Inhibitory potential of chemical substitutions at bioinspired sites of β-d-galactopyranose on neoglycoprotein/cell surface binding of two classes of medically relevant lectins

Article abstract of DOI:10.1016/j.bmc.2011.03.022

Galactose is the key contact site for plant AB-toxins and the human adhesion/growth-regulatory galectins. Natural anomeric extensions and 3′-substitutions enhance its reactivity, thus prompting us to test the potential of respective chemical substitutions

Full text of DOI:10.1016/j.bmc.2011.03.022

Bioorganic & Medicinal Chemistry 19 (2011) 3280–3287
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibitory potential of chemical substitutions at bioinspired sites
of b-D-galactopyranose on neoglycoprotein/cell surface binding
of two classes of medically relevant lectins
Denis Giguère a, Sabine André b, Marc-André Bonin a, Marc-André Bellefleur a, Alexandre Provencal a,
b,
Philipe Cloutier a, Bernard Pucci c, René Roy a, , Hans-Joachim Gabius
⇑
⇑
a PharmaQAM, Department of Chemistry, Université du Québec à Montréal, PO Box 8888, Succ. Centre-ville, Montreal (QC), H3C 3P8, Canada
b Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University, Veterinärstr. 13, D-80539 Munich, Germany
c Laboratoire de Chimie Bioorganique et des Systèmes Moléculaires Vectoriels, Université d’Avignon et des Pays du Vaucluse, Faculté des Sciences, 33 rue Louis Pasteur, F-84000
Avignon, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Galactose is the key contact site for plant AB-toxins and the human adhesion/growth-regulatory galec-
tins. Natural anomeric extensions and 30-substitutions enhance its reactivity, thus prompting us to test
the potential of respective chemical substitutions of galactose in the quest to develop potent inhibitors.
Biochemical screening of a respective glycoside library with 60 substances in a solid-phase assay was fol-
lowed by examining the compounds’ activity to protect cells from lectin binding. By testing 32 anomeric
extensions, 18 compounds with additional 30-substitution, three lactosides and two Lewis-type trisaccha-
rides rather mild effects compared to the common haptenic inhibitor lactose were detected in both
assays. When using trivalent glycoclusters marked enhancements with 6- to 8-fold increases were
revealed for the toxin and three of four tested galectins. Since the most potent compound and also 30-
substituted thiogalactosides reduced cell growth of a human tumor line at millimolar concentrations,
biocompatible substitutions and scaffolds will be required for further developments. The synthesis of
suitable glycoclusters, presenting headgroups which exploit differences in ligand selection in interlectin
comparison to reduce cross-reactivity, and the documented strategic combination of initial biochemical
screening with cell assays are considered instrumental to advance inhibitor design.
Received 2 December 2010
Revised 4 March 2011
Accepted 9 March 2011
Available online 30 March 2011
Keywords:
Agglutinin
Blood group
Glycocluster
Malignancy
Multivalency
Neoglycoconjugate
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
proteins need to be exploited toward the aim to develop selective
high-affinity inhibitors.
