Synthesis of bivalent lactosides and their activity as sensors for differences between lectins in inter- and intrafamily comparisons

Article abstract of DOI:10.1016/j.bmcl.2011.11.010

The synthesis of nine bivalent lactosides (based on ditriazoles, diamides, a glycocyclophane and an acyclic analogue of the glycocyclophane) and one monovalent lactosyl triazole facilitated the assessment of the sensitivity of plant/animal lectins to this

Full text of DOI:10.1016/j.bmcl.2011.11.010

Bioorganic & Medicinal Chemistry Letters 22 (2012) 313–318
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Bioorganic & Medicinal Chemistry Letters
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Synthesis of bivalent lactosides and their activity as sensors for differences
between lectins in inter- and intrafamily comparisons
Sabine André a, Dilip V. Jarikote b, Dandan Yan b,c, Lisa Vincenz b, Guan-Nan Wang b, Herbert Kaltner a,
a
Paul V. Murphy b, , Hans-Joachim Gabius
⇑
a Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, Veterinärstr. 13, 80539 Munich, Germany
b School of Chemistry, National University of Ireland, Galway, University Road, Galway, Ireland
c School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of nine bivalent lactosides (based on ditriazoles, diamides, a glycocyclophane and an acy-
clic analogue of the glycocyclophane) and one monovalent lactosyl triazole facilitated the assessment of
the sensitivity of plant/animal lectins to this type of ligand display. The inhibitory potency of the com-
pounds was determined in two assays of increasing biorelevance. These were solid-phase and cell bind-
ing set-ups. Hereby, the ability of the compounds to inhibit the binding of two plant agglutinins and the
entire set of adhesion/growth-regulatory galectins from one organism (chicken) to a glycoprotein or to
cell surfaces was systematically evaluated. Differential sensitivities were detected between plant and ani-
mal lectins and also between distinct galectin forms within the chicken series. Two of the bivalent probes
can be considered as sensors for interlectin differences. Most pronounced were the selectivities of N-gly-
cosyl 1,2,3-triazole derivatives for the chimera-type galectin and its proteolytically truncated version.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 18 September 2011
Revised 1 November 2011
Accepted 2 November 2011
Available online 10 November 2011
Keywords:
Agglutinin
Glycocluster
Glycocyclophane
Lectin
N-Glycosyl 1,2,3-triazoles
A broad range of physiological functions is emerging for the gly-
can chains of cellular glycoconjugates via carbohydrate-protein
(lectin) recognition.1 Distinct carbohydrate structures serve as tar-
get sites for receptors (exogenous agglutinins from plants or bacte-
ria, tissue lectins) and their interaction begins the conversion of
sugar-encoded information into cellular responses. If conse-
quences of this interplay are harmful for the organism, for exam-
ple, toxicity exerted by plant lectins or immune dysregulation/
tumor invasion by endogenous effectors,2 then blocking of the
docking onto cells by custom-made inhibitors becomes an attrac-
tive goal for medicinal chemistry. In principle, two general struc-
tural parameters can be varied with the aim of accomplishing
high-level specificity: (i) the carbohydrate headgroup including
any modification (additions such as sulfation or synthetic modifi-
cations at distinct sites to enhance complementary binding to tar-
get receptors) and (ii) the valency of presentation. Of course,
interpretation of experimental results benefits from a clear design
approach, avoiding too many parameter changes. Also, such stud-
ies gain impact if—besides revealing affinity of a carbohydrate
derivative in a binding assay for a single lectin—further compara-
tive binding analyses with a range of relevant targets are per-
formed to assess selectivity. Ideally, this should be accomplished
with cell assays where natural ligands are in their physiological
presentation.
