O-(β-D-galactopyranosyl)-(1-3)-2-acetamido-2-deoxy-α-D-galactopyranoside (8) was prepared in excellent yield from the corresponding galactosyl bromide (6, 7) and allyl 2-acetamido-4,6-benzylidine-2-deoxy-α-D-galactopyranoside (5) using Hg(CN)₂ as a promoter. Compound 5 was obtained from N-acetylglucosamine 1 following sequential protecting group strategy and C-4 epimerization as a key step. Carboxylic acid functionalized T-antigen derivative 15, obtained by radical addition of 3-mercaptopionic acid to allyl disaccharide 10, was conjugated to PAMAM dendritic cores 13-16 by an efficient amide coupling strategy using TBTU. GlycoPAMAM dendrimers having T-antigen residues with 4, 8, 16 and 32 valencies (17-20) were obtained in 73 to 99% yields. Their protein binding properties were demonstrated using peanut lectin from Arachis hypogaea and a mouse monoclonal IgG antibody. The higher valency conjugates generated stronger binding interactions indicating a cluster effect. The inhibitory potential of these glycoPAMAM conjugates toward antibody-coating antigen interactions was enhanced up to 3800 times over that of the monomeric T-antigen residue (10). Copyright

Full text of DOI:10.1016/S0968-0896(01)00248-6

Bioorganic & Medicinal Chemistry 10 (2002) 11–17
Synthesis and Protein Binding Properties of T-Antigen
Containing GlycoPAMAM Dendrimers
Myung-Gi Baeky and Rene Roy*
Centre for Research in Biopharmaceuticals, Department of Chemistry, University of Ottawa, Ottawa, ON, Canada K1N 6N5
Received 19 April 2001; accepted 21 June 2001
Abstract—Allyl O-(b-d-galactopyranosyl)-(1-3)-2-acetamido-2-deoxy-a-d-galactopyranoside (8) wasprepared in excellent yield
from the corresponding galactosyl bromide (6, 7) and allyl 2-acetamido-4,6-benzylidene-2-deoxy-a-d-galactopyranoside (5) using
Hg(CN)2 asa promoter. Compound 5 wasobtained from N-acetylglucosamine 1 following sequential protecting group strategy and
C-4 epimerization asa key step. Carboxylic acid functionalized T-antigen derivative 15, obtained by radical addition of 3-mercap-
topropionic acid to allyl disaccharide 10, wasconjugated to PAMAM dendritic cores 13–16 by an efficient amide coupling strategy
using TBTU. GlycoPAMAM dendrimers having T-antigen residues with 4, 8, 16 and 32 valencies (17–20) were obtained in 73 to
99% yields. Their protein binding properties were demonstrated using peanut lectin from Arachis hypogaea and a mouse mono-
clonal IgG antibody. The higher valency conjugates generated stronger binding interactions indicating a cluster effect. The inhibi-
tory potential of these glycoPAMAM conjugates toward antibody-coating antigen interactions was enhanced up to 3800 times over
that of the monomeric T-antigen residue (10). # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
Moreover, highly conjugated polymeric materialshave
been incorporated into biologically active species to
detect their role in biosystems.10 These new types of
conjugateshave great potential in the development of
biosensory devices for diagnostic purposes.
Cell surface carbohydrates are involved in critical bio-
logical phenomena such as cellular recognition, adhe-
sion, cell growth regulation, pathogenic infection,
cancer and xenotransplantation.1,2 The investigation of
novel carbohydrate-based biological interactions has
established a new platform of rational drug design for
the purpose of diagnostics and therapeutics. However it
isknown that individual carbohydrate–protein interac-
By virtue of glycodendrimersaschemically and geome-
trically well-defined monodisperse macromolecules, they
lend themselves as useful tools for medicinal and pha-
maceutical purposes.11À14 Poly(amidoamine) (PAMAM)
and related dendrimers15,16 have been employed in var-
iousbiochemical and medicinal applications. Examples
are complexation with DNA for efficient transfection17
and biological evaluation such as toxicity, immuno-
genicity and biodistribution to organs.18 The combina-
tion of PAMAM dendrimerswith different
carbohydrate moietieshasbroadened their potential
applications to biological systems.19À23 In spite of
potential biological applications, the systematic pre-
paration and biological evaluation of PAMAM based
T-antigen, bGal-(1-3)-aGalNAc, glycodendrimershave
not been reported to date.
3
tionsare of low affinity. Recent developmentsin the
multiple expression of these interactions by means of
cluster effects have been shown to improve the efficiency
of binding.4,5 Thisisa mimic of the multivalent nature
of cell surface carbohydrates, found on glycoproteins,
glycolipids, polysaccharides and proteoglycans where
cooperativity can increase the overall binding affinity.
The requirement of multivalency for strong binding has
stimulated the synthesis of several types of glycoconju-
gates with multiple carbohydrate binding sites, for
example,
glycodendrimers.6À9
glycopolymersand
more
recently,
T-antigen (Thomsen–Friedenrich antigen) has been
reported asa cancer-related epitope and asan impor-
tant antigen for the detection and immunotherapy of
carcinomasparticularly relevant in bretas cancer
patients.24À26 For pharmaceutical applications, T-antigen
*Corresponding author. Tel.: +1-613-562-5800 x 6055; fax: +1-613-
562-5170; e-mail: rroy@science.uottawa.ca
yCurrent address: Steacie Institute for Molecular Science, NRC,
Ottawa, Canada, ON K1A 0R6.