The complexity of cell surface glycoconjugates features all attri-
butes to cover a wide variety of biochemical signals.1 One route to
decode this information is via specific receptors. The emerging
functionality of the molecular recognition of distinct glycan deter-
minants by proteins (lectins) is therefore unraveling new targets
for rational drug design, a demanding challenge for chemical biol-
ogy.2,3 In fact, carbohydrates, their derivatives and mimetics offer
the promising perspective to block certain lectin effectors specifi-
cally, for example, in developing heart failure, inflammatory dis-
eases or progression of malignancy.4–7 To do so successfully, the
cross-reactivity of a lectin-blocking compound with other sugar
receptors, especially members from the same family, must be min-
imal, while reaching an optimal affinity for its target. Thus, any dif-
ferences in folding and sequence deviations among homologous
In the quest to explore to what extent substitutions of the core
contact site can improve inhibitory potency, we here focus on two
classes of galactose-binding lectins, that is, a plant toxin (Viscum
album agglutinin (VAA) with b-trefoil folding) and four human
adhesion/growth-regulatory galectins selected to represent the
three subgroups within this family (b-sandwich folding).8–10 Our
study thus investigates the relative inhibitory capacity of galactose
derivatives in interectin and intralectin family comparison. These
two classes of lectins are known to favor a natural 30-substitution
of galactose and also b-anomeric elongations beyond the core unit,
that is, by N-acetyllactosamine repeats, inspriring structural
additions at these sites.11–15 The concept for our study is put into
graphics in Figure 1, showing that these two types of substitutions
can contact the protein’s surface (here human galectin-3). Since
lactose binding is known to induce conformational changes in
these two lectin classes,16–19 high-affinity carbohydrate derivatives
will not only serve as sensors for inter- and intrafamily differences
but will also become tools to detect ligand-induced alterations
upon structural study. Their synthesis will also answer the
⇑
Corresponding authors. Tel.: +1 (514) 987 3000x2546; fax: +1 (514) 987 4054
(R.R.); tel.: +49 89 2180 2290; fax: +49 89 2180 2508 (H.-J.G.).
E-mail addresses: roy.rene@uqam.ca (R. Roy), gabius@tiph.vetmed.uni-muench-
en.de (H.-J. Gabius).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.022

D. Giguère et al. / Bioorg. Med. Chem. 19 (2011) 3280–3287
3281
Figure 1. Connolly surface of human galectin-3 cocrystallized with lactose (PDB 2NN8) with the depiction of the two bioinspired sites of substitution.
question to what extent chemical substitutions can compete with
natural glycans.
In this report, we tested a library of 60 synthetic compounds
covering four groups based on type of substitution and extension
Since the anomeric extension by a phenyl group is known to en-
hance binding for the toxin and galectin-1,25–30 we used a neogly-
coprotein with such a spacered lactose moiety as strong ligand
mimetic. Thus, we set rather restrictive conditions for blocking lec-
tin binding by the test compounds relative to lactose as internal
reference.
as well as degree of valency: 32 b-D-galactopyranoside derivatives
(1–32) with anomeric extension via C/O-glycosidic bonds
(Scheme 1), 18 S-linked derivatives (33–50) with additional 30-sub-
stitution of the galactose core (Scheme 2), three lactose derivatives
(51–53) and two Lewis-type trisaccharides (54, 55) (Scheme 3) as
well as three bi- to trivalent glycoclusters (56, 59, 60) with two
monovalent control derivatives (57, 58) bearing the long F-con-
taining spacer (Scheme 4). As noted above, the introduction of di-
verse chemical groups at the anomeric center and at the 30-site was
intended to mimic natural ligands and to exploit this region of the
lectins’ surface for affinity generation (Fig. 1). Besides substitutions
of the core ligand binding to lectins can further be enhanced by an
increase in valency, especially when the topologies of lectin-site/
glycan displays match or ligands are presented in microdo-
mains.20–23 To probe into the relative effect of bi- to trivalency in
the same experimental setting we included the three glycoclusters
56, 59, 60 into the test panel. The screening process was strategi-
cally designed in two levels of increasing biorelevance, starting
with an inhibition assay in which the labeled lectin (in solution)
in microtiter plate wells binds to a surface-presented ligand.24
In the second activity-assessment stage, we increased the bio-
relevance by testing the synthetic compounds as inhibitors of
lectin binding to human tumor cells, which present natural high-
affinity ligands. In addition to this two-step assay process we
addressed the issue whether such substitutions can convey cyto-
toxicity to galactose/lactose. This study thus answers the questions
on inter- and intrafamily differences inferred by these chemical
sensors, their activity as inhibitors of lectin binding to physiologi-
cal cell surface glycans and their potential for cytotoxicity, strate-
gically combining chemical synthesis with biochemical/cell
biological assays.