To meet these criteria we herein focused on characterizing the
impact of different modes of bivalent ligand presentation of a com-
mon sugar headgroup (lactose). Although the linker length (dis-
tance between two lactose residues) cannot always bridge the
distance between contact sites of bi- to oligovalent lectins in an
intramolecular sense, such a topological (bivalent) ligand display
has nonetheless previously indicated its potential for targeting
galactoside-specific lectins to varying extents.3 Three ditriazoles,
four diamides, a glycocyclophane and an acylic analogue of the lat-
ter were synthesized as the test panel (Fig. 1, 1–4, 6–10). Also in-
cluded was a control monomer (5), which contains structural
features of the aglycone found in 3 and 4, where lactose is con-
nected via a triazole to a benzylated glucuronic acid derivative. A
galactoside-binding plant toxin (Viscum album L., VAA; with low
(Tyr)- and high (Trp)-affinity sites in the dimer separated by 87 Å
or 15 Å, respectively, in the b-trefoil fold4), a leguminous b-sand-
wich agglutinin (Erythrina crystagalli agglutinin, ECA) and adhe-
sion/growth-regulatory galectins were purified by affinity
chromatography3a to give a range of suitable lectins for the bio-
chemical/cell biological part of the study. Of note, the family of
galectins encompasses three topological modes for binding-site
presentation (Fig. 2). Since it is appealing to comprehensively
determine the structure–activity profiles of these three types for
proteins from one organism, we have run the assays on the five
chicken galectins (CGs).5 Our panel thus comprises the three
homodimeric proto-type galectins (CG-1A, CG-1B, CG-2), the
chimera-type CG-3 along with its proteolytically truncated variant
⇑
Corresponding author. Tel.: +353 91 492465; fax: +353 91 495576.
E-mail address: paul.v.murphy@nuigalway.ie (P.V. Murphy).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.010

314
S. André et al. / Bioorg. Med. Chem. Lett. 22 (2012) 313–318
N
N
OH
N
HO
HO
O
OH
O
O
HO
O
OH
O
HO
OH
OH
OH
OH
O
HO
HO
N
HO
HO
HO
N
1
2
N
OH
O
OH
H
N
O
O
O
O
HO
HO
O
HO
N
O
HO
HO
H
OH
OH
OH
HO
O
HO
N
N
N
O
O
HN
OH
NH
OH
O
HO
HO
OH
O
O
O
O
HO
O
N
O
O
HO
HO
N
HO
OH
O
O
HO
N
HO
OBn
OH
HO
BnO
BnO
OBn
NH
3
4
OH
N
N
N
O
O
O
O
O
HN
HO
HO
O
OH
OH
HO
O
HO
O
O
OH
OH
O
HO
OH
N
HO
N
N
HO
O
HO
O
OBn
BnO
BnO
OBn
HO
HO
OH
O
OH
CO2H
O
O
O
O
N
HO
HO
BnO
HO
N
N
BnO
HO
HO
5
6
7
OH
OH
O
N
N
OH
O
N
O
HO
O
O
N
O
HO
O
HO
HO
N
O
N
HO
OH
OH
HO
HO
HO
OH
OH
OH
O
O
O
O
HO
N
HO
HO
N
N
N
OH
N
O
N
O
O
HO
O
HO
OH
OH
OH
HO
HO
HO
OH
O
O
OH
OH
H
N
O
O
O
O
N
H
HO
O
HO
HO
HO
O
OH
OH
HO
HO
OH
8
HO
HO
OH
OH
O
OH
H
N
O
O
O
HO
O
O
HO
N
N
H
O
HO
HO
OH
OH
OH
O
HO
HO
9
HO
HO
OH
O
O
O
OH
O
OH
O
HO
N
H
O
HO
H
O
HO
O
HO
HO
OH
OH
OH
HO
10
Figure 1. Compounds 1–10.
(trCG-3) and the two versions of tandem-repeat-type CG-8 with
natural variations in linker length (either 9 or 28 amino acids)
which arises from alternative splicing, together with a separate do-
main (the N-terminal domain, termed Gal-8N) (Fig. 2). Orthologs of
proteins from each group are present in mammals, and the poten-
tial for functional competition, documented between proto- and
chimera-type galectins,6 makes this broad screening approach
mandatory. Regarding potential for aggregation of the proteins in
solution, CG-8 behaves as monomer in gel filtration and ultracen-
trifugation, as does galectin-3, which can oligomerize in the pres-
ence of multivalent ligands.3c,5d The affinity of 1–10 was assessed
in inhibitory assays in two steps: firstly a measure of their potency
their ability to inhibit lectin binding to cultured cells was evalu-
ated. The latter experiment provides an in vitro setting with in-
creased relevance for the clinical situation.
The synthesis of ditriazoles began from the lactosyl azide 11
(Scheme 1), which was prepared as described previously3e and re-
acted with a variety of dialkynes (12–14) using copper-catalysed
azide alkyne cycloaddition reactions7,8 to give protected interme-
diates. Subsequent deacetylation using methoxide in methanol
gave compounds 1, 6 and 7.
