Modular synthesis of multivalent glycoarchitectures and their unique selectin binding behavior

Article abstract of DOI:10.1039/b813414f

Hyperbranched polyglycerols (HPGs) are ideal scaffolds for the multivalent presentation of saccharides, due to their biocompatible, carbohydrate-like properties; here, we report the conjugation of galactosesugar moieties to HPG, and the multivalent effect of these constructs on selectin binding. The Royal Society of Chemistry.

Full text of DOI:10.1039/b813414f

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Modular synthesis of multivalent glycoarchitectures and their unique
selectin binding behaviorw
Ilona Papp,a Jens Dernedde,b Sven Endersb and Rainer Haag*a
Received (in Cambridge, UK) 1st August 2008, Accepted 11th September 2008
First published as an Advance Article on the web 3rd October 2008
DOI: 10.1039/b813414f
Hyperbranched polyglycerols (HPGs) are ideal scaffolds for the
multivalent presentation of saccharides, due to their biocompatible,
carbohydrate-like properties; here, we report the conjugation of
galactose sugar moieties to HPG, and the multivalent effect of
these constructs on selectin binding.
and co-workers to prepare diverse dendritic structures.10
These reactions are quite efficient, as they proceed in high
yields, under aqueous conditions, and with complete regio-
selectivity. During the course of this work Riguera and
co-workers and Roy and co-workers5 have used the concept
of click-chemistry in combination with carbohydrate functio-
nalized dendrimers. Applying the click methodology, we first
introduced alkyne groups into the HPG 1 by reacting with
propargyl bromide in the presence of NaH. The obtained
polymer 2 was highly substituted, resulting in 88% function-
alization according to the 1H NMR calculations, with a
number average molar mass of 4.3 kDa. The sugar moiety,
galactose (Gal) was easily outfitted with an azido group using
a propylene spacer, based on a published procedure.11
Carbohydrates play a significant role in biological systems,
spreading from cellular recognition and adhesion to cell growth
and differentiation.1 Wide interest has been devoted to the
interplay of cell-surface receptors with their corresponding
binding carbohydrate moieties. Unfortunately, most carbo-
hydrate–receptor interactions are weak (dissociation constants
are in the millimolar concentration range)2 and in order to
compensate for their low binding affinity, different strategies
based on multivalent interactions have been designed, includ-
ing carbohydrate clusters, glycopolymers and glycopeptides.3
Multivalent carbohydrates are attractive synthetic targets as
they often bind much more strongly to the interacting protein
partner than their monovalent counterparts. Inspired by other
examples4 as well as recent breakthroughs in dendrimer syn-
thesis using click chemistry5 we developed a new class of
functional, multivalent glycoarchitectures based on HPG 1 as
a biocompatible building block.
Employing the click conditions polymer 2 was reacted with 3a–c
(protected, unprotected and sulfated carbohydrates) to furnish the
Hyperbranched polymers are highly branched, narrowly
disperse, three-dimensional macromolecules, with unique
structures and properties.6 More specifically HPGs 1 are
water-soluble, highly biocompatible polyether polyols, which
compared to their perfect dendritic analogs possess more
linear units and are more flexible.7 However, they can be
prepared in an efficient one-pot reaction, with predetermined
molecular weights and narrow molecular weight distribu-
tions.8 They show excellent biocompatible properties both
in vitro and in vivo.9 Their hyperbranched architectures and
high density of peripheral functional groups have prompted us
to explore multivalent glycodendritic polymers as mimics of
cell-surface glycans. For this purpose we developed a synthetic
strategy, which employs the Cu(I)-catalyzed azide–alkyne
cycloaddition reaction previously used by Sharpless, Hawker
a Freie Universitat Berlin, Institut fur Chemie und Biochemie,
¨
¨
Takustrasse 3, 14195 Berlin, Germany.
E-mail: haag@chemie.fu-berlin.de; Fax: +49 30 838 53357;
Tel: +49 30 838 52633
Scheme 1 Synthesis of multivalent HPG 4a–c and PE 7a,b architec-
tures. Reagents and conditions: (i) propargyl bromide, NaH, DMF,
16 h, 87%, functionalization 88% (2); (ii) CuSO4ꢀ5H2O, sodium
ascorbate, THF–water = 1 : 1, 24 h, 4a (89%), 4b (92%), 4c (86%),
7a (85%), 7b (94%); (iii) MeONa, MeOH, RT, 4 h, 90%; (iv) SO3ꢀPy,
DMF, 90%; HPG depicted as smaller partial structure (20 mer) for
clarity. HPG (1) used was average 3 kDa molecular weight (B40 mer),
Mw/Mn = 1.18, DB (degree of branching) = 0.57.
b
´
Universitatsklinikum Charite Berlin, Zentralinstitut fur
¨
¨
Laboratoriumsmedizin und Pathobiochemie, CBF Hindenburgdamm
30, 12200 Berlin. Fax: +49 30 844 54152; Tel: + 49 30 844 54639
w Electronic supplementary information (ESI) available: General
synthetic procedure and characterization of compounds. See DOI:
10.1039/b813414f
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desired glycopolymers 4a–c in relative high yields (85–90%)
(Scheme 1). In parallel, for low valency control, the commercially
available pentaerythritol (PE) scaffold was used for the prepara-
tion of tetrameric galactose derivates (7a,b) (see ESIw). The
resulting glycopolymers were isolated in relatively high yields,