2. Results and discussion
2.1. Chemistry
The test panel was divided into four groups (Schemes 1–4) to
enable dissection of the relative impact of the different types of
HO
HO
HO
HO
HO
HO
HO
HO
OMe
OH
O
OH
O
OH
O
OH
O
HO
HO
OH
O
CO2Me
CO2Me
HO
HO
HO
HO
HO
1
2
3
4
5
HO
HO
HO
HO
HO
HO
O
R
N
HO
HO
OH
O
OH
O
OH
O
OH
O
O
O
NO2
HO
HO
HO
HO
O
NNHR
6
7
8
10
R = H
R = Me
9 R = COPh
11 R = CMe3
HO
HO
HO
HO
14 R = H; 15 R = COMe; 16 R = CO2Me
OH
O
OH
O
NHR
17
19
21
18
R = SO2Ph;
R = SO2Ph-pMe
HO
HO
O
20
22
OH
O
R = SO2Ph-pBr;
R = SO2Ph-mCl;
R = SO2Ph-p-tBu
R = SO2β-naphthyl
O
N
S
O
N
HO
HO
O
23 R = SO2Ph-2,5-Br2
O
24 R = COPh; 25 R = COPh-pOMe
HO
26
28
27
R = COPh-pNO2;
R = COPh-pBr
12
13
29
R = COβ-naphthyl;
R = COCH2Ph
30 R = CH2Ph; 31 R = CH2Ph-pOMe
32 R = CH2Ph-pNO2
Scheme 1. Anomeric substitution of b-D-galactopyranoside derivatives.

3282
D. Giguère et al. / Bioorg. Med. Chem. 19 (2011) 3280–3287
HO
O
H
N
H
N
HO
O
OH
O
OH
O
CO2Me
CO2Me
N
N
N
O
O
HO
O
HO
O
MeO2C
OH
O
OH
O
S
N
S
S
S
S
NO2
HO
HO
HO
HO
33
34
35
36
H
N
R
HO
OH
O
O
HO
O
S
HO
N
RO
N
OH
O
OH
O
N
n
S
N
S
R
N
N
S
N
S
S
N
N
HO
N
HO
HO
N
N
37 R = CH2Ph
40 n = 1, R = SO3Na
41 n = 2, R = SO3Na
43 R = Ph
38 R = CH2Ph-pOH
39 R = CH2CH2OH
44 R = CH2OH
42
45 R = CH2CH2OH
46 R = CO2Me
n = 2, R = PO3Ph2
47
R = CO2H
H
H
H
N
O
N
O
N
O
N
HO
O
HO
O
HO
O
OH
O
OH
O
OH
O
S
S
S
S
NO2
OH
HO
HO
HO
48
49
50
Scheme 2. 30-Extension of b-
D-galactopyranoside derivatives.
HO
HO
HO
HO
OH
O
OH
O
CO2Me
OH
O
OH
O
N
O
R
O
H
N
O
O
O
S
HO
HO
HO
HO
HO
HO
N
51
53
R = Ph
52 R = CMe3
HO
HO
HO
OH
O
OH
O
OH
OH
O
N
N
O
HO
O
O
OMe
O
N3
O
N
OH
OH
OH
OH
O
O
O
OBn
OH
OBn
BnO
OH
HO
54
55
Scheme 3. Lactose derivatives.
substitution on lectin binding. In the first category, diverse
aglycones were added to the galactopyranose core in b-anomeric
linkage, mostly with non-hydrolyzable C-glycosidic linkage
(Scheme 1).28–37 For instance, C-allylated acrylate 136 served as a
versatile starting material for most of the derivatizations in the
first series (Scheme 1), while the methyl ketone 7 provided access
to the aminothiazole series Scheme 5).36 In order to probe for the
effects of the bioinspired modification at the 30-hydroxyl group
of galactose a further panel of 18 derivatives (33–50)34,35 (see Sup-
plementary data) with different types of substitution at this site in
addition to those at the anomeric center was synthesized
(Scheme 2). These compounds will thus answer the question
whether the prepared mimetics of natural glycans can surpass
their affinity. In our cases, 2-(methoxycarbonyl)phenyl 3-O-
spacers of compounds 57–59 are known to form micelles at the
tested concentration41,42, adding a topological aspect to the di-
(56)36 and trivalent glycoclusters (60)29 (Scheme 4).
