Evidence suggests that these ligands are poor initiators of
cell signaling because they aggregate receptors predominantly
in the form of stable cyclic dimers.12 Modeling suggests that
cyclic dimers, in which two ligand molecules connect two
IgE-FcꢀRI complexes to form a closed ring, prevent chain
elongation, limit the size of ligand-induced aggregates, and
may generate an inhibitory signal.13
In contrast to divalent antigens, trivalent antigens are
predicted to form larger, highly branched aggregates (Figure
1B) leading to strong responses by IgE-FcꢀRI presenting
cells (Figure 1C). Recently, Whitesides and co-workers14
reported the preparation of a trivalent antigen capable of
aggregating IgE. They found that aggregates formed with
stoichiometries of 3:2 (IgE/trivalent ligand). In this manu-
script we describe the synthesis of a series of di- and trivalent
ligands and comparison of their relative ability to stimulate
degranulation in IgE sensitized RBL cells. We find that the
trivalent ligands are potent antigens causing degranulation
at nanomolar concentrations, while analogous divalent
antigens are inactive by comparison.
In allergic responses resulting in release of inflammatory
mediators, IgE targeting a nearly limitless variety of antigens
may be involved. To simplify experiments, monoclonal IgE
that associates with dinitrophenyl groups is used, and
multivalent antigens therefore contain multiple dinitrophenyl
amines. RBL cells are readily culturable and respond strongly
to receptor aggregation and consequently are useful in
measuring responses to polyvalent antigens. Measurement
of degranulation is made by spectroscopic determination of
the activity of granule-stored â-hexosaminidase secreted into
the supernatant on p-nitrophenyl-N-acetyl-â-D-glucosamine.
Design considerations of the multivalent antigens included
the length of the tether between dinitrophenylamine groups
and an attachment point for an additional fluorophore. In
earlier work, we determined that separation of antigenic
epitopes of more than 40 Å was sufficient to allow efficient
binding to IgE and subsequent aggregation of FcꢀRI.15
To ensure that a tether of adequate length was generated,
we prepared tethers derived from both tetra- and pentaeth-
ylene glycol. On the basis of the modeling of the tethers
(MMFF with Spartan) a maximum separation of 43.3 and
49.5 Å between dinitrophenylamine groups was predicted
for the tetraethylene and pentaethylene glycol-based com-
pounds, respectively. The tethers were prepared using
Schwabacher’s effective desymmetrization procedure of a
diazide16 giving 1a and 1b (Scheme 1), followed by reaction
with fluorodinitrobenzene and reduction providing 2a and
2b.
Figure 1. (A) Schematic representation of ligands, antibodies and
receptors involved in degranulation; (B) IgE aggregates formed with
di- and trivalent antigens; (C) aggregation of FcꢀRI leading to
aggregation.
For example, it has been observed that aggregation caused
by IgE dimers are less effective than those caused by larger
IgE oligomers at stimulating cellular responses6 and that
cellular responses are inhibited when an optimal degree of
aggregation is exceeded.9 The factors that cause an aggregate
to be a robust signaling unit, an inhibitor of signal trans-
duction, or a nonsignaling unit remain to be fully elucidated.
The importance of influences of ligand-induced receptor
aggregates on cellular signals has prompted significant effort
in developing means for quantitative analysis of the interac-
tions between multivalent ligands and cell surface receptors.10
An attractive approach to studying IgE-FcꢀRI aggregation
involves the use of synthetic symmetric divalent ligands,
which are the simplest type of ligand capable of aggregating
receptors. The interaction of divalent IgE antibodies with a
divalent ligand can result in an IgE-FcꢀRI aggregate
spectrum that contains only linear chains or rings of various
sizes (including one-to-one complexes) (Figure 1B). This
limited spectrum of aggregates makes divalent ligands much
easier to study theoretically than ligands of valences higher
than two, which can form highly branched aggregates. A
primary goal of studies performed with divalent ligands has
been to measure or predict the number of receptors in
aggregates on the cell surface, so that this quantity then can
be compared and correlated with cellular responses.11
However, a major limitation of divalent ligands studied to
date is their inability to activate strong cellular responses.
Design of trivalent antigens was based on a tris-hy-
droxymethylaminomethane (Tris) core because this com-
Scheme 1. Preparation of Dinitrophenyl-Appended Glycol
Linkers.
(9) Seagrave, J.; Oliver, J. M. J. Cell Physiol. 1990, 144, 128.
(10) Hlavacek, W. S.; Perelson, A. S.; Sulzer, B.; Bold, J.; Paar, J.;
Gorman, W.; Posner, R. G. Biophys. J. 1999, 76, 2421.
(11) Posner, R. G.; Subramanian, K.; Feder, T.; Thomas, J.; Holowka,
D.; Baird, B.; Goldstein, B. J. Immunol. 1995, 155, 3601.
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Org. Lett., Vol. 9, No. 18, 2007
Posner, Richard G.