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00248-6

12
M.-G. Baek, R. Roy / Bioorg. Med. Chem. 10 (2002) 11–17
containing linear glycopolymershave been employed to
develop solid-phase glycosyltransferase assays for high-
throughput screening in drug discovery research.27
Recently, we have reported that mouse monoclonal
antibodies recognize breast cancer tissues selectively.28
The corresponding vaccine was generated from a T-
antigen-Bovine Serum Albumin (BSA) conjugate which
did not contain the O-linked Ser/Thr residues of the
natural T-antigen, thus demonstrating the sole carbo-
hydrate binding requirement of our antibodies.28 Con-
sequently, multiple O-linked T-antigensdeprived of O-
Ser/Thr aglyconswere synthesized. Recently, a new type
of water soluble T-antigen containing linear conjugates
(Scheme 1).32 In brief, selective benzoylation of 1 at the
C-3 and 6 positions was performed at À60 ꢀC to give
compound 2 in 92% yield. Compound 2 was succes-
sively treated with Tf2O (CH2Cl2/Py, À15 ꢀC) and
NaNO2 (DMF, rt) to give compound 3 in 85% overall
yield. De-benzoylation (NaOMe/MeOH) and benzyli-
dene acetal formation of
PhCH(OMe)2/p-TSA, o.n.) afforded compound 5 in
3
(PhCHO/ZnCl2 or
87% overall yield.
Compound 5 wascoupled with different glycosyl donors
(6, and 7) in two strategies to give the desired T-Ag, 8, 9
and 12 (Scheme 1). Perbenzoylated (or peracetylated)
Gal-Br (6 or 7) were employed under Helferich condi-
tionsusing HgCN 2 to give 8 and 9 in 98 and 78% yields,
respectively. Successive deprotection (i. NaOMe/
MeOH, ii. aq AcOH, 60 ꢀC) afforded T-Ag 10 in 95%
yield. Acid functionalization of 10 wasaccomplihsed
using 3-mercaptopropionic acid (11) (254 nm, degassed
water) to give compound 12 in 83% yield.
29
that have high lipophilicity hasbeen ysntheiszed.
These biological and immunochemical results motivated
the synthesis of chemically well-defined multivalent T-
antigen-glycoPAMAM dendrimersreported here. Their
binding propertiesagaints our moues monoclonal IgG
antibody were also evaluated.
T-Ag-GlycoPAMAM dendrimerswere prepared by
amide bond formation between T-Ag-acid (12) and
PAMAM dendritic cores( 13, 14, 15 and 16). Briefly,
commercially available PAMAM dendritic coresin
MeOH were dried under vacuum and the the residue
was dissolved in dimethyl sulfoxide (DMSO). To a
slight excess of 12, 2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium tetra-fluoroborate (TBTU) and
N,N-diisopropylethylamine (DIPEA) (pH=9) were
successively added and the resulting solution was stirred
overnight at room temperature (Scheme 2). The solu-
tion was lyophilized to dryness and the residue was
purified by either gel permeation chromatography (P-2
or P-4, H2O) or dialysis (H2O, 2000 molecular cut-off)
to afford glycoPAMAM conjugates, 17, 18, 19 and 20
having 4, 8, 16 and 32 T-Ag residues in 73, 81, 99 and
79% yields, respectively. Conjugation was judged com-
plete based upon negative ninhydrin tests and high field
1H NMR spectral data (D2O) (sensisitivityꢁ2–3%; Æ1
residue/30). The ratios of the signals 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 ppm for 17 and 18,
2.77 ppm for 19 and 3.05 ppm for 20, respectively) to
those of the two anomeric protons of the T-antigen
(Fig. 1) (d 4.96 ppm for H-1 and 4.56 ppm for H-10).
Results and Discussion
For the synthesis of T-antigen (T-Ag), the glycosyl
acceptor, 4,6-benzylidene-a-d-GalNAc (5) wasprepared
from GalNAc (1) by a C-4 epimerization method
Immunochemical assays with proteins (peanut lectin from
arachis hypogaea and mouse monoclonal igg antibody).
