Strong inhibition of cholera toxin binding by galactose dendrimers

Article abstract of DOI:10.1039/b711070g

Galactose-containing dendrimers with long spacer arms inhibit cholera toxin binding as strongly as the natural ganglioside GM1 oligosaccharide does. The Royal Society of Chemistry.

Full text of DOI:10.1039/b711070g

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Strong inhibition of cholera toxin binding by galactose dendrimers{
Hilbert M. Branderhorst,a Rob M. J. Liskamp,a Gerben M. Visserb and Roland J. Pieters*a
Received (in Cambridge, UK) 19th July 2007, Accepted 7th September 2007
First published as an Advance Article on the web 24th September 2007
DOI: 10.1039/b711070g
Galactose-containing dendrimers with long spacer arms inhibit
cholera toxin binding as strongly as the natural ganglioside
GM1 oligosaccharide does.
Cholera toxin (CT) is the causative agent of cholera. This disease
still causes major problems in the developing world, with over
100 000 reported cases per year.1 The cholera toxin is a member of
the AB5 toxin family, and its B-subunits bind to the oligosacchar-
ide portion of the GM1 ganglioside (GM1os) molecules present on
Fig. 1 Structure of the galactose building block used here (bottom) to
mimic some of the features of the GM1os building block (top, ref. 7), such
as the terminal galactose moiety, the spacer length and its hydro- and
lipophilicity.
the cell surfaces of the intestines. The binding process leads to
toxin internalization2 followed by disease initiation caused by the
A-subunit.3 Inhibitors of B-subunit binding have therapeutic
potential and they may also be useful for toxin detection either in
patient samples4 or in materials suspected of having a terrorist
origin.5 Considering the pentameric architecture of the B-subunit
(CTB5), to which up to five GM1 oligosaccharide ligands can bind
simultaneously, a multivalency approach is particularly attractive
for inhibitor design. Several multivalent inhibitors have been
designed for cholera toxin and related proteins of the AB5 family.6
Recently we reported on a series of GM1os conjugated
dendrimers that showed unprecedented affinity enhancements of
up to 380 000-fold beyond GM1os derivatives.7 The strong
binding was attributed to the combined use of the strong
GM1os ligand and its multivalent presentation on a dendrimer
with long spacer arms.6e While these GM1os–dendrimer con-
jugates were very potent, the goal still remains to prepare inhibitors
based on cheap bulk sugars like galactose with potencies equal to
or better than the endogenous GM1os ligand. GM1os is very
complex and difficult to prepare on a large scale. The design
lessons learned in the GM1os study7 were applied to the
development of galactose-based multivalent CTB5 inhibitors. We
here report such inhibitors that exhibited similar CTB5 inhibitory
potencies to GM1os derivatives.
with 1,11-dibromo-undecane and the trityl group was cleaved by
p-TsOH in MeOH to give 3. Introduction of the galactose moiety
was achieved with galactosyl donor 4 and BF3?OEt2 as the
promoter. An azide function for ‘click’ conjugation9 was
introduced by reaction of the coupling product with NaN3 in
DMF at elevated temperature and as the final step the acetyl
protecting groups were removed by NaOMe in MeOH to afford
the desired compound 5a. The monovalent reference compound 5b
was prepared from per-acetylated galactose (see ESI{).
Ligation of 5a to dendrimers 6a, 7a and 8a7 (Scheme 2) was
performed using ‘‘click’’ chemistry.9 Our recently developed
protocol involving microwave heating at 80 uC was used10 and
the products 6b, 7b and 8b were obtained after preparative HPLC
purification in good yields and purity.
The CTB5 ligand, galactose, was used since this is by far the
most important residue for binding within GM1os.8 Galactose was
outfitted with a poly(ethylene glycol) unit, to crudely mimic the
other sugar rings of the GM1os (Fig. 1). Furthermore, a lipophilic
part was attached in order to keep this factor the same as in our
GM1os-based system.7
Building block 5a was prepared as shown in Scheme 1. The
synthesis started with penta(ethylene glycol), which was mono
tritylated to give 2. The chain was elongated via an SN2 reaction
aDepartment of Medicinal Chemistry and Chemical Biology, Utrecht
Institute for Pharmaceutical Sciences, Utrecht University, PO Box
80082, 3508 TB Utrecht, The Netherlands. E-mail: R.J.Pieters@uu.nl;
Fax: +31 30-2536655
Scheme 1 Synthesis of galactose building block 5a and the structure of
5b; reagents and conditions: (a) trityl chloride, pyridine, 72%; (b) (i) NaH,
Br(CH2)11Br, DMF, 66%; (ii) TsOH, MeOH, 95%; (c) (i) 4, BF3?Et2O,
toluene, 44%; (ii) NaN3, DMF, 84%; (ii) NaOMe, MeOH, 72%.
bLaboratory of Organic Chemistry, Wageningen University, Dreijenplein
8, 6703HB Wageningen, The Netherlands
{ Electronic supplementary information (ESI) available: Synthesis and
characterization details. See DOI: 10.1039/b711070g
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Chem. Commun., 2007, 5043–5045 | 5043

