Optical sensing of sulfate by polymethinium salt receptors: Colorimetric sensor for heparin

Article abstract of DOI:10.1039/b718492a

Polymethinium salts based on substituted malondialdehyde have been prepared; the salt 9 with PEG substitution showed high selectivity for sulfate anions and heparin in aqueous medium at physiological condition; intracellular imaging of heparin-rich subcel

Full text of DOI:10.1039/b718492a

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Optical sensing of sulfate by polymethinium salt receptors: colorimetric
sensor for heparinw
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d
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Tomas Brıza,* Zdenek Kejık, Ivana Cısarova, Jarmila Kralova,
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Pavel Martasek and Vladimır Kral*
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b
ae
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Received (in Austin, TX, USA) 29th November 2007, Accepted 31st January 2008
First published as an Advance Article on the web 20th February 2008
DOI: 10.1039/b718492a
Polymethinium salts based on substituted malondialdehyde have
been prepared; the salt 9 with PEG substitution showed high
selectivity for sulfate anions and heparin in aqueous medium at
physiological condition; intracellular imaging of heparin-rich
subcellular compartments was achieved with our polymethinium
novel receptor 9 for cancer cell lines.
conditions. Recently, we reported the synthesis of chromo-
phoric trimethinium 1,10-binaphthyls with benzothiazolium
units;15 now we test them as sensors for recognition sulfate
over phosphate. We have observed affinity of the binaphthyls
for sulfate (log K = 4.7) and for phosphate (log K = 3.2) in
1 : 1 complexation. This study has showed promising potential
of polymethinium compounds for sulfate/phosphate recogni-
tion. Therefore we decided to test other methinium systems for
selective anion recognition. The synthetic protocol of new
polymethinium sensors with benzothiazolium units is
described in Scheme 1.
Anions and anionic biopolymers play a fundamental role in a
wide range of chemical and biological processes. Development
of receptors, which are designed for these analytes is an
important branch of modern chemistry.1–7 One of the major
challenges in supramolecular chemistry is the design of recep-
tors for selective anion recognition. Nature has developed
selective protein receptors even for structurally very similar
biologically important anions, e.g., including phosphate and
sulfate binding proteins.8
The structure of polymethinium chromophoric receptor 7
was determined by 1D and 2D NMR spectroscopy and single-
crystal X-ray analysis (Fig. 1).z
Initial study revealed that salt 7 (Scheme 1) exhibits a strong
interaction with sulfate anion, but no significant spectral
change in the presence of chloride, fluoride, nitrate, acetate
and phosphate was observed as shown in Fig. 2. Because of the
low water solubility of receptor 7, mixed solvent was used in
order to avoid aggregation effects. Multiple binding modes
were determined in the case of sulfate anion. For sulfate anion
and salt 7 the 2 : 2 and 1 : 2 complexes had K values of 2 ꢀ 1014
and 5 ꢀ 1011, respectively; values of K of other tested anions
were below 10.
The design of synthetic receptors for selective sulfate over
phosphate recognition in aqueous media has been a significant
challenge. In the case of polysulfates, such as heparin, two
types of optically-responsive synthetic receptors have been
studied. One of these is using cationic boronic sensors.9–11
Limitations of the boronic acid sensors include pH dependent
operation modes. Another reported method involves the
colorimetric detection of anionic polymers,12 mainly DNA13
by cationic heteroaromatic systems—cyanine dyes. This allows
for the preparation of sensors with larger extinction coeffi-
cients that can exhibit significant spectral changes in the visible
region.14 We intend to prepare new types of these salts as
receptors and to explore their properties as optical (colori-
metric) sensors.11 Our goal was the preparation of a receptor,
which can display a significant spectral change in the visible
spectral region for the determination of sulfate and polysulfate
even in presence of phosphate anions under physiological
In the next synthetic step, in order to increase water solubility,
the salt 8 substituted with two hydroxy groups on benzothiazo-
lium unit was prepared (Scheme 1). However, this synthetic
a Department of Analytical Chemistry, Institute of Chemical
´
Technology in Prague, Technicka 5, Prague 6, 16628, Czech
Republic. E-mail: ftor@seznam.cz; Fax: +420 224 310 352; Tel:
+420 224 310 352
First Faculty of Medicine, Charles University in Prague, Katerˇinska
b
´
32, Prague 2, 12108, Czech Republic
c Department of Inorganic Chemistry, Charles University in Prague,
Hlavova 8, Prague 2, 12108, Czech Republic
d Institute of Molecular Genetics, Academy of Sciences, Flemingovo
sq. 2, 166 10 Prague 6, Czech Republic
e Zentiva R & D, U Kabelovny 130, 10237 Prague 10, Czech Republic.