To demonstrate the binding ability of the conjugates
toward proteins, turbidimetric analysis was initially
performed with peanut lectin from Arachis hypogaea
(Fig. 2). In the presence of the same amount of T-Ag,
conjugate 20 (n=32) showed the highest OD value
(0.41) where other conjugates, 17, 18 and 19 showed
valuesof 0.17, 0.26, and 0.28, repsectively. Thees
microquantitative precipitation experimentsfirts con-
firmed the direct binding (cross-linking) abilities of the
glycoPAMAM conjugateswith protein, thusgenerating
glycodendrimer–protein complexesthat could be
Scheme 1. (a) Allyl alcohol, BF3OEt2, reflux, 3 h, 65%; (b) BzCl (2.2
equiv), Py/CHCl3, À60 ꢀC, 92%; (c) (i) Tf2O (1.5 equiv), Py (3.3
equiv)/CH2Cl2, À15 ꢀC; (ii) NaNO2 (10 equiv), DMF, 25 ꢀC, 85% (2
steps); (d) NaOMe/MeOH; (e) MeONa, MeOH, quantitative; (f)
PhCHO, ZnCl2 (2 equiv) or PhCH(OMe)2 (5 equiv), 25 ꢀC, o.n., 87%;
(g) peracylated Gal-Br [4 (Bz) or 5 (Ac)] (1.5 equiv), Hg(CN)2 (1.5
equiv), benzene/MeNO2, 25 ꢀC 78–98%; (h) (i) MeONa, MeOH; (ii)
60% aq AcOH, 60 ꢀC, 93%; (i) HSCH2CH2CO2H (11, 1.1 equiv, UV
(254 nm), deoxygenated water, 83%. BzCl=benzoyl chloride,
Py=pyridine, Tf2O=trifluoroacetic anhydride, Gal-Br=peracylated
galactopyranosyl bromide, AcOH=acetic acid.

M.-G. Baek, R. Roy / Bioorg. Med. Chem. 10 (2002) 11–17
13
visualized with the naked eye and quantified as a
function of time by optical density measurements.
better to the hydrophobic polystyrene plate surfaces.
For instance, dendrimer 20 (32-mer) showed 18-fold
better sensitivity when compared to that of 17 (4-mer).
These results represent enhanced coating ability as the
glycoPAMAMs’ density increased.
The efficiency of these conjugates toward protein bind-
ing interactions were further substantiated by a solid-
phase enzyme linked lectin assay (ELLA) where the
glycoPAMAM conjugateswere employed ascoating
antigens(Fig. 3). Clearly, and in psite of apparent
increase in surface group hydrophilicity, the more sub-
stituted glycoPAMAMs generated species that bound
The relative efficiency of the glycodendrimersto inhibit
the binding of mouse monoclonal IgG antibody28
to coating antigen (T-Ag-copolyacrylamide)27,29 was
determined using goat anti-mouse monoclonal IgG
Scheme 2. (a) TBTU, DIPEA, DMSO, 25 ꢀC; (b) gel permeation chromatography (P-2, or P-4, water) or dialysis (2000 molecular weight cut-off) 73–
99%. TBTU=O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, DIPEA=diisopropylethylamine, DMSO=dimethyl sulfoxide.
Figure 1. Structure of 32-valent glycoPAMAM dendrimer (20).

14
M.-G. Baek, R. Roy / Bioorg. Med. Chem. 10 (2002) 11–17
antibody by a competitive solid-phase enzyme linked
immunosorbent assay (ELISA). Antibody (50 mL/well,
0.25 mmol in phosphate buffer saline containing tween
(PBST) waspre-incubated with varying concentration
(257–1.07 nmol) of glycoPAMAMs. This solution was
added to a plate coated with T-Ag-copolyacrylamide.
The inhibitory potential of each conjugate wasmea-
sured at 410 nm. The degree of inhibition was propor-
tional to the conjugate valenciesand showed maximum
inhibition when conjugate 20 (32 T-antigen surface
residues) was used. These results are consistent with the
evaluation using turbidimetric analysis and ELLA. The
concentrationsof conjugatesto give 50% inhibition of
the antibody-binding to T-Ag copolymer interaction
were 5.0, 2.4, 1.4 and 0.6 nmol for conjugates 17, 18, 19
and 20 where monomeric T-Ag, 10 required 2.3 mmol.
These values represent a 460, 960, 1700 and 3800 fold
enhancement of inhibitory potentialsover that of
monomeric 10 (IC50 2300 nM) when considered as a
whole. However, each T-Ag epitope is, on average, only
20 fold more efficient than the monomer irrespective of
the dendrimer valency (IC50/valency). These results
confirmed earlier findings33,34 demonstrating that tetra-
meric T-Ag clusters may represent the optimum size for
antibody-glycocluster inhibition.
From a series of bioassays, the most remarkable finding
isthat artificial T-Ag glycoPAMAMsshowed excellent
protein binding potential in the presence of competitive
antigens and are successfully employed as inhibitors for
antibody-coating antigen interactions.
Conclusions
Chemically and geometrically well-defined T-Ag glyco-
PAMAM dendrimerswith valenciesof 4, 8, 16 and 32
were synthesized via efficient amide bond formation.
Successive bioassays with peanut lectin from Arachis
hypogaea which haspotent anti-T activity analogousto
our mouse monoclonal IgG antibody showed strong
protein binding propertiesdemontsrating an excellent
cluster effect. GlycoPAMAMs are strong candidates for
biological and immunochemical applicationsusch as
inhibition of cancer cell metastasis.35
Materials and Methods
General methods
Melting pointswere determined on a Gallenkamp
apparatus. Optical rotation values were measured on a
Perkin–Elmer 241 polarimeter and were run at 25 ꢀC.