Next, the glycocyclophane 3 was prepared, also from the azide
11 (Scheme 2). Reaction of 11 with glucuronic acid derivative 15
by copper-catalysed azide alkyne cycloaddition reaction gave tria-
zole 16. The coupling of 16 with p-xylylenediamine, subsequent
ring closure metathesis and removal of the acetates gave the final
in interfering with lectin binding to
a glycoprotein matrix
(asialofetuin (ASF), a pan-galectin ligand) was evaluated; secondly

S. André et al. / Bioorg. Med. Chem. Lett. 22 (2012) 313–318
315
result of binding of the labeled lectin to the glycoprotein, depended
on amount of glycoprotein coated and was saturable. The concentra-
tion of thelectinsto be usedin theexperimentswas henceoptimized
in each case to yield OD-readings in the linear range during the titra-
tions. All compoundsproved tobe able to inhibitlectin bindingtothe
glycans presented on the matrix to some degree. As a relative mea-
sure of the inhibitory potency of each compound, the concentration,
which reduced the signal for bound lectin by 50% (the IC50-value),
was obtained from titration curves, as shown in Figure 3. Normaliza-
tion of concentrations to lactose enabled to directly identify in-
creases in inhibitory potency of the dilactosides relative to free
lactose, which were obviously at work for CG-3 and compounds 1
and 4 (Fig. 3). Preliminary experimental runs for the ditriazoles (1,
6, 7) and diamides (2, 8–10) revealed rather gradual changes within
a rather narrow range, so we decided to focus the titrations on the
representative compounds 1 and 2, with inter-lactose distances of
around 12.5 Å and 8.4 Å, respectively, and compare these with the
glycocyclophane 3 and the more flexible analogue 4 where the in-
ter-lactose distances would be greater. In several cases, the presence
of the triazole was associated with an enhanced level of inhibition.
Forhumanorthologs, galectin-1wasratherinsensitivetothevarious
structural analogues, while susceptibility of galectin-3 to a triazole
in the anomeric position has already emerged.12 In our series, this
factor appeared to be most pronounced for CG-3 (Table 1). In this
special case, the monomeric compound 5 was between three and
fourfold more potent than lactose. Although the triazole and/or glu-
curonic acid residue contributes to the binding, bivalency is clearly
required to observe highest inhibitory activity against CG-3, even
though the triazole and possibly also the glucuronic acid residue
are contributing to the binding to CG-3. A tendency for increased
binding was also seen when comparing CG-1A versus CG-1B/CG-2.
The comparison between CG-3 and its truncated version (trCG-3)
was of particular interest. Binding of trCG-3 to ASF was considerably
less susceptible to inhibition with the tested dilactosides. This is
because trCG-3 is less prone to form oligomers as it is a product of
the proteolytic processing of CG-3, which leads to removal of the
collagenase-sensitive stalk.
Figure 2. Representation of the three types of structural organization of chicken
galectins based on crystallographic data,18b,c experimental evidence in solution and
homology considerations.5c–e Proteolytic degradation of the collagen-like stalk of
CG-3 with its 10 Gly/Pro-rich sequence repeats consisting of either five (one repeat),
seven (five repeats) or eight (four repeats) amino acids turns the full-length version
(CG-3) into trCG-3. The numbers of amino acids constituting the linker is given for
CG-8S/L.
product 3. The comparatively flexible analogue 4 was also pre-
pared by coupling and deacetylation, avoiding the macrocyclisa-
tion step. Deacetylation of 16 gave the monomer 5.9
Finally,
a series of dilactosyl diamides was synthesized
(Scheme 3). Coupling of the lactosyl amine 17, prepared by reduc-
tion of azide 11 as previously described,3e,7c,10 with a variety of dia-
cyl chlorides 18–21 and subsequent deacetylation gave 2 as well as
8–10, completing the test panel (Fig. 1). In addition to compound
characterization (please see Supplementary data) modeling for
the bivalent ditriazoles and diamides, as described previously,3e,7c
yielded inter-lactose (Glc C-1 to Glc C-1) distances ranging from
4.1 Å (8) to 19.6 Å (7).
