Combining carbohydrate substitutions at bioinspired positions with multivalent presentation towards optimising lectin inhibitors: Case study with calixarenes

Article abstract of DOI:10.1039/c1cc11163a

Carbohydrate derivatisation and glycocluster formation are both known to enhance avidity for lectin binding. Using a plant toxin and human adhesion/growth-regulatory lectins (inter- and intrafamily comparisons) the effect of their combination is examined. In detail, aromatic substituents were introduced at the 2-N or 3′-positions of N-acetyllactosamine and the products conjugated to two types of calix[n]arenes (n = 4, 6) via thiourea-linker chemistry.

Full text of DOI:10.1039/c1cc11163a

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COMMUNICATION
Combining carbohydrate substitutions at bioinspired positions with
multivalent presentation towards optimising lectin inhibitors: case study
with calixareneswz
a
b
Sabine Andre,y Cyrille Grandjean,y Franc¸
ois-Moana Gautier,b Silvia Bernardi,c
´
Francesco Sansone,c Hans-Joachim Gabiusa and Rocco Ungaro*c
Received 26th February 2011, Accepted 31st March 2011
DOI: 10.1039/c1cc11163a
Carbohydrate derivatisation and glycocluster formation are both
known to enhance avidity for lectin binding. Using a plant toxin
and human adhesion/growth-regulatory lectins (inter- and
intrafamily comparisons) the effect of their combination is
examined. In detail, aromatic substituents were introduced at
the 2-N or 30-positions of N-acetyllactosamine and the products
conjugated to two types of calix[n]arenes (n = 4, 6) via thiourea-
linker chemistry.
Galectins share affinity to N-acetyllactosamine (LacNAc)
but exhibit differing degrees of reactivity for the histo-blood
group A epitope with its 30-extension.4 Analyses by crystallo-
graphy and flexible docking provided a detailed structural
rationalization for this preference in the case of human
galectin-3 and prompted synthetic work to capitalize on it.5
However, cross-reactivity of such derivatives between the
chimera-type galectin-3 and at least the tandem-repeat-type
galectins-4 and -9 was delineated. In addition to this bioinspired
site for exchange of a hexopyranose by an aromatic moiety the
2-N-position of LacNAc, held in its place by three hydrogen
bonds with the 3-hydroxyl group,5a,c is a target. Aromatic
substitution at this point has attracted interest as source for
affinity generation.6 Due to its impact on solubility aromatic
bisubstitution is impeded, as is introduction of bulky substituents.
In contrast to galectins, the plant toxin is less permissive for
substitutions at these sites, therefore furnishing an inherent
negative control.5b,7 Having on this ground reached the
decision on where to enter structural additions, the previously
proven compatibility of the linker chemistry with lectin
activity could be applied.8 We selected the cone calix[4]arene
and the conformationally mobile calix[6]arene as scaffolds to
obtain clusters 1–4 (for structures, please see Scheme 1) owing
to their graded activity profiles to galectins-1, -3 and -4 [with
IC50-values (inhibitor concentration to reach 50%-level of
signal reduction) of lactoside clusters of 10 mM/0.6 mM for
galectin-1, 0.2 mM/0.4 mM for galectin-3, 20 mM/5 mM for
galectin-4].8 By testing the toxin and five human galectins
against the free LacNAc derivatives and the glycoclusters in
two assays of increasing biorelevance the following question is
answered: will combination of ligand substitution with valency
improve target properties?
The growing realisation of the high-density coding capacity of
glycans gives reason to devise routes for medical applications.1
Thus, sugar receptors (lectins), which translate the carbo-
hydrate-encoded message into cellular responses,1,2 become
targets for drug development. Custom-made inhibitors would
protect cells from harmful lectin activities.3 This concept can
be turned into compounds by teaming up carbohydrate and
supramolecular chemistry as follows: (i) tailoring the carbo-
hydrate for best-possible contact complementarity to the lectin
and (ii) acquiring lectin-matching multivalent presentation using
suitable conjugation chemistry and scaffold selection. This
perspective defines the aim of our study, i.e. to determine the
effects of exploiting changes in a core unit together with clustered
presentation. We used members of the family of human
adhesion/growth-regulatory galectins2 and a potent plant toxin
akin to ricin (Viscum album agglutinin, VAA) as models.
a Institut fur Physiologische Chemie, Tierarztliche Fakultat,
¨
¨
Ludwig-Maximilians-Universitat, Veterinarstr. 13, 80539 Munchen,
¨
¨
¨
¨
Germany. E-mail: gabius@tiph.vetmed.uni-muenchen.de;
Fax: +49-89-2108-2508; Tel: +49-89-2108-2290
b Laboratoire des Glucides, UMR CNRS 6219, Institut de Chimie de
´
Picardie, Universite de Picardie Jules Verne, 33 rue Saint-Leu,
F-80039 Amiens, France. E-mail: cyrille.grandjean@u-picardie.fr;
Fax: +33 322827560; Tel: +33 322828812
`
Dipartimento di Chimica Organica e Industriale, Universita degli
c
A modified Danishefsky’s iodosulfonamidation/azidation
process,9 accompanied or not by a tin-mediated, regioselective
arylation carried out on D-lactal derivatives, led to the key
1-azido-2-sulfonamido intermediates 5 and 6, as precursors of
2-amido-1-isothiocyanato lactopyranoses (Scheme 2, see ESI
for full detailsz). Acylation, removal of the sulfonamide
protecting groups followed by selective reduction of the azides
produced amines 7 and 8, which were not isolated, but turned
into the corresponding isothiocyanates 9 and 10, or trapped
Studi, Parco Area delle Scienze 17/A, 43124 Parma, Italy.
E-mail: rocco.ungaro@unipr.it; Fax: +39 0521 905472;
Tel: +39 0521 905458
w This article is part of a ChemComm ‘Supramolecular Chemistry’
web-based themed issue marking the International Year of Chemistry
2011.
z Electronic supplementary information (ESI) available: Experimental
procedures and compounds characterization. See DOI: 10.1039/
c1cc11163a
y Contributed equally.
c
6126 Chem. Commun., 2011, 47, 6126–6128
This journal is The Royal Society of Chemistry 2011