Mass spectral data were obtained on a VG 7070-E
spectrometer (CI, ether) and Krotas Concept IIH for
FAB-MS (glycerol). Thin layer chromatography (TLC)
was performed using silica gel 60 F-254 and column
chromatography on silica gel 60. The 1H and 13C NMR
spectra were obtained on a Bruker 500 MHz AMX
NMR spectrometer. The chemical shifts of protons (d)
were given relative to internal chloroform (7.24 ppm)
for CDCl3 solutions, to internal DMSO (2.49 ppm) for
DMSO-d6 solutions, and to internal HOD (4.65 ppm)
for D2O solutions. 13C chemical shifts were given rela-
tive to CDCl3 (77.0 ppm) or DMSO-d6 (39.5 ppm). The
assignments were based on COSY, HMQC, and DEPT
experiments. Absorbance for the turbidimetric, ELLA,
and ELISA tests were performed on a Dynatech MR
600 Microplate Reader. a-d-glucosamine was purchased
from Sigma. Peanut lectin from Arachis hypogaea and
labeled peanut lectin-peroxidase were purchased from
Sigma (product number L 0881 and L 7759). Mouse
monoclonal IgG antibodieswere generated using bovine
Figure 2. Turbidimetric analysis of glycoPAMAM dendrimers, 17
(&), 18 (~), 19 (^) and 20 (*), with peanut lectin from Arachis
hypogaea.
serum
albumin
(BSA)-T-antigen
conjugates.28
PAMAMswere kindly donated by Dr R. Spindler
(Dendritech Inc., Midland, MI).
Allyl (2,3,4,6-tetra-O-benzoyl-ꢀ-D-galactopyranosyl)-(1-
3)-2-acetamido-4,6-O-benzylidene-2-deoxy-ꢁ-D-galacto-
pyranoside (8). A solution of allyl 2-acetamido-4,6-O-
benzylidene-2-deoxy-a-d-galactopyranoside (5)30 (1.67
g, 4.78 mmol) in a mixture of nitromethane/benzene (60
mL, 1:1 v/v) wasstirred for 2 h at room temperature. To
Figure 3. Enzyme linked lectin assays (ELLA) of peanut lectin to
coated glycoPAMAM dendrimers, 17 (&), 18 (~), 19 (^) and 20 (*).

M.-G. Baek, R. Roy / Bioorg. Med. Chem. 10 (2002) 11–17
15
ensure dryness, the solution was concentrated under
reduced pressure. This process was repeated 3 times.
The same volume of solvent was added and then con-
centrated until half the volume remained. The tempera-
ture wasadjusted to 25 ꢀC and 2,3,4,6-tetra-O-benzoyl-
a-d-galactopyranosyl bromide (6)31 (4.70 g, 7.13 mmol)
and Hg(CN)2 (1.80 g, 7.13 mmol) were added succes-
sively under N2 atmosphere. The resulting solution was
stirred at room temperature for 18 h. The solvent was
removed under vacuum and 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, distilled water, and then dried
over Na2SO4. After concentration, the residue was pur-
ified by silica gel column chromatography (benzene/
EtOAc, 15:1) to give a white foamy solid in 98% yield
(4.31 g, 4.67 mmol): mp 109.7–111.0 ꢀC; Rf 0.59 (ben-
3.71 (m, 1H, H-50), 3.69 (dd, 1H, J230=10.0, J340=3.4
Hz, H-30), 3.59 (dd, 1H, J120=7.7, J230=10.0 Hz, H-20),
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 C17H29O11N1: C, 45.30; H, 6.92;
N, 3.30. Found: C, 45.30; H, 6.93; N, 3.07
3-(2-Carboxyethylthio)propyl ꢀ-D-galactopyranosyl-(1-
3)-2-acetamido-2-deoxy-ꢁ-D-galactopyranoside (12). To
a solution of allyl b-d-Gal-(1-3)-a-d-GalNAc (13) (100
mg, 0.24 mmol) in deoxygenated distilled water (2.5
mL) wasadded 3-mercaptopropionic acid ( 11, 21 mL, 1
equiv) and the resulting solution was irradiated (254
nm) for 7 h under N2 atmosphere. The reaction solution
wasthen loaded onto an anion exchange resin column
(Amberite IRA 400 OHÀ) and washed with water. Elu-
ent wasthen changed gradually to aqueousacetic acid
with an increasing acetic acid gradient. The fractions
containing 12 were collected and evaporated under
reduced pressure followed by multiple coevaporation
with ethanol. A small amount of water was added and
lyophilized to afford a white spongy solid in 83% yield
(105.9 mg, 2.0 mmol): mp 90.0–92.5 ꢀC; aD +76.0ꢀ (c 1,
zene/EtOAc, 1:2); [a]D+116.0ꢀ (c 1, CHCl3); H NMR
1
(CDCl3) d 8.06–7.19 (m, 25H, Ar), 5.98 (dd, 1H,
J340=3.3, J450 ꢁ1 Hz, H-40), 5.85–5.78 (m, 2H, CH, H-
20), 5.60 (dd, 1H, J230=10.2, J340=3.4 Hz, H-30), 5.48
(broad 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-10), 5.10 (d, 1H, J12=3.4 Hz, H-1), 4.68 (dd,
1H, J560=6.9, J6ab0=11.4 Hz, H-60), 4.63–4.58 (m, 1H,
H-2), 4.46–4.36 (m, 3H, H-4, H-50, H-60), 4.15–4.07 (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)
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 C52H49O15N1: C,
67.20; H, 5.31; N, 1.53. Found: C, 66.94; H, 5.27; N,
1.48.
