Mannose-substituted PPEs detect lectins: A model for Ricin sensing

Article abstract of DOI:10.1039/b416587j

The interaction of a mannose-substituted poly(para phenyleneethynylene) (mPPE) with a lectin, Concanavalin A (ConA), is reported; the ConA causes fluorescence quenching of the mPPE with a K_SV of 5.6 × 10 ⁵. The Royal Society of Chemi

Full text of DOI:10.1039/b416587j

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Mannose-substituted PPEs detect lectins: A model for Ricin sensing{
Ik-Bum Kim, James N. Wilson and Uwe H. F. Bunz*
Received (in Columbia, MO, USA) 25th October 2004, Accepted 2nd December 2004
First published as an Advance Article on the web 20th January 2005
DOI: 10.1039/b416587j
An aqueous solution of 6 was exposed to ConA but no distinct
change in the fluorescent properties of 6 were observed, suggesting
only weak binding of 6 to ConA. When an aqueous solution of
polymer 5 in phosphate buffer was exposed to ConA, efficient
fluorescence quenching occured at low concentrations of the lectin.
The Stern–Volmer relationship15
The interaction of a mannose-substituted poly(para phenyl-
eneethynylene) (mPPE) with a lectin, Concanavalin A (ConA),
is reported; the ConA causes fluorescence quenching of the
mPPE with a KSV of 5.6 6 105.
Sugar binding proteins, lectins, play a crucial role in cell-surface
recognition, cell signalling and pathogen and toxin docking.1–3
While lectins such as Concanavalin A (ConA) are harmless, Ricin,
a toxic protein is perhaps the best known representative of its class,
due to a bizarre assassination episode involving a toxin-spiked
umbrella.4 To detect pathogens5 and toxins2 on a broader base, it
would be of interest to have simple fluorescence sensing methods
for Ricin, Botulinum toxin, E. coli toxin(s), and other lectins of
importance.5 At the moment, lectin–sugar interactions are studied
by agglutination of erythrocytes,1 by surface plasmon resonance
studies of carbohydrate-carrying polynorbornene derivatives,6,7 or
by colorimetric reaction with sugar-coated polydiacetylene vesi-
cles.8,9 We disclose herein the synthesis of the fluorescent mannose-
substituted poly(para phenyleneethynylene) (mPPE) 5 and its
interaction with Concanavalin A (ConA), the lectin of the jack
bean. Detection of ConA by fluorescence quenching of the
multivalent mannoside 5 is effective and sensitive.
(F0/F[Q]) = 1 + KSV [Q] or KSV = {(F0/F[Q]) 2 1}/[Q]
quantitatively correlates the loss of fluorescence (F0/F[Q]) with the
concentration [Q] of added quencher. The slope of the graph of [Q]
vs. (F0/F[Q]) is the Stern–Volmer constant. There are broadly two
mechanisms for quenching of fluorophores: a static and a dynamic
one. In dynamic (collisional) quenching, the excited state of the
fluorophore forms a complex with the quencher, and the excited
Reaction of 1 with 8-chloro-3,6-dioxaoctanol in the presence of
potassium carbonate in DMF furnished the diiodide 2 (see
Scheme 1). Coupling of 2 to trimethylsilylacetylene in the presence
of a Pd-catalyst gave rise to the formation of the monomer 3
after removal of the trimethylsilyl groups by tetrabutylammonium
fluoride in THF. To attach the mannose substituents to the
monomer core, 2 was treated with mannose pentaacetate and BF3-
etherate in dichloromethane analogous to a preparation described
by van Doren for glycosylation of phenols.10 The mannosylation
of 2 is stereospecific under these conditions and furnishes 4 as the
double a-anomer. In the last step 3 and 4 are coupled in a
piperidine/THF mixture with copper iodide and (Ph3P)2PdCl2 to
form the PPE 5 in excellent yield and with a high degree of
polymerization, according to gel permeation chromatography
(yield 95%, Pn = 19, Mn = 22 6 103 by 1H NMR; Mn = 62 6 103,
Mw/Mn = 1.5 by gel permeation chromatography).11–14 The
nucleophilic solvent, piperidine, leads to the convenient in situ
stripping of the acetate groups and the deacetylated polymer 5 is
directly obtained. For a monomeric model, 4 was coupled under
standard conditions to 4-methoxyphenyl acetylene; 6 formed in