These compounds were first tested in the solid-phase assay set-
up, which was performed as described previously.11 In detail, the
glycoprotein (ASF) was adsorbed to the surface of microtiter plate
wells, and its three N-glycans were ligands for carbohydrate-
dependent binding of the two labeled plant agglutinins and the
galectins. The signal (optical density, OD) intensity, observed as a
The relative flexibility, too, matters for compounds 3 and 4 and
influences their binding affinities. The more rigid 3 was more po-
tent for the plant agglutinins than its acyclic analogue 4, albeit
with only a slight increase relative to free lactose. It was also more
potent for the tandem-repeat-type CG-8 and its N-terminal do-
main (Table 1). In contrast, CG-3 binding is clearly more sensitive
for the more flexible 4, whereas only minor differences between
3 and 4 occur among the proto-type proteins (Table 1). Thus, the
different ratio of binding affinity of the glycocyclophane 3 and its
more flexible analogue 4 appears to disclose disparities in lectin-
site properties and/or reactivity to structural differences between
1. CuOAc, sodium ascorbate, dialkyne,
AcO
AcO
OAc
O
MeOH, THF, H2O, sonication, 1h
OAc
O
or
O
AcO
N3 CuSO4, sodium ascorbate, dialkyne,
CH3OH-H2O >85%
OAc
OAc
1, 6 & 7
11
2. NaOMe, MeOH, >60%
O
O
O
O
13
12
14
Dialkynes:
Scheme 1. Synthesis of 1, 6 and 7.

316
S. André et al. / Bioorg. Med. Chem. Lett. 22 (2012) 313–318
AcO
AcO
OAc
O
BnO
OAc
O
+
BnO
O
O
O
N3
CO2H
AcO
AcO
AcO
15
11
Cu(OAc)2, sodium ascorbate
dialkyne, MeOH, THF, H2O
sonication, 1h, 82%
AcO
AcO
OAc
O
OAc
NaOMe
MeOH
CO2H
O
O
O
O
5
N
AcO
AcO
BnO
AcO
N
N
BnO
16
1. p-xylylenediamine, PyBop,
DIPEA, DMF, 48h, 76%
2. Grubbs-II, CH2Cl2, 91%
3. NaOMe, MeOH, 70%
1. p-xylylenediamine, PyBop
DIPEA, DMF, 48h, 76%
2. NaOMe, MeOH, 70%
3
4
Scheme 2. Synthesis of 3 and 4
1. coupling with diacyl
chlorides
AcO
AcO
OAc
O
OAc
O
2. protecting group
removal
O
NH2
Cl
AcO
OAc
2, 8-10
OAc
17
O
O
O
O
O
Cl
Cl
Cl
Cl
Cl
Cl
Cl
O
O
O
21
18
Diacyl chlorides
19
20
Scheme 3. Synthesis of 2 and 8–10.
3 and 4 for the tandem-repeat-type (bivalent) CG-8 when com-
pared to the homodimeric proteins. In other words, this pair of
synthetic compounds is a sensor to distinguish between the galec-
tin subgroups depicted in Figure 2. The monomeric N-terminal do-
main of CG-8 (CG-8N) is rather equally well blocked by
dilactosides, irrespective of the nature of the scaffold. In line with
this result, proteolytic processing of CG-3, which abolishes oligo-
mer formation via the collagen-like stalk, impairs this lectin’s
marked sensitivity to dilactosides (cf. CG-3 and trCG-3, Table 1).
The acyclic compound 4 had highest relative potency of all the li-
gands for CG-3. Lectin binding to the surface-presented glycopro-
tein in this case was effectively impaired, with a reasonably high
level of selectivity.
In order to establish whether the solid-phase assay has predic-
tive value for a compounds’ ability to inhibit the binding of a lectin
to cells we next performed such bioassays. On cell surfaces, a nat-
ural panel of high-affinity ligands for lectins is presented in the
physiologically relevant mode. Experimentally, the labeled lectin,
in the absence or presence of inhibitors, was incubated with ali-
quots of cell suspensions from the same passage, in order to avoid
glycophenotype changes upon prolonged periods in culture, and
the extent of binding was quantitated by cytofluorometry, as re-
ported previously when testing aglyconic extensions of lactose
and dilactosides with human galectins.3e,12
Figure 3. Titration curves for extent of binding of biotinylated CG-3 (1 lg/ml) to
surface-immobilized glycoprotein (ASF) in the presence of increasing amounts of
lactose (top) and compounds 1 and 4 (bottom), respectively. For compilation of
IC50-values, please see Table 1.