1
H2O); Rf 0.33 (CHCl3/MeOH/H2O, 10:9:1); H NMR
(D2O) d 4.96 (d, 1H, J12=3.7 Hz, H-1), 4.54 (d, 1H,
J120=7.7 Hz, H-10), 4.39 (dd, 1H, J12=3.7, J23=11.0
Hz, H-2), 4.31 (broad d, 1H, H-4), 4.10 (dd, 1H,
J23=11.1, J34=3.1 Hz, H-3), 4.07 (broad s, 1H, H-5),
3.98 (broad d, 1H, J340=3.5 Hz, H-40), 3.89–3.79 (m,
5H, H-6, H-60, 1H of CH2), 3.74–3.72 (m, 1H, H-50),
3.69 (dd, 1H, J230=10.0, J340=3.4 Hz, H-30), 3.65–3.57
(m, 2H, H-20, 1H of CH2), 2.89 (t, 2H, J=7.5 Hz, CH2),
2.80–2.73 (m, 4H, 2 CH2), 2.10 (s, 3H, Ac), 2.01–1.96
(m, 2H, CH2); 13C NMR (D2O) d 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; (+) FAB-MS (glycerol)
m/z: 530.3 (M+1).
Allyl O-(ꢀ-D-galactopyranosyl)-(1-3)-2-acetamido-2-deoxy-
ꢁ-D-galactopyranoside (10). Compound 8 (or 9) (1 g,
2.0 mmol) was dissolved in MeOH (10 mL) and
MeONa/MeOH (cat.) wasadded to adjust the pH to 9.
The resulting solution was stirred for 30 min at room
temperature. Solvent wasremoved under vacuum and
the residue was dissolved in water (10 mL), the aqueous
layer wasseparated from the organic, and then lyophi-
lized to give the de-benzoylated intermediate. Subse-
quently, this intermediate was dissolved in 60%
aqueous acetic acid (15 mL) and the resulting solution
wastsirred for 1.5 h at 60 ꢀC. Solvent wasevaporated
under reduced pressure and the crude product was pur-
ified by silica gel column chromatography (CHCl3/
MeOH/H2O, 11:6:1) to give white solid in 93% yield
(0.79 g, 1.86 mmol): mp 230–232 ꢀC; Rf 0.53 (CHCl3/
Synthesis of glycoPAMAM dendrimers
General procedure. PAMAM in MeOH (13, 14, 15 and
16, n=4, 8, 16 and 32) wasadded to a round bottom
flask (25 mL) and dried under vacuum. The residue was
dissolved in DMSO and compound 12 (1.1 equiv per
amino group), TBTU (1.2 equiv per acid) and DIPEA
(pH=9) were added. The resulting solution was stirred
overnight at room temperature. The extent of coupling
wasconfirmed by a negative ninhydrin test. DMSO was
eliminated by lyophilization and the residue was pur-
ified by gel permeation chromatography (P-2, P-4, H2O)
or dialysis (2000 molecular weight cut-off). The product
portion wascollected and lyophilized to afford the cor-
responding glycoPAMAM dendrimers (17–20) in 73, 81,
99 and 79% yields, respectively.
MeOH/H2O); [a]D+120.0ꢀ (c 1, H2O); H NMR (D2O)
1
d 6.08–6.01 (m, 1H, CH), 5.42 (dd, 1H, Jgem=1.6,
Jtrans=17.3 Hz, CH), 5.33 (dd, 1H, Jgem=1.7, Jcis=10.4
Hz, CH), 5.01 (d, 1H, J12=3.8 Hz, H-1), 4.53 (d, 1H,
J120=7.8 Hz, H-10), 4.41 (dd, 1H, J12=3.7, J23=11.2
Hz, H-2), 4.31–4.27 (m, 2H, H-4, 1H of CH2), 4.13–4.07
(m, 3H, H-3, H-5, 1H of CH2), 3.98 (dd, 1H, J340=3.4,
J450=0.8 Hz, H-40), 3.86–3.78 (m, 4H, H-6, H-60), 3.74–
Tetrameric glycoPAMAM dendrimer 17. PAMAM (13,
n=4) dendritic core (7.2 mg, 13.9 mmol) and T-antigen
derivative (12, 32.4 mg, 61.2 mmol) were dissolved in
DMSO (6 mL) and TBTU (23.6 mg, 73.5 mmol) and
DIPEA (pH=9) were added. After purification, glyco-
PAMAM dendrimer 17 wasobtained in 73% yield (26
mg, 10.2 mmol). 1H NMR (D2O) d 4.96 (d, 4H, J12=3.8

16
M.-G. Baek, R. Roy / Bioorg. Med. Chem. 10 (2002) 11–17
Hz, H-1), 4.55 (d, 4H, J120=7.8 Hz, H-10), 4.40 (dd, 4H,
J12=3.8, J23=11.1 Hz, H-2), 4.32 (broad d, 4H,
J34=2.8, J45ꢁ1 Hz, H-4), 4.10 (dd, 4H, J23=11.1,
J34=3.0 Hz, H-3), 4.07 (broad t, 4H, J45ꢁ1, J56=6.2
173.1, 172.3 (60 C), 104.2 (16 C), 102.8 (16 C), 76.9 (16
C), 74.7 (16 C), 72.1 (16 C), 70.4 (16 C), 70.2 (16 C),
68.2 (16 C), 68.2 (16 C), 66.0 (16 C), 60.8 (16 C), 60.57
(16 C), 53.9, 51.6, 49.3, 48.8 (40 C between 53.9 and
48.8), 49.3, 48.8, 48.3, 43.0, 38.4, 38.3 (44 C between
49.3 and 38.3), 48.8 (16 C), 35.3 (16 C), 31.50, 29.4,
28.05 (46 C between 31.5 and 28.0), 27.6 (16 C), 26.7 (16
C), 21.7 (16 C).