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Scheme 2 Synthetic galactose dendrimers; reagents and conditions: (a) 5a, CuSO4, sodium ascorbate, DMF–H2O, 80 uC, 20 min, yields: 70% for 6b, 78%
for 7b, 77% for 8b.
The glycodendrimers were tested for cholera toxin inhibition in
a well established ELISA-like assay.7,11 A GM1 ganglioside coated
96 well plate was allowed to bind horseradish peroxidase labelled
CTB5 (CTB5-HRP) and this binding was inhibited with a
concentration range of dendrimeric inhibitors (Table 1). In this
assay the monovalent galactose and its derivative 5b exhibited the
expected weak inhibitory power with IC50s of 240 and 80 mM
respectively. The divalent dendrimer 6b showed a large increase in
potency with an IC50 value of 130 mM. Tetravalent compound 7b
showed a further increase in potency (IC50 25 mM) and the affinity
of octavalent compound 8b increased only by another factor of ca.
2 (IC50 12 mM).
derivative. The compounds of higher valency, 7b and 8b,
showed IC50s in the same range as the GM1os derivative. The
multivalency effect, as expressed by the relative potency per sugar,
still increased from di- to tetravalent (923 vs. 2400), while it
remained basically the same at the octavalent stage (2500).
Additional experiments, including the use of complementary
experimental methods, will likely uncover the nature of the
multivalency effects, i.e. chelation, aggregation or a combination
thereof. In comparison to reported multivalent galactose-based
CTB5 inhibitors, the present system compares favourably. A
reported pentavalent galactose-based system still fell short by a
factor of ca. 80 relative to GM1.6e Another decavalent system
seemed to come close, but no direct comparison with GM1os or its
derivatives was made.6c
The potency increase of divalent 6b is remarkable. However, the
compound still binds more weakly than the monovalent GM1os
To conclude, a galactose containing building block was
synthesized in a straightforward synthesis from bulk galactose.
This building block was efficiently coupled to di-, tetra- and
octavalent dendrimers via ‘click’ chemistry. The potencies are very
high, considering that 7b and 8b showed roughly equal IC50 values
to the GM1os derivative in this assay. This result is an important
step towards low cost potent ligands for cholera toxin with
applications in therapy and detection.
Table 1 Inhibitory potencies of the CTB5 inhibitors
Relative potency
(per sugar)
Compound
Valency IC50a/M
Galactose
5b
6b
7b
8b
GM1os–C11H21
1
1
2
4
8
1
2.4 (¡ 0.5) 6 1021 1 (1)
8.0 (¡ 0.2) 6 1022 3 (3)
1.3 (¡ 0.2) 6 1024 1846 (923)
2.5 (¡ 0.4) 6 1025 9600 (2400)
1.2 (¡ 0.3) 6 1025 20 000 (2500)
1.9 (¡ 0.6) 6 1025 12 632 (12 632)
The Utrecht Institute for Pharmaceutical Sciences (UIPS) is
gratefully acknowledged for support. We thank the EU for
fostering collaboration in this area: EU COST D34 working group
‘Multivalent ligands for cholera toxin inhibition and sensing’.
(ref. 7)
a
Determined in an ELISA experiment with 0.43 nM CTB5-HRP
and wells coated with 0.2 mg GM1.
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