The binding data obtained by cytofluorometry is documented as
the percentage of positive cells and the mean fluorescence inten-
sity for each experiment shown in Figure 4; the two determined
variables will range between two controls, the first obtained in

S. André et al. / Bioorg. Med. Chem. Lett. 22 (2012) 313–318
317
Table 1
IC50-values of four bivalent lactosides and free lactose (Lac) for blocking binding of biotinylated lectins to surface-immobilized ASF (in mM)a
Lectin
inhibitor
VAA
(1.5
ECA
(0.2
CG-1A
(8 g/ml)
CG-1B
(3 g/ml)
CG-2
(4 g/ml)
CG-3
(1 g/ml)
trCG-3
(1.5 g/ml)
CG-8S
(0.75
CG-8L
(0.75
CG-8N
(15 g/ml)
l
g/ml)
lg/ml)
l
l
l
l
l
lg/ml)
lg/ml)
l
1
2
3
4
0.13 (4.6)
0.55 (1.1)
0.24 (2.5)
1.3 (0.5)
0.6 (1)
0.6 (1.8)
1.2 (0.9)
0.8 (1.4)
1.3 (0.8)
1.1 (1)
0.22 (2.3)
0.36 (1.4)
1.6 (0.3)
1.4 (0.5)
0.5 (1)
1.5 (2.7)
1.3 (3.1)
0.6 (6.7)
0.46 (8.7)
4 (1)
1.2 (5.0)
1.4 (4.3)
0.7 (8.6)
0.65 (9.2)
6 (1)
0.07 (15.7)
0.6 (1.8)
0.08 (13.8)
0.014 (78.6)
1.1 (1)
3.1 (1.5)
4.2 (1.1)
5.3 (0.8)
5.0 (0.9)
4.5 (1)
0.7 (2.6)
2.5 (0.7)
0.4 (4.5)
3 (0.6)
1.8 (2.8)
4.5 (1.1)
0.6 (8.3)
2.4 (2.1)
5 (1)
0.5 (3.2)
0.8 (2.0)
0.3 (5.3)
0.4 (4.0)
1.6 (1)
Lactose
1.8 (1)
a
For structures of 1–4, see Figure 1; assays at a constant amount of 0.5 lg ASF used for coating of microtiter plate wells were routinely done in triplicates for up to five
independent series with standard deviations not exceeding 12.4%. The lectin concentration is given in each case. Numbers in brackets denote the inhibitory potency relative
to free lactose. Concentration values are normalized to lactose in all cases.
also in this context, whereas simultaneous reactivity (intramolec-
ular binding) with extended sites, as discussed for selectins,14 ap-
pears less likely, despite binding of histo-blood group
tetrasaccharides, digalactosides or poly-N-acetyllactosamine re-
peats presenting two galactose units to the extended contact site
of chicken and human galectins.15 In general, density of ligand pre-
sentation appears to be a key factor for galectins when targeting
distinct counter-receptors from the glycome complexity, at the le-
vel of N-glycan branching and presentation in microdomains, also
seen between CG-1A and CG-1B when testing polyvalent glycopro-
teins.15a,16 The example of ‘non-spanning’ bivalent ligands and the
cholera toxin B—pentamer teaches the lesson that transient, non-
specific binding can also have a significant bearing on affinity.17
Dilactosides with a triazole at the anomeric carbon appeared more
favorable than those with an amide in most cases. Screening in two
assay types with increasing biorelevance were in agreement and
revealed notable differences in binding within the set of galectins,
separating the two natural forms of the chimera-type galectin as
well as the tandem-repeat-type protein and the non-covalently
associated homodimers.
In summary, the compounds described herein block glycopro-
tein and cell binding to various lectins, with differences in potency
and selectivity in some cases. This provides a basis for further re-
search with a view to accomplish selectivity enhancement. Explo-
ration of the impact of (i) substitutions in the sugar headgroup,
Figure 4. Cell surface staining (percentage of positive cells/mean fluorescence
intensity) by labeled lectins and the impact of presence of inhibitors. Binding curves
for CG-3 (a) and trCG-3 (b) at 5 lg/ml in the absence of inhibitor (100%-value) and
(listed from bottom to top) 1 mM lactose, 1 mM 2 and 0.25 mM 1 (a, gray area: 0%-
with guidelines provided by chemical mapping, glycan testing,
crystallographic analysis and molecular modeling15,16,18 and (ii)
increasing valency would both be of interest in this regard.