0
Hz, H-5), 3.99 (broad d, 4H, J340=3.3, J450 ꢁ1 Hz, H-4 ),
3.89–3.76 (multi, 20H, H-6, H-60, CH2), 3.75–3.72
(multi, 4H, H-50), 3.70 (dd, 4H, J230=9.9, J340=3.4 Hz,
H-30), 3.66–3.58 (multi, 8H, CH2, H-20), 3.41 (broad s,
16H, CH2, CH2), 3.29 (broad t, 8H, CH2), 2.89 (t, 8H,
J=6.9 Hz, CH2), 2.80 (s, 4H, CH2), 2.77 (t, 8H, J=7.1
Hz, CH2), 2.72 (t, 8H, J=6.7 Hz, CH2), 2.62 (t, 8H,
J=6.9 Hz, CH2), 2.11 (s, 12H, CH3), 2.01–1.95 (multi,
8H, CH2); 13C NMR (D2O) d 174.2, 174.0, 172.6 (12 C
between 174.2 and 172.6), 104.2 (4 C), 96.8 (4 C), 76.8 (4
C), 74.5 (4 C), 72.1 (4 C), 70.2 (4 C), 70.1 (4 C), 68.2 (4
C), 68.1 (4 C), 66.0 (4 C), 60.7 (4 C), 60.6 (4 C), 48.6 (4
C), 48.3 (4 C), 38.4 (8 C), 38.0 (2 C), 35.3 (4 C), 29.9 (4
C), 28.0 (4 C), 27.6 (4 C), 26.7 (4 C), 21.6 (4 C).
32 Mer glycoPAMAM dendrimer 20. PAMAM (16,
n=32) dendritic core (12.4 mg, 1.80 mmol) and T-anti-
gen derivative (12, 33.5 mg, 63.4 mmol) were dissolved in
DMSO (8 mL) and TBTU (24.4 mg, 76.0 mmol) and
DIPEA (pH=9) were added. After purification, glyco-
PAMAM dendrimer 20 wasobtained in 79% yield (32.2
mg, 1.38 mmol). 1H NMR (D2O) d 4.96 (d, 32H,
J12=3.7 Hz, H-1), 4.56 (d, 32H, J120=7.7 Hz, H-10),
4.40 (dd, 32H, J12=3.7, J23=1.1 Hz, H-2), 4.32 (broad
d, 32H, J34=2.9, J45ꢁ1 Hz, H-4), 4.10 (dd,32H,
J23=11.1, J34=3.0 Hz, H-3), 4.06 (broad t, 32H,
J56=6.1 Hz, H-5), 3.99 (broad d, 32H, J340=3.4 Hz, H-
40), 3.95–3.79 (multi, 160H, H-6, H-60, CH2), 3.78–3.69
(multi, 64H, H-30, H-50), 3.65–3.58 (multi, 64H, H-20,
CH2), 3.40 (broad s, 192H, CH2), 2.92 (broad s, 112H,
CH2), 2.88 (t, 64H, J=7.0 Hz, CH2), 2.77 (multi, 128H,
CH2), 2.62 (t, 64H, J=7.0 Hz, CH2), 2.56–2.51 (broad
s, 112H, CH2), 2.10 (s, 96H, CH3), 2.00–1.39 (multi,
64H, CH2); 13C NMR (D2O) d 174.2, 174.0, 173.9 (124
C), 104.20 (32 C), 96.8 (32 C), 76.8 (32 C), 74.6 (32 C),
72.1 (32 C), 70.2 (32 C), 70.1 (32 C), 68.2 (32 C), 68.1
(32 C), 66.0 (32 C), 60.7 (32 C), 60.55 (32 C), 50.9 (32
C), 48.7 (56 C), 48.3 (32 C), 38.2, 36.2 (98 C between
38.2 and 36.2), 35.3 (32 C), 32.1 (56 C), 28.1 (32 C), 27.6
(32 C), 26.7 (32 C), 21.7 (32 C).