after purification of the reaction mixture by ultrafiltration or
dialysis. The completion of the conjugation reactions, and the
degree of ligand loading (galactose) on the polyglycerol were
1
established in all cases by H NMR spectroscopy, as well as by
DLS measurements. IR data also confirmed that neither alkyne
(3289 cmꢂ1; 2114 cmꢂ1) nor azide (2098 cmꢂ1) residues remain in
the final glycostructures. These new multivalent glycoarchitectures
were then analysed for in vitro inhibition of selectins. The term
‘‘selectin’’ is employed to designate a general class of receptor
which displays a selective adhesive function and which includes a
lectin-like domain responsible for such selective adhesive function.
Known selectins include E-selectin, P-selectin and L-selectin.
Galactose is an important binding moiety in selectin inhibition
of the tetrasaccharide sialyl Lewis X (SLex) and its isomer sialyl
Lewis a (SLea).
Selectins share the ability to recognize and bind the SLex or
SLea and other related oligosaccharides bound to different
glycoprotein scaffolds.12
All galactose derivatives were tested by surface plasmon reso-
nance (SPR) in a competitive binding assay, a well established
selectin-ligand interaction assay,13 in order to analyze their
potency in selectin recognition (Scheme 2).z
These galactose functionalized HPGs 4b,c (35 terminal
galactose per molecule) showed strong inhibition of leukocytic
L-selectin and platelet derived P-selectin, with IC50 values in
the nanomolar range (nM). Only in case of 4b a moderate
inhibition of the endothelial E-selectin was observed (mM).
Hyperbranched polyglycerol itself showed no inhibition to any
selectins. The Gal multimer 4b showed binding in the nano-
molar range (70 and 1000 nM, respectively, for L- and
P-selectin), which represents an affinity enhancement of up
to 103-fold over the tetrameric counterpart. Furthermore,
functionalizing the HPG with previously sulfated galactose
moieties (88% functionalization, with a number of average
molar mass of 24.4 kDa), the binding affinity to L- and
especially to P-selectin was dramatically enhanced, resulting
in IC50 values in the low nanomolar range (1 and 7 nM,
respectively). (Fig. 1) This result is in accordance with prior
findings that polyanionic carbohydrate ligands show a marked
binding enhancement to selectins.14,15 It is interesting to note,
however, that galactose sulfation appears to hinder binding to
E-selectin.
Fig. 1 Inhibition curves to (a) P-, (b) L- and (c) E-selectins: percent
inhibition (y axis) is plotted against the inhibitor concentration in
(x axis). Inhibition is shown.
The results (Table 1) from the selectin inhibition studies
indicate that the hyperbranched glycopolymers prepared in
this work act as multivalent ligands. Compared on a per
Scheme 2 SPR based competitive selectin-ligand binding assay; RU = resonance unit; details in Notes section.
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Table 1 IC50 values (nM) of the compounds towards binding of L-, P- and E-selectins (N.I.: no inhibition below 1 mM)
7b
4b
4c
Compound
Total
Per Gal
Total
Per Gal
Total
Per Gal
L-Selectin
P-Selectin
E-Selectin
60 000
30 000
N.I.
240 000
120 000
N.I.
70
1000
70 000
2450
35 000
2 450 000
1
7
N.I.
35
245
N.I.
with respect to the inhibitor concentration was recorded and calculated as
x% of the control. The concentration of inhibitor, that causes 50%
reduction in the binding of the labelled reference ligand, was referred to
as IC50 value. Each concentration was applied at least in duplicate.
galactose basis the IC50 value for structure 4b shows in case of
L-selectin a 100-fold enhancement of the inhibitory power
over the tetravalent counterpart 7b. This dependence of affi-
nity increase on the number of Gal residues per polymer
clearly is a result of these Gal residues being engaged in
binding at multiple binding sites. Around a four-fold increase
in affinity per sugar was observed in case of P-selectin inhibi-
tion when the number of Gal moieties was increased from 4 to
35 per molecule of HPG. The identity of the binding elements,
structure of the scaffold and numbers of binding groups are all
parameters that influence the mechanism by which a ligand
acts. These multivalent HPG-Gal architectures contain
elements of structural complexity which are not present in
the tetravalent counterpart.
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selectin proteins,15,16 it was surprising for us that polyvalent
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sulfated carbohydrates). Additional work is underway to
further increase the inhibitory effect and to more thoroughly
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This study has been carried with financial support from the
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Notes and references
z SPR assay description in brief: Selectin functionalized Au-nanoparticles
were passed over a standard ligand (20 mol% sLex and 5 mol%
sulfotyrosine conjugated to polyacrylamide) of all selectins, immobilized
on the sensor chip. The resulting binding signal was set to 100% and
serves as a control. To evaluate selectin binding of potential inhibitors, a
defined preincubation step with the selectin nanoparticles was performed
before its passage over the sensor chip. Reduction of the binding signal
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Chem. Commun., 2008, 5851–5853 | 5853