2.2. Inhibition assays (glycoprotein)
This library of 60 synthetic compounds was tested for activity to
block lectin binding to a ligand. Besides considering different
folding (b-trefoil and b-sandwich) we studied the aspect of intra-
family reactivity pattern by selecting galectins from the three sub-
groups, that is, homodimeric proto-type (galectin-1), chimera-type
(galectin-3) and tandem-repeat-type (galectins-8 and -9) proteins.
The functional competition, up to now detected for galectins-1 and
-3,43,44 signifies differential cross-linking properties, an indication
for disparate reactivity with glycoclusters. Each lectin was labeled
and rigorously checked for maintained binding properties. A neo-
glycoprotein with p-isothiocyanatophenyl lactosides24 as ligand
was adsorbed to the plastic surface of microtiter plate wells to
establish a lectin-binding matrix. The parameters of coating den-
sity and lectin concentration were systematically examined to de-
fine the conditions, which yielded a linear range for dependence of
signal intensity from lectin concentration and a sugar-inhibitable
binding. Controls with non-cognate sugars (mannose, maltose) ex-
cluded non-specific effects. Under these subsaturating conditions
systematic titrations for each derivative and each lectin were
performed to determine the compound concentration required to
(prop-2-enyl)-1-thio-b-D
-galactopyranoside (33)35 served as the
starting material for this series, while a 3-azido galactoside deriv-
ative31 started the route to compounds 40–47.34,35 In order to
determine the effect of anomeric extension of lactose relative to
galactose, thus probing into a further distal region of the lectin
sites, five such derivatives (51–55)32 were included (Scheme 3).
Compound 53 has been reported previously in the literature38
and served as comparison already used for construction of lectin-
reactive glycodendrimers.39,40 Comparison between the three
groups of monovalent compounds and the glycoclusters is a means
to infer the relative potential of these two synthetic strategies to
generate potent inhibitors. Of note, the long-chain fluorinated

D. Giguère et al. / Bioorg. Med. Chem. 19 (2011) 3280–3287
3283
HO
OH
OH
O
HO
OH
OH
O
HO
O
HO
HO
O
F
F F F F F F F
HO
HO
O
56
F
OH
S
N
H
F
F
F F F F F F
HO
HO
57
OH
OH
OH
OH
HO
OH
F F F F F F F F
O
HO
HO
OH
O
F
O
O
N
N
N
HO
H
HO
F
F
F F F F F F
OH
N
O
HO
HO
O
OH
O
OH
O
HO
58
NH
O
HO
O
OH
HO
N
HO
H
N
OH
N
O
N
HN
O
HO
OH
O
HO
N
N
60
O
N
OH
HO
HO
O
O
F
F F F F F F
F
OH
F
HO
HO
O
S
N
O
O
OH
HO
H
F
O
F
F F F F F F
OH
HO
HO
O
OH
59
O
OH
HO
HO
HO
Scheme 4. b-D-Galactopyranoside and lactoside clusters with a monovalent control.
NH2
N
OAc
O
OR
O
OH
O
1. Br2 / DCM
(quant)
AcO
AcO
RO
RO
1. 2,4-pentanedione
HO
HO
S
NaHCO3, H2O, Δ
O
OH
2. Thiourea
DIPEA
2. Ac2O, DMAP, Pyr
OAc
14b
OR
OH
14a
MeCN, Δ
14c
R = Ac
(65%)
14 R = H
3. NaOMe, MeOH
NHR
NHR
NH2
N
OAc
O
OH
S
OAc
AcO
AcO
AcO
AcO
HO
HO
S
S
O
E+
O
N
NaOMe
MeOH
N
OAc
OAc
15a-32a
Scheme 5. Synthetic strategy to obtain the aminothiazole series.