excellent yield after washing with an ethyl acetate–hexane mixture
(see Scheme 2). As for 5, the acetate groups are removed by
piperidine in the course of the reaction.
{ Electronic Supplementary Information (ESI) available: Synthesis of
polymer 5 and model compound 6, and details of the quenching
experiments. See http://www.rsc.org/suppdata/cc/b4/b416587j/
*uwe.bunz@chemistry.gatech.edu
Scheme 1 Synthesis of the mannose-substituted polymer 5 by Pd-
catalyzed coupling of 3 to 4.
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Scheme 2 Synthesis of the mannose-substituted model compound 6 by
Pd-catalyzed coupling of methoxyphenylacetylene to 4.
Fig. 2 Aggregates of 5 and ConA shown in the TEM.
state is quenched. In static quenching the ground state of the
fluorophore forms a complex with the quencher, and KSV
represents the stability constant of the ground state complex.
For PPEs as fluorophores static quenching is prevalent.16–18 The
short (0.3 ns) emissive lifetime of PPEs12,16,17 makes dynamic
quenching of PPEs difficult; KSV here therefore equals the constant
of complex formation between PPE 5 and the quencher, ConA.
Fig. 1 shows fluorescence spectra and Stern–Volmer plot of the
exposure of 5 to ConA. The quenching of 5’s fluorescence is linear
at low ConA concentrations (Fig. 1 inset), but deviates from
linearity at higher quencher concentrations. The KSV based on the
linear part of the curve is 5.6 6 106; 5 binds tightly to ConA. A
control experiment with bovine serum albumin shows no
quenching of the fluorescence and therefore no unspecific binding
of 5 occurs; neither does the galactose-binding lectin jacalin (see
ESI{) elicit a response, showing that 5 is a specific sensor for
mannose binding lectins. The interaction of ConA with 5 leads
finally to the precipitation of the complex. We examined the
aggregates of ConA and 5 by transmission electron microscopy.
Fig. 2 shows the spherical fluorescent aggregates that are
approximately 300–500 nm in diameter. To make the assay
more sensitive we induced aggregation of 5 with biotinylated
ConA (Fig. 3) and find a similar fluorescence quenching as in Fig. 1.
Upon addition of commercially available streptavidin-coated
Fig. 3 Fluorescence of aggregates of 5 and biotinylated ConA before
and after addition of streptavidin-coated microspheres.
polystyrene spheres, however, the fluorescence decreases signifi-
cantly further, by precipitating the ConA?5-complex through the
formation of a super-aggregate. The formation of the super-
aggregate is important, because it significantly enhances the
sensitivity of the assay. At the moment we are exposing solutions
of 5 and its biotinylated congener to a mannose-binding strain of
E. coli to examine the agglutination of bacteria by 5.
In conclusion we have demonstrated that PPE 5 is an excellent
fluorescent biosensor for lectins and multivalent interactions can
be exploited in this scheme to obtain high sensitivities for lectin
sensing by sugar-substituted PPEs, particularly when using the
formation of a super-complex. Our results complement a recent
study19 that utilizes a postfunctionalization route to sugar-coated
carboxy-substituted PPEs to sense ConA and E. coli. Our
approach uses well-defined and anomerically pure building blocks
and avoids problems such as partial functionalization and
introduction of defects that are common in postfunctionalization
schemes. The polymer 5 is well characterized by NMR and IR and
is easily available on a 300-mg-scale.
The authors thank the National Institute of Health (NIH 1U01-
AI5-650-301) for generous support.
Ik-Bum Kim, James N. Wilson and Uwe H. F. Bunz*
School of Chemistry and Biochemistry, Georgia Institute of Technology,
Atlanta, GA 30332, USA. E-mail: uwe.bunz@chemistry.gatech.edu;
Fax: +1(404)385-1795; Tel: +1(404)385-1795
Fig. 1 Emission spectrum and Stern–Volmer plot (inset) of 5 in the
presence of ConA.
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J. Chem. Soc., Perkin Trans. 1, 1996, 2873–2877.
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