value, background control in the absence of lectin) or lactose, 2 and 1 at 2 mM using
CHO cells (b). Low level of sensitivity was recorded for ECA (2
SW480 colon adenocarcinoma cells for 2, 3, lactose and 1 at 0.2 mM (c), whereas
marked differences were present for interfering of binding of CG-8S (5 g/ml) to
CHO cells by 2, lactose and 1 tested at 1 mM (d). Aliquots of the same batch were
analyzed in triplicates in at least three independent series with standard deviations
not exceeding 11.3% after normalization of the data.
lg/ml) and human
Acknowledgments
l
The authors are grateful to the reviewers for their helpful ad-
vice. The work described herein was generously funded by IRCSET
(D.Y.), Science Foundation Ireland (PI/IN1/B966, 08/SRC/B1393)
and the EU (MC-EIF to D.V.J.; GlycoHIT, Grant agreement no.
260600 to H.-J.G.).
the absence of lectin (0%-value; gray area in each panel of Fig. 4)
and the second obtained in the presence of the lectin without addi-
tion of inhibitor (100%-value; black line in each panel of Fig. 4).
Blocking lectin binding by an inhibitor will reduce the 100%-value
according to its potency. The results of these assays, when carried
out with the compounds, were consistently in agreement with the
results obtained from the solid-phase binding assays; this is
exemplarily documented for the two forms of CG-3 (Fig. 4a, b),
the leguminous lectin and its low level of sensitivity (Fig. 4c) as
well as for CG-8 (Fig. 4d). At the same time, the experiments ex-
tend the data basis for differences in binding affinity toward the
tested lectins to cell surfaces as the assay platform and hereby af-
ford a determination of the potential of the compounds to reduce
lectin binding to cell surfaces.
Supplementary data
Supplementary data (experimental details for synthesis of the
compounds and analytical data) associated with this article can
be found, in the online version, at doi:10.1016/j.bmcl.2011.11.010.
References and notes
1. (a)The Sugar Code. Fundamentals of Glycosciences; Gabius, H.-J., Ed.; Wiley-VCH:
Weinheim, 2009; (b) Gabius, H.-J.; André, S.; Jiménez-Barbero, J.; Romero, A.;
Solís, D. Trends Biochem. Sci. 2011, 36, 298.
2. (a) Read, R. J.; Stein, P. E. Curr. Opin. Struct. Biol. 1993, 3, 853; (b) Gabius, H.-J.;
Darro, F.; Remmelink, M.; André, S.; Kopitz, J.; Danguy, A.; Gabius, S.; Salmon, I.;
Kiss, R. Cancer Invest. 2001, 19, 114; (c) Ulbrich, H.; Eriksson, E. E.; Lindbom, L.
Trends Pharmacol. Sci. 2003, 24, 640; (d) Hartley, M. R.; Lord, J. M. Biochim.
Biophys. Acta 2004, 1701, 1; (e) Audi, J.; Belson, M.; Patel, M.; Schier, J.; Osterloh,
J. J. Am. Med. Assoc. 2005, 294, 2342; (f) Villalobo, A.; Nogales-González, A.;
Gabius, H.-J. Trends Glycosci. Glycotechnol. 2006, 18, 1; (g) Gabius, H.-J. Biochem.
Soc. Trans. 2008, 36, 1491; (h) Norling, L. V.; Perretti, M.; Cooper, D. J. Endocrinol.
Overall, the combined data shown in this communication leads
to the conclusion that bivalency contributes to inhibitory activity.
Kinetic aspects of binding affecting on/off rates to increase affinity,
as reported for mucin loading,13 may be a reasonable explanation

318
S. André et al. / Bioorg. Med. Chem. Lett. 22 (2012) 313–318
2009, 201, 169; (i) Osborn, H. M. I.; Turkson, A. In The Sugar Code. Fundamentals
10. (a) Peto, C.; Batta, G.; Györgydeák, Z.; Sztaricskai, F. Liebigs Ann. Chem. 1991, 5,
505; (b) Aravind, S.; Park, W. K. C.; Brochu, S.; Roy, R. Tetrahedron Lett. 1994, 35,
7739.