OH
Conditions
14c
15-32
reach 50% inhibition (IC50) of binding, using lactose as internal
reference.
The systematic testing revealed a remarkable relative activity
level of the natural disaccharide. Two monovalent galactose deriv-
atives and three 30-modified galactosides surpassed its inhibitory
capacity (Table 1, Fig. 2). The contact area in the plant toxin for
an anomeric extension is thus rather limited, in accord with the
mapping of contacts via saturation transfer difference NMR spec-
troscopy, and the synthetic core modifications did not appear to
Table 1
Relative potency of synthetic compounds based on IC50-values relative to lactosea
Lectin compound
VAA
Gal-1
Gal-3
Gal-8
Gal-9
24
29
33
40
47
52
56
59
60
1.2
1.3
––
––
––
––
––
––
––
––
––
––
1.8
––
1.3
1.1
1.8
––
1.4
1.6
––
––
––
––
––
––
2.2
––
4.6
6.9
––
––
––
1.4
––
1.7
––
5.1
7.7
reach the level of activity of the natural
a1,3-linked digalacto-
side.12,25,45,46 The Lewis-type trisaccharides were not active, cor-
roborating previous chemical mapping of the relative importance
of hydroxyl groups with methyl b-lactoside analogues.17,47 An ano-
meric extension beyond lactose showed slight improvements for
three of four galectins, here the chimera-type galectin-3 and the
two tandem-repeat-type galectins-8 and -9 (Table 1, Fig. 2). This
result is in line with our previous report under identical conditions
that anomeric extensions, especially the S-lactosides of b-naphthyl
sulfone and 2-benzothiazolyl, can improve reactivity, pointing to
––
1.9
2.2
6.2
6.2
8.4
a
The concentrations yielding a 50%-level of inhibition of lectin binding to lac-
tosylated neoglycoprotein were calculated as sugar content and normalized to free
lactose; all data are given for compounds surpassing the potency of the internal
control. Data represent assay results routinely done in triplicates for up to five
independent series with nine concentrations, standard deviations not exceeding
15.5%.

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D. Giguère et al. / Bioorg. Med. Chem. 19 (2011) 3280–3287
Figure 2. Relative potencies of the compounds of the 60-membered galactoside library (compounds 1–23 not shown) compared to the disaccharide lactose in the solid-phase
inhibition assay (please see footnote to Table 1 for details).
an extended contact site for several galectins.24 The most notable
effects were yet seen with bi- to trivalent compounds 56, 59, 60
(Table 1, Fig. 2). The two trivalent clusters 59, 60 reached enhance-
ments up to 6- or 8-fold, respectively, with galectins-3, -8 and -9
(Fig. 2, Table 1). Thus, while the anomeric extensions tested, even
in combination with 30-substitutions at the galactose core, did not
markedly surpass the capacity of lactose as inhibitor, clustered li-
gand presentation has a comparatively strong effect. Of note, intra-
family cross-reactivity can occur within this library under these
assay conditions using a carrier-immobilized lactose derivative as
ligand. In the medical context, the ligands will be natural glycans
with variations in structure and in topological presentation. To
what extent the synthetic compounds will interfere with the bind-
ing of lectins to their natural high-affinity ligands on cell surfaces
was an open question. To address this issue we performed cell as-
says by using each constituent of the glycoside library as inhibitors
of binding of the labeled lectin to human tumor cells.
concentration (normalized to sugar content) as the free sugar
(Fig. 3B). The two thiazolyl galactoside derivatives 24 and 29 listed
in Table 1 also passed the inherent activity threshold in the cell as-
say (Fig. 3C). Further focusing on the identification of best-activity
cases, it is clear that the increase in valency, as best exemplified by
the trivalent glycocluster 60, is quantitatively more suited to gen-
erate enhanced inhibitory capacity than synthetic derivatization
within this panel. Because galectins can share common ligands
such as the pentasaccharide of ganglioside GM1 but also have
the potential for cell- and lectin-type-specific binding,7,43,44 inhib-
itory capacity and extent of cross-reactivity were analyzed with
four members of this family in parallel.
