Angewandte
Chemie
DOI: 10.1002/anie.200805238
Supramolecular Probes
Pattern-Based Recognition of Heparin Contaminants by an Array of
Self-Assembling Fluorescent Receptors**
Richard B. C. Jagt, Rodolfo F. Gꢀmez-Biagi, and Mark Nitz*
During late 2007 and early 2008, 81 patients in the USA and
Germany died and hundreds were seriously injured after
being administered contaminated unfractionated heparin
sulfate (UFH) while undergoing anticoagulation therapy.[1]
Multiple orthogonal analytical techniques, including exten-
sive high-field NMR spectroscopy, HPLC, and capillary
electrophoresis, were needed to identify the contaminant as
the semisynthetic glycosaminoglycan, oversulfated chondroi-
tin sulfate (OSCS).[2] Even though some batches of heparin
were found to contain up to a third of this non-natural form of
chondroitin sulfate, its presence was masked in standard
quality-control assays owing to the inherent anticoagulant
activity of OSCS.[3] The development of quick and reliable
tests for heparin contaminants is currently of great interest.[4]
Herein we report the design and evaluation of a fluorescent
receptor array that is able to assess the quality of a heparin
sample by quickly differentiating UFH from OSCS and other
commonly encountered negatively charged polymers.
The design of selective receptors for biological macro-
molecules, such as UFH, poses a significant challenge.[5] As an
alternative, chemists have turned towards differential arrays
which do not rely on receptors that are specific for a particular
molecule, but on a unique diagnostic pattern that is derived
from an array of receptors with broad specificity.[6] Many of
the most successful examples of solution-phase differential
receptor arrays are based on indicator-displacement assays
(IDAs).[7] The power of IDAs lies in their modular nature,
which enables many unique receptors to be constructed
rapidly with a minimum number of synthetic steps. Herein we
describe an alternative modular receptor array that does not
rely on dye displacement, but on the binding of an environ-
mentally sensitive fluorophore proximal to the analyte-
recognition site.
receptor array capable of differentiating between negatively
charged polymers.
To design a modular receptor with a high propensity for
binding negatively charged biopolymers, we employed the
cyclodextrin (CD) 1a as a scaffold.[13] The primary amine
groups of 1a were functionalized to provide the amide and
guanidino derivatives 1b–e as a small collection of polycat-
ionic receptors (Scheme 1). The different modes of electro-
static recognition of CDs 1a–e towards the polyanionic
biopolymers provides the diversity that is needed to generate
specific response patterns in the receptor array for each
analyte.
Conveniently, 1a also forms a remarkably stable inclusion
complex (KD = 18 nm) with lithocholic acid (LCA).[14] The
stability of LCA–b-cyclodextrin complexes has previously
enabled the innovative application of this complex in direct-
ing protein–protein interactions.[15] According to previous
NMR spectroscopic studies, LCA binds to 1a with the
carboxylic acid oriented through the primary rim of the
CD.[13,16] This binding orientation provides a simple and
modular way to position a fluorophore near the positively
charged analyte-binding site of CDs 1a–e (Figure 1). The
quinolinium fluorophore employed is an efficient fluorescent
reporter of heparin-binding events driven by electrostatic
interactions in aqueous solution.[9a] Thus, the synthesis of a
quinolinium fluorophore tethered through a variable spacer
to LCA (to give 2a–c), followed by complexation with CDs
1a–e, rapidly generated fifteen potential fluorescent recep-
tors for the desired analytes.
An initial evaluation of complexes 1a·2a–c was carried
out with UFH as the analyte. The optimum conditions with
respect to both fluorescence response and binding affinity for
A wide variety of different colorimetric and fluorescent
heparin indicators have been reported with mechanisms of
heparin sensing based on boronic acids,[8] heparin-specific
peptides,[9] labeled heparin-binding proteins,[10] changes in
polymer conformation,[11] and fluorophore aggregation.[12]
However, none of these strategies provide a modular design
that enables the facile generation, and optimization, of a
[*] Dr. R. B. C. Jagt, R. F. Gꢀmez-Biagi, Prof. M. Nitz
Department of Chemistry, University of Toronto
80 St. George Street, Toronto, ON, M5S 3H6 (Canada)
E-mail: mnitz@chem.utoronto.ca
[**] We gratefully acknowledge financial support from The Netherlands
Organization for Scientific Research (NWO) and the Natural
Sciences and Engineering Research Council of Canada (NSERC).
Scheme 1. Modular self-assembling fluorescent receptors for poly-
anionic biopolymers. The receptors consist of a polycationic cyclo-
dextrin host (1a–e) and a fluorescent reporter tethered to a lithocholic
acid guest (2a–c).
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2009, 48, 1995 –1997
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1995