Octameric glycoPAMAM dendrimer 18. PAMAM (14,
n=8) dendritic core (8.37 mg, 5.85 mmol) and T-antigen
derivative (12, 27.3 mg, 51.6 mmol) were dissolved in
DMSO (5 mL) and TBTU (20 mg, 62.3 mmol) and
DIPEA (pH=9) were added. After purification, glyco-
PAMAM dendrimer 18 wasobtained in 81% yield (26.1
1
mg, 4.7 mmol). H NMR (D2O) d 4.96 (d, 8H, J12=3.7
Hz, H-1), 4.55 (d, 8H, J120=7.8 Hz, H-10), 4.39 (dd, 8H,
J12=3.7, J23=11.1 Hz, H-2), 4.32 (d, 8H, J34=2.7,
J45ꢁ1 Hz, H-4), 4.10 (dd, 8H, J23=11.1, J34=2.9 Hz,
H-3), 4.06 (broad t, 8H, J56=6.6 Hz, H-5), 3.99 (d, 8H,
J340=3.3 Hz, H-40), 3.89–3.77 (multi, 40H, H-6, H-60,
CH2), 3.74–3.69 (multi, 24H, H-50, H-30, CH2), 3.66–
3.52 (multi, 24H, H-20, CH2), 3.47 (broad t, 8H, CH2),
3.41 (broad s, 32H, CH2), 3.28 (broad t, 16H, CH2),
2.90–2.83 (multi, 24H, CH2), 2.80 (s, 4H, CH2), 2.79–
2.75 (multi, 32H, CH2), 2.62 (t, 16H, J=6.8 Hz, CH2),
2.10 (s, 24H, CH3), 1.99 (multi, 16H, CH2); 13C NMR
(D2O) d 174.2, 173.7, 173.3, 171.42 (28 C), 104.2 (8 C),
96.8 (8 C), 76.8 (8 C), 74.5 (8 C), 72.1 (8 C), 70.2 (8 C ),
70.1 (8 C), 68.2 (8 C), 68.1 (8 C), 66.0 (8 C), 60.8 (8 C),
60.6 (8 C), 51.7 (4 C), 49.6 (4 C), 48.7 (8 C), 38.4, 38.0
(18 C between 38.4 and 38.0), 35.3 (8 C), 34.0 (4 C), 30.2
(8 C), 29.8 (8 C), 28.1 (8 C), 28.0 (4 C), 27.6 (8 C), 26.7
(8 C), 21.6 (8 C).
Turbidimetric analysis between peanut lectin from Ara-
chis Hypogaea and glycoPAMAM dendrimers (17, 18,
19 and 20). Turbidimetric experimentswere performed
in Linbro (Titertek) microtitration plateswhere 50 mL/
well of stock lectin solutions prepared from peanut lec-
tin (2 mg/mL in PBS) were mixed with 50 mL of a stock
solution of glycodendrimers, 17, 18, 19 and 20 (2.1, 1,
0.54 and 0.27 nmol/well in PBS) and incubated at room
temperature for 3 h. The turbidity of the solutions
wasmonitored by reading the optical denisty (OD) at
16-Mer glycoPAMAM dendrimer 22. PAMAM (18,
n=16) dendritic core (11.5 mg, 3.52 mmol) and T-anti-
gen derivative (15, 32.8 mg, 62.0 mmol) were dissolved in
DMSO (10 mL) and TBTU (24.0 mg, 74.7 mmol) and
DIPEA (pH=9) were added. After purification, glyco-
PAMAM dendrimer 22 wasobtained in 99% yield (40
mg, 3.5 mmol). 1H NMR (D2O) d 4.96 (d, 16H, J12=3.7
Hz, H-1), 4.56 (d, 16H, J120=7.8 Hz, H-10), 4.40 (dd,
16H, J12=3.7, J23=11.1 Hz, H-2), 4.32 (d, 16H,
J34=3.0, J45ꢁ1 Hz, H-4), 4.10 (16H, J23=11.1,
J34=3.1 Hz, H-3), 4.06 (broad t, 16H, J56=6.4 Hz, H-
5), 3.99 (d, 16H, J340=3.3 Hz, H-40), 3.89–3.76 (multi,
80H, H-6, H-60, CH2), 3.75–3.69 (multi, 32H, H-30, H-
50), 3.65–3.58 (multi, 64H, H-20, CH2), 3.41–3.30 (multi,
120H, CH2) 3.28–3.06 (multi, 80H, CH2), 2.89 (t, 32H,
J=7.0 Hz, CH2), 2.79–2.75 (multi, 92H, CH2), 2.62 (t,
32H, J=7.0 Hz, CH2), 2.10 (s, 48H, CH3), 2.09–1.95
(multi, 32H, CH2); 13C NMR (D2O) d 174.1, 173.9,
490 nm at regular time intervalsuntil no noticeable
36
changeswere obesrved.
triplicate.
Each test was performed in
Enzyme linked lectin assays (ELLA) between peanut
lectin and PAMAM based T-antigen dendrimers (17, 18,
19 and 20). Linbro (Titertek) microtitration plateswere
coated with 100 mL/well of glycoPAMAM dendrimers
(17–20) in a stock solution (1.4 nmol in 0.01M PBS,
pH=7.3) and incubated overnight at room temperature.
Previous studies with lactose-based dendrimers showed
34
equimolar amount of dendrimersbound to the plates.