As noted above, inhibitory potency for compounds active in the
solid-phase assay was maintained in the competition with the cell
surface glycans. The trimeric glycoclusters 59 and 60 surpassed all
other compounds significantly for galectin-3, reaching an about 7-
fold enhancement relative to free lactose (Fig. 3D, E). To exclude
that this effect was attributable to the nature of the cell line, we
tested a T-cell leukemia and a colon adenocarcinoma line and
determined a rather similar extent of avidity increase (Fig. 3E, F).
Similar levels of potency, a clear sign for intergalectin cross-reac-
tivity, were measured for galectins-8 and -9, corroborating the so-
lid-phase data (Fig. 3G, H). These results indicate that an initial
screening step by the biochemical assay is feasible and that the
hereby identified compounds also stand out from the panel in cell
assays, where lectins home in on physiologic ligands. However,
this activity does not necessarily qualify such compounds as med-
ically relevant lectin inhibitors. An essential prerequisite is to show
lack of cytotoxicity.
2.3. Inhibition assays (cells)
Concentration dependence of the signal intensity was tested for
each combination of lectin and cell line at a constant cell number
(5 ꢀ 104) per assay. A representative plot is exemplarily shown in
Figure 3A in the case of the plant toxin VAA. Lectin binding could
completely be blocked by lactose in each case, while maltose or
mannose used as osmolarity control did not affect staining proper-
ties (not shown). Monitoring of positivity in the cell population (in
percent) and the mean fluorescence intensity was always run in
parallel with inherent positive and negative controls on aliquots
of cell suspensions of the same passage. When systematically using
galactose/lactose concentrations with weak to medium effects on
extent of staining and the test compounds at the same concentra-
tion in parallel, several substances could be singled out to interfere
with lectin binding to the cell surface more potently than the
unsubstituted sugars. This set gave a complete match to the data
from solid-phase screening. The two assays thus identified activity
for the same panel of compounds. The assay sensitivity is
underscored by picking up the increased reactivity of lactose com-
pared to galactose (Fig. 3B, C). In correlation with the results of the
solid-phase assays, toxin binding to cells was reduced most
potently by the glycoclusters 59 and 60 when used at the same
2.4. Cytotoxicity
To address this issue, we monitored cell proliferation in aliquots
of suspensions of human SW480 colon adenocarcinoma cells after
a period of 48 h in culture, adding experimental series with galact-
ose and lactose as controls. Six synthetic compounds were added
to medium at concentrations of 1, 2, 5, and 10 mM. The substituted
galactosides 3, 22 and 25 did not affect proliferation at 2 mM, an
increase to 5 mM decreasing growth to 50–70% values. The 30-
substituted galactoside 47 reduced growth to about 40% at 2 mM
(70% at 1 mM), the 30-substituted S-galactoside 33 most potently

D. Giguère et al. / Bioorg. Med. Chem. 19 (2011) 3280–3287
3285
to about 10% at 1 mM. Both compounds are listed in Table 1 with
activity on galectin-3. The most active compound, that is, the tri-
meric lactoside 60, also had an effect on proliferation, allowing
75% growth at 1 mM and about 58% at 2 mM.