11. (a) André, S.; Pei, Z.; Siebert, H.-C.; Ramström, O.; Gabius, H.-J. Bioorg. Med.
Chem. 2006, 14, 6314; (b) André, S.; Maljaars, C. E. P.; Halkes, K. M.; Gabius, H.-
J.; Kamerling, J. P. Bioorg. Med. Chem. Lett. 2007, 17, 793.
12. (a) André, S.; Giguère, D.; Dam, T. K.; Brewer, C. F.; Gabius, H.-J.; Roy, R. New J.
Chem. 2010, 34, 2229; (b) Giguère, D.; André, S.; Bonin, M.-A.; Bellefleur, M.-A.;
Provencal, P. C.; Pucci, B.; Roy, R.; Gabius, H.-J. Bioorg. Med. Chem. 2011, 19,
3280.
13. Dam, T. K.; Brewer, C. F. Adv. Carbohydr. Chem. Biochem. 2010, 63, 139.
14. Maaheimo, H.; Renkonen, R.; Turunen, J. P.; Penttilä, L.; Renkonen, O. Eur. J.
Biochem. 1995, 234, 616.
of Glycosciences; Gabius, H.-J., Ed.; Wiley-VCH: Weinheim, 2009; pp 469–483;
(j) Schwartz-Albiez, R. In The Sugar Code. Fundamentals of Glycosciences; Gabius,
H.-J., Ed.; Wiley-VCH: Weinheim, 2009; pp 447–467; (k) Osorio, F.; Reis e
Sousa, C. Immunity 2011, 34, 651.
3. (a) André, S.; Ortega, P. J. C.; Perez, M. A.; Roy, R.; Gabius, H.-J. Glycobiology
1999, 9, 1253; (b) André, S.; Liu, B.; Gabius, H.-J.; Roy, R. Org. Biomol. Chem.
2003, 1, 3909; (c) Ahmad, N.; Gabius, H.-J.; André, S.; Kaltner, H.; Sabesan, S.;
Roy, R.; Liu, B.; Macaluso, F.; Brewer, C. F. J. Biol. Chem. 2004, 279, 1081; (d)
André, S.; Specker, D.; Bovin, N. V.; Lensch, M.; Kaltner, H.; Gabius, H.-J.;
Wittmann, V. Bioconjugate Chem. 2009, 20, 1716; (e) Leyden, R.; Velasco-
Torrijos, T.; André, S.; Gouin, S.; Gabius, H.-J.; Murphy, P. V. J. Org. Chem. 2009,
74, 9010.
4. (a) Olsnes, S.; Stirpe, F.; Sandvig, K.; Pihl, A. J. Biol. Chem. 1982, 257, 13263; (b)
Niwa, H.; Tonevitsky, A. G.; Agapov, I. I.; Saward, S.; Pfüller, U.; Palmer, R. A. Eur.
J. Biochem. 2003, 270, 2739; (c) Jiménez, M.; André, S.; Siebert, H.-C.; Gabius, H.-
J.; Solís, D. Glycobiology 2006, 16, 926; (d) Jiménez, M.; André, S.; Barillari, C.;
Romero, A.; Rognan, D.; Gabius, H.-J.; Solís, D. FEBS Lett. 2008, 582, 2309.
5. (a) Beyer, E. C.; Zweig, S. E.; Barondes, S. H. J. Biol. Chem. 1980, 255, 4236; (b)
Oda, Y.; Kasai, K.-I. Biochem. Biophys. Res. Commun. 1983, 761, 237; (c) Kaltner,
H.; Solís, D.; Kopitz, J.; Lensch, M.; Lohr, M.; Manning, J. C.; Mürnseer, M.;
Schnölzer, M.; André, S.; Sáiz, J. L.; Gabius, H.-J. Biochem. J. 2008, 409, 591; (d)
Kaltner, H.; Solís, D.; André, S.; Lensch, M.; Manning, J. C.; Mürnseer, M.; Sáiz, J.
L.; Gabius, H.-J. Biochemistry 2009, 48, 4403; (e) Kaltner, H.; Kübler, D.; López-
Merino, L.; Lohr, M.; Manning, J. C.; Lensch, M.; Seidler, J.; Lehmann, W. D.;
André, S.; Solís, D.; Gabius, H.-J. Anat. Rec. 2011, 294, 427.