After washing with PBST and blocking with BSA (1%
PBS), peanut lectin (10 mL of supernatant (1 mg/mL) in
PBS) wasadded and incubated for 1 h at 37 ꢀC. The
plate waswahsed with PBST and the wellswere filled
with 100 mL/well of Arachis hypogaea-peroxidase
supernatant (0.1 mg/mL) with a serial dilution by a

M.-G. Baek, R. Roy / Bioorg. Med. Chem. 10 (2002) 11–17
17
factor of 10 in PBST and incubated at 37 ꢀC for 1 h. The
References and Notes
plateswere washed and 50 mL/well of 2,20-azinobis(3-
ethylbenzothiazoline-6-sulfonic acid) diammonium salt
(ABTS) (1 mg/4 mL) in citrate-phosphate buffer (0.2 M,
pH=4.0 with 0.015% H2O2) wasadded. The reaction
wasstopped after 20 min by adding H 2SO4 (1 M, 50 mL/
well) and the optical density was measured at 410 nm
relative to 570 nm. A blank well contained citrate-
phosphate buffer. Each test was performed in triplicate.
1. Varki, A. Glycobiology 1993, 3, 97.
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and Applications; Lee, Y. C., Lee R. T., Eds.; Academic Press,
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Competitive double sandwich inhibition ELISA using
mouse monoclonal antibody JAA-F11 IgG3 and T-anti-
gen dendrimers, 17, 18, 19 and 20 as inhibitors. Linbro
(Titertek) microtiter plateswere coated overnight with
T-antigen containing co-polyacrylamide28,29 with 100
mL of a polymer stock solution, [10 mg/mL in 0.01 M
phosphate buffer (pH=7.3)] at room temperature. Each
well contained 1 mg/well of polymer which corresponds
to 0.36 mg (0.85nmol) T-antigen. The wellswere then
washed 3 times with 400 mL of PBST (0.01 M phosphate
buffer (pH=7.3) containing 0.05% (v/v) Tween 20).
BSA solution (1% in PBS 150 mL/well) wasadded to
each well and incubated for 1 h at 37 ꢀC. While, 50 mL
of mouse monoclonal IgG antibody solution in PBST
(10 timesdilution of ascitic fluid, 0.25 mmol/50 mL) and
50 mL of inhibitor solution in PBST with varying con-
centrationsfrom 275 to 1.07 nmol/well of T-antigen
prepared by twofold serial dilution were mixed in Nun-
clon (Delta) microtiter platesand preincubated for 1 h
at 37 ꢀC. After excess BSA was washed out with with
PBST, each well wasfilled with 100 mL/well of pre-
incubated mouse IgG MAb/inhibitor solution and
incubated again for 1 h at 37 ꢀC. The wellswere washed
with PBST asdescribed above and then filled with 100
mL of goat anti-mouse IgG in PBST (1,000 times dilu-
tion of ascitic fluid) followed by incubation for 1 h at
37 ꢀC. The wellswere washed with PBST and 50 mL of
14. Jayaraman, N.; Nepogodiev, S. A.; Stoddart, J. F. Car-
bohydr. Eur. 1998, 20, 30.
15. Tomalia, D. A.; Durst, H. D. Topics Curr. Chem. 1993,
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¨
dritic Molecules: Concepts Syntheses and Perspectives; VCH:
Weinheim: Germany, 1996. (b) Bosman, A. W.; Janssen,
H. M.; Meijer, E. W. Chem. Rev. 1999, 99, 1665. (c) Zeng, F.;
Zimmerman, S. C. Chem. Rev. 1997, 97, 1681. (d) Chow, H. F.;
Mong, T. K. K.; Nongrum, M. F.; Wan, C. W. Tetrahedron
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17. Bielinska, U.; Chen, C. J.; Baker, J. J. R., Jr. Bioconjugate
Chem. 1999, 10, 843.
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2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic
acid)
diammonium salt (ABTS) (1 mg/4 mL of citrate-phos-
phate buffer (0.2 M, pH=4.0 with 0.015% H2O2) was
added. The reaction wastsopped after 20 min by the
addition of 50 mL/well of 1 M aqueoususlfuric acid
solution. Optical density was measured at 410 nm rela-
tive to 570 nm. The percent inhibition wascalculated as
follows:
27. Donovan, R.; Datti, S. A.; Baek, M. G.; Wu, Q. Q.; Sas,
I.; Korczak, B.; Berger, E. G.; Roy, R.; Dennis, J. W. Glyco-
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% inhibition ¼ ½Aðno inhibitorÞ ꢀ A
ꢂ 100
=Aðno inhibitorÞꢁ
ðwith inhibitorÞ
IC50swere calculated asthe concentration required for
50% inhibition of the coating antigen. All test were
performed in triplicate.
Acknowledgements
34. Andre, S.; Ortega, P. J.; Perez, M. A.; Roy, R.; Gabius,
H.-J. Glycobiology 1999, 9, 1235.
We thank Dr LouisCuccia for hisvaluable comments
and proof reading. We also thank the National Science
and Engineering Research Council of Canada (NSERC)
for financial support.
35. Gabius, H. J.; Schroter, C.; Brinck, G. U.; Tietze, L. F. J.
Histochem. Cytochem. 1990, 38, 1625.
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