3. Conclusions
The presented data contribute to characterize the potential of
carbohydrate derivatization to produce inhibitors of binding for
two classes of galactoside-specific lectins. The assessment was
based on testing a surface-presented neoglycoprotein and cell sur-
faces as ligands, the latter assay increasing the biorelevance. Using
lactose as internal standard, our results reveal the inherent limits
of the tested substitutions, although activity improvements were
noted here and also in other cases.24,48,49 In addition, and even
more important, interlectin cross-reactivity occurred. This finding
is a strong argument to turn efforts to identify the natural substi-
tutions favorable for high-affinity and selective binding and then
to exploit them using the presented strategy. As binding assays
with glycosylation mutants documented recently, galectins-8 and
-9, to give an instructive example, can distinguish cell surfaces
based on the level of a
2,3-sialylation of N-glycans,50 and the doc-
umented bioactivity of glycopeptides, too, can aid in increasing
affinity and selectivity.51,52 The lack of cytotoxicity of natural gly-
cans, derived from synthesis,53,54 should not be extrapolated to
substituted derivatives, as our study clearly shows. Of further note,
compared to the tested substitutions in monovalent compounds,
an increase of valency appears to promote inhibitory potency more
than a derivatization. Combining the slight activity increase by the
p-isothiocyanate derivatization of lactose with multivalent display
in starburst dendrimers had even resulted in up to 10,000-fold
enhancement for the plant toxin.55 Conformational flexibility/
rigidity of the scaffold, distinct cluster design and the structural
context of the presentation of the carbohydrate headgroup can also
make its mark on selectivity.39,56–60 In the family of galectins, the
three different types of display of the lectin sites are thus becoming
a target for suited glycocluster design, with the aim to team up
headgroup design with these topological parameters for optimal
affinity and selectivity. The availability of diverse scaffolds, whose
biocompatibility has to be rigorously tested, as done for O-glycosyl
carbamates,49 gives respective research a clear direction.61,62 Our
strategically combined screening strategy comprising biochemical
and cell biological assays will be helpful along this route.
4. Materials and methods
4.1. Chemistry
Figure 3. Semilogarithmic representation of fluorescent surface staining of human
tumor cells by labeled lectins. The control value representing marker-independent
staining by incubation with the second-step reagent is given as shaded area, the
100%-value obtained by lectin binding in the absence of test compound is shown
as black line. Quantitative data on percentage of positive cells and mean
fluorescence intensity are given in each panel (numbers for controls printed in
bold). They are listed in increasing level of inhibitory potency from bottom to top
as given in this text, and lines connect these data with the respective peak. The
following pairs of human tumor cell line/biotinylated lectin were processed for
the given set of compound/concentration: (A) T-cell leukemia cell line Jurkat and
4.1.1. General chemistry
All reactions involving water-sensitive chemicals were carried
out in flame-dried glassware with magnetic stirring under a nitro-
gen atmosphere. Anhydrous DCM was distilled from CaH2 and
anhydrous THF from Na/K prior to use. All non-aqueous reactions
were carried out under anhydrous conditions within a nitrogen
atmosphere in distilled solvents. Other solvents and reagents were
used as received. TLC was performed on aluminum plates (Silica
gel 60 F254) with detection by UV or by coloration with ammo-
nium molybdate in acid solution. Column chromatography was
performed on silica gel (230–400 mesh) with the indicated eluent.
1H and 13C NMR spectra were recorded at 300 (75) MHz with a Var-
iant apparatus. Chemical shifts (ppm) are reported relative to
CHCl3 (7.27), D2O or CD3OD as internal standard. Optical rotations
were measured on a Polarimeter JASCO P-1000 and melting point
on a Fisher–Johns Melting Point Apparatus. ESI-MS analyses were
carried out on MICROMASS Quattro LC equipment.
