suggesting that this CCI may have functions beyond simply
mediating preliminary cell adhesion.4
Table 1. Activity of G4 PAMAM Glycodendrimers
[lactose]b
(µM)
entry
P-G4-LacxCapy
% lac ∆πa (mN/m)
1
2
3
4
5
6
7
8
9
P-G4-Lac0Gly56
P-G4-Lac7Gly48
P-G4-Lac17Gly27
P-G4-Lac25Gly30
P-G4-Lac35Gly9
0
13
31
45
64
75
45
45
48c
42d
0.9 ( 0.4
1.2 ( 0.8
5.3 ( 0.7
8.1 ( 2.0
6.2 ( 1.0
3.2 ( 0.3
1.1 ( 1.7
1.7 ( 1.0
2.5 ( 0.2
3.2 ( 0.9
0.0
7.3
18.5
26
38
44
P-G4-Lac42Gly2
P-G4-Lac25Cell22Gly5
P-G4-Lac25Malt21Gly2
P-G4-Cell26Gly21
P-G4-Malt23Gly30
26
26
29c
25d
Biophysical studies of the lactose (Lac)·GM3 CCI have
been carried out using model systems to probe the interaction
between these two oligosaccharides. We and others have
reported on the use of Langmuir monolayers to study the
interaction between GM3-containing monolayers and mul-
tivalent glycoconjugates.5,6 These interactions are manifested
as changes in surface pressure when the glycoconjugates are
injected into the aqueous subphase beneath the monolayer.
We have shown that monolayers containing GM3 selectively
interact with glycolipid micelles containing lactose but do
10
a Binding to 1:1 GM3-DPPC monolayer in 1 mM CaCl2 was measured.
See Supporting Information for details. b All dendrimer concentrations in
the subphase were kept at 1.0 µM. c Refers to [cellobiose]. d Refers to
[maltose].
We have followed well-established protocols to function-
alize amine-terminated PAMAM dendrimers using glycosyl
isothiocyanates.9 Carbohydrates that are attached to a dieth-
yleneglycol isothiocyanate linker have been extensively used
in glycodendrimer synthesis.9c,10 The use of an isothiocyanate
linker has the advantage that no additional coupling reagents
are required for the reaction, facilitating purification. Gly-
codendrimers with different amounts of lactose (expressed
as % lac in Table 1) were prepared by reaction of P-G4 with
the appropriate amount of lactosyl isothiocyanate 3 (Scheme
1). The degree of lactose functionalization was determined
using 1H NMR and MALDI-TOF MS. The remaining amines
on the dendrimer were capped with the glycol isothiocyanate
4 or glycosyl isothiocyanates 5 and 6. The carbohydrate
ligands are functionalized randomly on the dendrimer scaf-
fold, giving rise to a homogeneous distribution of carbohy-
drate epitopes on the dendrimer surface.10a Acetyl groups
on the sugars were removed using sodium methoxide, and
the final products were purified by dialysis with water
followed by lyophilization. The molecular weights of the final
glycoconjugates were determined by MALDI-TOF MS.
Glycodendrimers functionalized with the disaccharides cel-
lobiose or maltose in place of lactose were prepared as
controls.11
not interact with structurally similar disaccharides.6d
A
limitation of using micelles to examine interactions with
monolayers is that most glycolipids are also surface-active,
and therefore the effects of non-specific insertion into the
monolayer need to be taken into account. The CCI of GM3
has also been examined with surface plasmon resonance
(SPR) experiments.7
To circumvent non-specific insertion into the monolayer,
we explored the use of glycodendrimers, which have been
extensively employed in studies of carbohydrate-protein
interactions.8 We report here that glycodendrimers can also
be used to examine the CCI of lactose with GM3 monolayers.
We have used polyamidoamine (PAMAM) dendrimers, a
well-studied multivalent platform that is commercially avail-
able. Although it is possible to vary carbohydrate multiva-
lency by increasing or decreasing the dendrimer generation
number, we elected to vary carbohydrate density for a single
generation of PAMAM generation-4 (P-G4) (vide infra).
We describe here the synthesis of P-G4 glycodendrimers
with varying carbohydrate valency and the results of their
interaction with mixed monolayers of GM3 and dipalmitoyl
phosphatidylcholine (DPPC). We have also investigated the
influence of subphase and monolayer composition on the
CCI.
Langmuir monolayer binding studies were carried out by
injecting glycodendrimers into a 1 mM CaCl2 subphase
underneath a monolayer of GM3 and DPPC (1:1) that was
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(11) A complete list of all glycodendrimers prepared, along with
characterization information, is found in Supporting Information.
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Org. Lett., Vol. 11, No. 3, 2009