Table 1 IC50-values of the free sugar derivatives and the sugar-
bearing calix[4/6]arenes on binding for lectins to the glycoproteina
Type of
VAA
Galectin-3
(3 mg mlꢀ1
Galectin-4
(5 mg mlꢀ1
Galectin-9
(10 mg mlꢀ1
)
inhibitor (0.5 mg mlꢀ1
)
)
)
Lactose
300
4800
800
2800
420
200
45
2500
250
360
0.15
0.4
1200
800
1250
2.2
3000
900
300
2.8
11
12
1
2
3
0.8
2.5
1.2
1.4
2.0
0.8
1.3
1.5
4
a
Coating with constant amount of 0.5 mg asialofetuin per well; assays
to determine the IC50-value given in mM sugar (please see Fig. 1 in ESI
for examplez) were routinely performed in triplicates for up to five
independent series with standard deviations not exceeding 14.2%; for
structures of the glycoclusters, please see Scheme 1.
Scheme
1 Structures of the calixarene glycoclusters based on
2/30-substitutions in the LacNAc core.
Fig. 1 Inhibition of lectin binding to cells by the test compounds: the
background control given as gray area, 100% -value as solid black
line; data on percentage of positive cells/mean fluorescence intensity;
data in each panel in the order of compound listing from bottom to
top. (a, b) Galectin-4 (20 mg mlꢀ1) in the presence of 0.5 mM 12, 10 mM
3 and 10 mM 4 (a), 0.5 mM 11, 10 mM 1 and 10 mM 2 (b) for
tumor-suppressor p16INK4a-expressing pancreatic carcinoma Capan-1
cells. Assays were routinely performed in duplicates with up to five
independent series on aliquots of cell suspensions of the same or next
passage with standard deviations not exceeding 13.7%.
Scheme 2 Preparation of isothiocyanates and reference ligands.
Reagents and conditions: (i) thiophosgene (2 equiv.), CaCO3 (3 equiv.),
THF,
0
1C to r.t., overnight or 1,10-thiocarbonyldiimidazole
(1.5 equiv.), CH2Cl2, r.t., 6 h; (ii) Ac2O (3 equiv.), Et3N (2 equiv.),
CH2Cl2, 0 1C to r.t., 2 h; (iii) MeONa, MeOH, r.t., 2 h.
with excess acetic anhydride and deprotected under Zemplen
´
conditions to provide monovalent reference compounds 11
and 12. Isothiocyanates 9 and 10 were then conjugated at
the upper rim of amino-alkoxycalix[4/6]arenes followed by
deprotection,8 to yield the glycoclusters 1–4 (Scheme 1). These
glycoclusters, along with the monovalent controls, established
the test panel. Physiologically, lectin functionality rests on
binding to multivalent cellular glycoconjugates so that a
matrix of a glycoprotein (here asialofetuin with up to nine
terminal LacNAc units reactive with these lectins10) is an
adequate model for testing.8 It served as reactant for the
labelled lectins, and the inhibitory capacity of the compounds
was determined by co-incubation of increasing amounts with
the lectin in solution, as described in detail previously.8
noted previously.8 An about 10-fold increase in inhibition
ensued from 30-substitution relative to lactose, and tetra-
valency lowered the IC50-value to 0.15 mM (in sugar), relative
to 200 mM reached for lactose-bearing cone-4-type calixarene.8
Both synthetic processes thus substantially add up to a
significant gain in lectin-blocking capacity.
Whether the level of intrafamily selectivity is increased is
answered by assays using galectins-1 and -8. Parallel testing
disclosed rather weak inhibitory activity of the test panel with
IC50-values at least above 2 mM (not shown).
As exemplarily shown for galectin-3 (Fig. 1 in ESIz),
titrations revealed successively increasing extent of inhibition.
They enabled the determination of IC50-values. Used as
control with predictive value, the tested substitutions were
not favorable for VAA, as expected, although especially
hexavalent presentation was still capable to establish some
activity (Table 1). On the other hand, the inhibitory capacity
was markedly increased by both types of substitution for
galectin-3 (Fig. 1 in ESIz) and also galectin-9, less for galectin-4
(Table 1). The chimera-type galectin-3 maintained its characteristic
sensitivity for cone-type tetravalent presentation (Table 1),
These results thus encouraged to proceed to examine these
compounds in an in vitro system, i.e. challenging them with the
task to protect cells from lectin binding. Cell surface glycans
present a wide array of terminal galactosides along with
particular spatial arrangements such as microdomains.
A recent study on galectins had proven their significance
to form high-affinity contact sites for galectins-1 and -3.11
Routinely, sugar-dependent lectin binding was ascertained and
working concentrations for lectins in the linear range of signal
intensity were set for comparative analysis, as described for
initial calixarene testing previously.8
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 6126–6128 6127