VAA at increasing concentrations of 1
lg/ml, 2 lg/ml and 4 lg VAA/ml without an
inhibitor; (B, C) respective results at the constant lectin concentration of 2.5
lg
VAA/ml in the presence of galactose and compounds 56, 59 and 60 (B) as well as
lactose and compounds 29 and 24 (C) at 1 mM; (D, E) T-cell leukemia cell line
Jurkat and 5
compound 59 and 1 mM 60 (D) and in the presence of 15
100 M lactose (E); (F) colon adenocarcinoma line SW480 and 2
in the presence of 100 lactose and 15 compound 60; (G, H) colon
adenocarcinoma line SW480 and 1 g galectin-8/ml (G) or 40 g galectin-9/ml (H)
l
g galectin-3/ml in the presence of 2 mM compound 6, lactose and
M compound 60 and
g galectin-3/ml
l
l
l
lM
lM
l
l
in the presence of lactose and compounds 52, 59, and 60 at 2 mM (G) and
compound 52, lactose and compounds 59 and 60 at 2 mM (H). Assays were
performed in triplicates with up to five independent series using aliquots of cell
suspensions at the same day with standard deviations not exceeding 10.8% after
normalization of the data.

3286
D. Giguère et al. / Bioorg. Med. Chem. 19 (2011) 3280–3287
4.1.2. Sources of the compounds of the chemical library
Compounds in Schemes 1, 3 and 4 were part of the chemical li-
brary collection in one of the author laboratory (R. R.) and have
been published elsewhere as follows: 1–2,36 33,3 4–9,36 10–11,29
12,32 13,37 14–32,36 41,45–46,34 51–55,32 56,36 57–58,41 59,42 and
finally compound 60.29 Compounds 33–50 are new derivatives
(see Supplementary data for a detailed account).
4.5. Cell proliferation assay
The extent of growth of human SW480 colon adenocarcinoma
cells after 48 h in culture was determined for aliquots of cell sus-
pensions in the absence (control) and in the presence of test com-
pounds including galactose and lactose spectrophotometrically
using the blue chromogen 3-(4,5-dimethyl-thiazol-3-yl)-2,5-di-
phenyl-tetrazolium bromide (Sigma; 0.5 mg/ml).67 Assays were
performed in triplicates and in three independent series with stan-
dard deviations not exceeding 12.6%.
4.2. Lectins
Recombinant expression in the case of galectins and extraction
of dried mistletoe leaves in the case of the plant toxin provided the
starting material for lectin purification by affinity chromatography
on lactosylated Sepharose 4B, obtained by divinyl sulfone activa-
tion, as crucial step, followed by purity controls using one- and
two-dimensional gel electrophoresis, gel filtration and mass spec-
trometry and activity assay by haemagglutination as de-
scribed.50,51,63,64 Biotinylation with the N-hydroxysuccinimide
ester derivative of biotin (Sigma, Munich, Germany) under activ-
ity-preserving conditions and product analysis to determine extent
of label incorporation by two-dimensional gel electrophoresis/
mass spectrometry were carried out as described.24 The labeled
products were tested in standard solid-phase/cell assays to ascer-
tain bioactivity and absence of non-specific interactions due to la-
bel incorporation (for experimental assay details, see below).
Acknowledgements
The generous financial support by a Canadian Research Chair in
Therapeutic Chemistry grant from the NSERC, the EC GlycoHIT pro-
gram and the Verein zur Förderung des biologisch-technologischen
Fortschritts in der Medizin e. V. (Heidelberg, Germany), inspiring
discussions with Drs. B. Friday, S. Namirha, Y. Nekcic and W. Not-
elecs as well as the excellent assistance of Tze Chieh Shiao are
gratefully acknowledged.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.03.022.
4.3. Solid-phase inhibition assay
References and notes
The lectin-binding matrix was established by adsorption of
lactosylated bovine serum albumin, exposing the p-isothiocyana-
tophenyl derivative (26 sugar units on average per carrier pro-
tein65), to the surface of microtiter plate wells from phosphate-
buffered saline at 4 °C overnight. Following systematic titrations
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an optimal quantity of 0.25
lg neoglycoprotein was routinely
applied in 50 l for coating. Residual sites on the plastic surface
l
with capacity for protein binding were saturated with a solution
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Lectin-containing solutions (1
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l
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