E-mail: vladimir.kral@vscht.cz
w Electronic supplementary information (ESI) available: Syntheses of
compounds 7–9 and details of binding studies. See DOI: 10.1039/
b718492a
Scheme 1 Synthetic strategy for the preparation of receptors 7–9.
Chem. Commun., 2008, 1901–1903 | 1901
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Fig. 1 Single-crystal X-ray structure of methinium salt 7.
modification of the basic skeleton did not lead to the desired
properties; the salt 8 showed very strong precipitation in aqu-
eous medium, probably due to self-aggregation of individual
molecules by means of p–p stacking and ion pairing.
For generation of a receptor with potential to be applied
under physiological conditions, we appended triethyleneglycol
chains to the benzothiazolium unit. Thus we have prepared
salt 9 (Scheme 1), which was sufficiently soluble in aqueous
medium and showed strong interactions with sulfate anion,
higly selective over other anions, including chloride, fluoride
and nitrate. The salt 9 was studied in a mixture of water–PEG
(n = 9) where PEG was added as a cosolvent.16 The notable
sulfate selectivity led us to use our novel receptor for the
colorimetric sensing of biopolymers with sulfate moieties. We
chose heparin as a model substrate. We have found that
compound 9 showed significant spectral changes in the pre-
sence of heparin (hypsochromic shift from 663 to 485 nm)
(Fig. 3).
Fig. 3 Titration of 9 (c = 4.6 ꢀ 10ꢂ6 M) with heparin in a solution of
85% H2O–15% PEG (n = 9), 1 mM phosphate buffer pH = 5.53; no.
of equivalents are from 0 to 100.
of synthetic ligands to biological receptors. The advantage of
our colorimetic molecular probe 9, is, that can recognize the
unique molecular signatures of cancer cells (localized on the
surface, or on the subcellular structure)—sulfonated sacchar-
ides with very high selectivity and efficiency (based on very
high binding constant under physiological conditions, see
Table 1).
We have observed relatively fast into-cell transport of probe
9 for mammary gland tumor tissue cell culture; and because of
high lysosome levels of polysulfated polysaccharides17 for this
cancer cell line, we used these cells as a model for an
UV-Vis study with other anionic polysaccharides, such as
pectin, polygalacturonic acid and also 2-sulfoglucosamine
showed, in comparison to heparin, relatively weak interactions
(Fig. 4). K values for 9 with a series of anionic polysaccharides
are summarized in Table 1.
The next question was whether our receptor 9 could be
applied to the in vivo detection of sulfonated biopolymers,
namely cancer markers.
The main obstacle for molecular recognition of cancer
biomarkers and/or cancer cells is the relatively weak binding
Fig. 4 Titration curves of the salt 9 with acid saccharides. Concentra-
tion of the salt 9 = 4.6 ꢀ 10ꢂ6 M in a solution of 85% H2O–15% PEG
(n = 9), pH = 5.53, 1 mM phosphate buffer, wavelength = 663 nm.
Table 1 Log K values (error o15%) and stoichiometry of salt 9 at
various pH values
Analyte : 9
stoichiometry pH 5.53 pH 6.22 pH 7.35
Analyte
Heparin
1 : 2
1 : 3
10.7
18.4
4.8
10.7
18.1
3.6
10.8
17.8
4
Polygalacturonic acid 1 : 1
Fig. 2 Selectivity study of salt 7 for simple anions. Concentration of 7
= 2.2 ꢀ 10ꢂ6 M in a solution of 68% H2O–30% MeOH–2% DMSO,
pH = 5.53, medium = 1 mM phosphate buffer, wavelength = 663 nm.
Sulfate anion
2 : 2
1 : 2
14.7
11.7
11.3
7.8
13.5
3.8
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reflections, 6262 with I 4 2s(I), R(all) = 0.0308, R(I 4 2s(I)) =
0.0274, wR(all) = 0.0666, wR(I 4 2s(I)) = 0.0645. CCDC 671040.
For crystallographic data in CIF or other electronic format see DOI:
10.1039/b718492a
1 P. Piatek, V. M. Lynch and J. L. Sessler, J. Am. Chem. Soc., 2004,
126, 16073.