6. (a) Kopitz, J.; von Reitzenstein, C.; André, S.; Kaltner, H.; Uhl, J.; Ehemann, V.;
Cantz, M.; Gabius, H.-J. J. Biol. Chem. 2001, 276, 35917; (b) Sanchez-Ruderisch,
H.; Fischer, C.; Detjen, K. M.; Welzel, M.; Wimmel, A.; Manning, J. C.; André, S.;
Gabius, H.-J. FEBS J. 2010, 277, 3552.
7. (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004;
(b) Meldal, M.; Tornoe, C. W. Chem. Rev. 2008, 108, 2952; (c) André, S.; Velasco-
Torrijos, T.; Leyden, R.; Gouin, S.; Tosin, M.; Murphy, P. V.; Gabius, H.-J. Org.
Biomol. Chem. 2009, 7, 4715.
15. (a) Wu, A. M.; Singh, T.; Liu, J. H.; Krzeminski, M.; Russwurm, R.; Siebert, H.-C.;
Bonvin, A. M. J. J.; André, S.; Gabius, H.-J. Glycobiology 2007, 17, 65; (b)
Krzeminski, M.; Singh, T.; André, S.; Lensch, M.; Wu, A. M.; Bonvin, A. M. J. J.;
Gabius, H.-J. Biochim. Biophys. Acta 2011, 1810, 150; (c) Martín-Santamaría, S.;
André, S.; Buzamet, E.; Caraballo, R.; Ferná, B.; Cañada, F. J.; Menéndez, M.;
Ramström, O.; Jiménez-Barbero, J.; Solís, D.; Gabius, H.-J. Org. Biomol. Chem.
2011, 9, 5445; (d) Miller, M. C.; Ribeiro, J. P.; Roldós, V.; Martín-Santamaría, S.;
Cañada, F. J.; Nesmelova, I.; André, S.; Pang, M.; Klyosov, A.; Baum, L. G.;
Jiménez-Barbero, J.; Gabius, H.-J.; Mayo, K. H. Glycobiology 2011, in press,
doi:10.1093/glycob/cwr083.
16. (a) Wu, A. M.; Wu, J. H.; Tsai, M.-S.; Kaltner, H.; Gabius, H.-J. Biochem. J. 2001,
358, 529; (b) Wu, A. M.; Wu, J. H.; Liu, J.-H.; Singh, T.; André, S.; Kaltner, H.;
Gabius, H.-J. Biochimie 2004, 86, 317; (c) Morelle, W.; Stechly, L.; André, S.; van
Seuningen, I.; Porchet, N.; Gabius, H.-J.; Michalski, J.-C.; Huet, G. Biol. Chem.
2009, 390, 529; (d) Stechly, L.; Morelle, W.; Dessein, A. F.; André, S.; Grard, G.;
Trinel, D.; Dejonghe, M. J.; Leteurtre, E.; Drobecq, H.; Trugnan, G.; Gabius, H.-J.;
Huet, G. Traffic 2009, 10, 438; (e) Kopitz, J.; Bergmann, M.; Gabius, H.-J. IUBMB
Life 2010, 62, 624.
17. Liu, J.; Begley, D.; Mitchell, D. D.; Verlinde, C. L. M. J.; Varani, G.; Fan, E. Chem.
Biol. Drug Des. 2008, 71, 408.
18. (a) Solís, D.; Romero, A.; Kaltner, H.; Gabius, H.-J.; Díaz-Mauriño, T. J. Biol. Chem.
1996, 271, 12744; (b) Varela, P. F.; Solís, D.; Díaz-Mauriño, T.; Kaltner, H.;
Gabius, H.-J.; Romero, A. J. Mol. Biol. 1999, 294, 537; (c) López-Lucendo, M. F.;
Solís, D.; Sáiz, J. L.; Kaltner, H.; Russwurm, R.; André, S.; Gabius, H.-J.; Romero,
A. J. Mol. Biol. 2009, 386, 366.
8. For synthesis of other bivalent ditriazole derivatives see: Ziegler, T.; Hermann,
C. Tetrahedron Lett. 2008, 49, 2166.
9. Details for preparation of 5 are provided in the Supplementary data section. See
also: (a) Mereyala, H. B.; Gurrala, S. R.; Mohan, S. K. Tetrahedron 1999, 55,
11331; (b) Jarikote, D. V.; O’Reilly, C.; Murphy, P. V. Tetrahedron Lett. 2010, 51,
6776.