To interpret the scans presented in Fig. 1 (and Fig. 2 in
ESIz) the binding of a labelled lectin to cells will shift the
signal from the position of the control curve (in gray) to the
right. An inhibitor will then interfere with lectin association
(measured in percentage of positive cells and mean fluorescence
intensity), moving the fluorescence profile back into the
direction of the control. This principle can now readily be
applied to the scans. In full accord with Table 1, the
monovalent derivative 12 was not inhibitory at this tested
concentration on VAA, whereas lactose and the two glyco-
clusters 3 and 4 reduced lectin binding with increasing potency
(Fig. 2a in ESIz). As noted previously,8 the cone-4-type
presentation showed increased reactivity relative to the
hexavalent compound in this system. This was further under-
scored by testing a different cell line, i.e. Chinese hamster
ovary (CHO) cells with defective a2,3-sialylation (Lec2
mutant12) (not shown). The tetravalent glycocluster 1 proved
most active against galectin-3 (Fig. 2b in ESIz). In addition to
the colon carcinoma cells, CHO cells with normal and reduced
levels of a2,3-sialylation and decreased b1,6-branching of
N–glycans (wild-type cells; Lec2/Lec4 mutant cells12) as well
as pancreatic carcinoma cells, in which galectin-3 can act as
competitive inhibitor against galectin-1 which induces anoikis
Interdipartimentale Misure ‘‘G. Casnati’’ for use of NMR
facilities, the Conseil Regional de Picardie for a post-doctoral
´
fellowship to FMG and Drs B. Friday and S. Namirha for
insightful discussions.
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under the control of the tumor suppressor p16INK4a 13
were
,
processed, yielding comparable results. These data provide
further evidence for the cone-4-type calixarene as suitable
scaffold to hit galectin-3, irrespective of changes in the glycomic
profile. Of note, galectin-1 binding was not affected at 10 mM
sugar concentration by this calixarene (not shown), and
galectin-4 remained more sensitive to the hexa- than the
tetravalent glycoclusters (Fig. 1). The same tendency with
rather similar quantitative data was observed for galectin-9,
with optimal activity for the 2-N-substituted hexavalent 4 (not
shown), in full accord with the data from the solid-phase
assays (Table 1).
´
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Overall, our results illustrate the benefit to combine core
derivatisation with conjugation of the biomimetic product to
distinct scaffolds for multivalent display. Practically, the
documented interference of galectin-3 with the pro-anoikis
effector of galectin-1 as e.g. in pancreatic cancer in vitro,13 can
be precluded by using such a selective blocking reagent, i.e.
cone-4-type calixarene with 30(or 2-N-)-substituted LacNAc.
Further tailoring the nature of the substituent to fully match
the galectin’s individual microenvironment in the contact site,
comprising Arg144, His158, Asn160, Lys176 and Trp181,5a,c
affords the possibility for iterative improvements. Using
bivalent glycophanes with/without conformational flexibility
the attained selectivity increases could then be exploited
to affect galectin-3 and its proteolytically processed form
differently.14 20-Substitution and starburst glycodendrimers
will be worthy of testing VAA,7,15 and the sulfatide headgroup
is a commendable candidate for galectin-4.4c,16 In general
terms, the presented strategy to amalgamate carbohydrate
and supramolecular chemistry can have relevance beyond
the particular lectins tested in this study.
´
´
´
,
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We thank the EC GlycoHIT program for generous
funding, the Ministero dell’Istruzione, Universita e Ricerca
(MIUR, PRIN-project no. 200858SA98), the Centro
G. Grard, O. Moreau-Hannedouche, E. Maes, A. Pons, S. Andre,
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c
6128 Chem. Commun., 2011, 47, 6126–6128
This journal is The Royal Society of Chemistry 2011