2 N. J. Singh, E. J. Jun, K. Chellappan, D. Thangadurai, R. P.
Chandran, I. C. Hwang, J. Yoon and K. S. Kim, Org. Lett., 2007,
9, 485.
Fig. 5 Partial co-localization of salt 8 and 9, compared with staining
3 F. P. Schmidtchen, Top. Curr. Chem., 2005, 255, 1.
4 Anion Receptor Chemistry, ed. J. L. Sessler, P. Gale and A. W. S.
Cho, Royal Society of Chemistry, Cambridge, 2006.
5 Supramolecular Chemistry of Anions, ed. A. Bianchi, K. B. James
and E. G. Espana, Wiley-VCH, Weinheim, 1997.
6 J. F. Callan, A. Prasann and D. C. Magri, Tetrahedron, 2005, 61,
8551–8588.
7 E. A. Katayev, N. V. Boev, V. N. Khrustalev, Y. A. Ustynyuk, I.
G. Tananaev and J. L. Sessler, J. Org. Chem., 2007, 72, 2886.
8 S. Shrestha, L. E. Salin, C. M. Ensor and S. Daaunert, Biotechnol.
Bioeng., 2002, 78, 517.
9 A. T. Wright, Z. Zhong and E. V. Anslyn, Angew. Chem., Int. Ed.,
2005, 44, 5679.
10 Z. Zhong and E. V. Anslyn, J. Am. Chem. Soc., 2002, 124, 9014.
11 W. Sun, H. Bandmann and T. A. Schrader, Chem.–Eur. J., 2007,
13, 7701.
with commercial lysosome specific probe LysoTracker Green.
intracellular distribution study.18 Indeed, lysosome localiza-
tion of the polymethinium salt 9 was observed. Intracellular
lysosome imaging is summarized on Fig. 5.
In conclusion, we have developed highly selective sulfate
receptors (based on a substituted polymethine scaffold) and
demonstrated their application as optical sensors for heparin.
These colorimetric sensors can recognize heparin over other
acidic polysaccharides such as pectin and polygalacturonic
acid in the presence of high phosphate concentration, as well
as other anions present under physiological conditions. In-
tracellular localization of novel receptors opens the possibility
for imaging of highly sulfonated compartments as has been
demonstrated on lysosome imaging for cancer cells.
This work was supported by grants from MSMT of Czech
Republic-MSMT 1M 6837805002, LC 512, KAN200200651,
MSM0021620857 and Grant Agency of the Czech Republic
No. 203/06/1038.
¨
12 T. D. Slavnova, A. K. Chibisov and H. Gorner, J. Phys. Chem. A,
2002, 106, 10985.
13 M. K. Pal and J. K. Ghosh, J. Photochem. Photobiol., A, 1994, 78,
31.
14 R. Bean, W. Shepherd, R. Kay and E. Walwick, J. Phys. Chem.,
1965, 69, 4368.
15 T. Brı
´
za, Z. Kejık, P. Vasek, J. Kralova, P. Martasek, I. Cısarova
´ ´ ´ ´ ´ ´
l, Org. Lett., 2005, 17, 3661–3664.
and I. V. Kra
´
16 P. Chaudhari, P. Sharma, N. Barhate, P. Kulkarni, C. Mistry and
D. Y. Padmashree, Curr. Sci., 2007, 92, 1586.
17 B. J. Burbach, A. Friedl, C. Mundhenke and A. C. Rapraeger,
Matrix Biol., 2003, 22, 163.
Notes and references
18 4T1 cells were sequentially loaded with 1 mM TB-30 and then with
500 nM LysoTracker Green (Molecular Probes). Staining with
methinium salt (violet) was monitored in regular light and green
fluorescence of LysoTracker using a bandpass filter BP450-490 for
excitation and a long pass filter LP 515 for emission.
z Crystal data for 7: C31H30IN3O2S2, M = 667.60, triclinic, space
ꢀ
group P1, a = 10.1420(2), b = 10.3469(2), c = 15.9680(3) A, a =
95.4235(13), b = 108.3581(9), g = 108.4221(11)1, V = 1473.42(5) A3,
Z = 2, Dc = 1.505 g cmꢂ3, m = 1.261 mmꢂ1, T = 150(0) K, 6771
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Chem. Commun., 2008, 1901–1903 | 1903