A colorimetric sensing ensemble for heparin

Article abstract of DOI:10.1021/ja020505k

A receptor possessing ammoniums and a novel amino acid with a boronic acid side chain was designed and synthesized. The receptor shows good affinity and selectivity for heparin over similar polysaccharides possessing lower anionic charge density. The affi

Full text of DOI:10.1021/ja020505k

Published on Web 07/11/2002
A Colorimetric Sensing Ensemble for Heparin
Zhenlin Zhong and Eric V. Anslyn*
Department of Chemistry and Biochemistry, The UniVersity of Texas at Austin, Austin, Texas 78712
Received April 8, 2002
Scheme 1. Synthesis of 1a
Anticoagulation therapy is used during cardiopulmonary bypass
surgery to prevent thrombosis of the extracorporeal circuit and to
mediate activation of the hemostatic system.1 Heparin is the agent
of choice because it is reversible, well-tolerated, inexpensive, and
very effective. During surgery, it is commonly monitored using a
process which entails determining the activated clotting time
(ACT).2 Typically, the ACT is measured by a physical means, such
as mass deposition, plunger movement, or viscokinetic changes.
More recently, some chemical-based sensing schemes have emerged.
One measures heparin indirectly by monitoring its inhibition of
thrombin activity on a flourogenic substrate,3 while another uses a
potentiometric signal generated from deposition of heparin into a
membrane.4 Although these assays are already in practice or
undergoing clinical testing, a simple single-step colorimetric method
could be useful and competitive with these other methods. Herein,
we report the use of a designed receptor, possessing a novel amino
acid with a boronic acid side chain, that when used in an indicator
displacement assay5 shows strong binding to heparin with good
selectivity over glycosaminoglycans with lower anionic charge
density.
a Conditions: (a) n-BuLi, THF, -98 °C, (b) B(OMe)3, -98 °C to rt, (c)
neopentyl glycol, toluene, reflux, 88%, (d) NBS, AIBN, CCl4, reflux, (e)
dimethylamine, ether, 78% (two steps), (f) 500 psi H2, Raney-Ni, MeOH/
NH3, quantitative, (g) N-Fmoc-Asp(O-t-Bu)-OH, DCC/HOBt, CH2Cl2, 77%,
(h) TFA, CH2Cl2/thioanisole, 93%, (i) 1,3,5-triaminomethyl-2,4,6-triethyl-
benzene, PyBOP, DIEA, DMF, (j) piperidine, DMF, 68%.
plays a dominant role in heparin-protein interactions.7 Boronic
acids rapidly and reversibly form cyclic esters with diols in aqueous
media and are widely used for binding and sensing of saccharides.8
A peptide possessing both cationic and boronic acid binding sites
should be a potential receptor for heparin and other oligomeric
anionic saccharides. Thus, we designed and synthesized 1, which
contains ammoniums and an amino acid with a boronic acid side
chain (Scheme 1). The hexasubstitution of the benzene in 1 enforces
adjacent groups alternating up and down,9 creating a preorganized
cavity for binding interactions.10
Indicator displacement assays are used to convert synthetic
receptors into sensors without requiring the covalent introduction
of a reporter moiety.5 With these systems, analyte binding leads to
indicator displacement from the binding cavity, which in turn yields
an optical signal modulation. By using pyrocatechol violet as an
indicator, the binding and sensing ability of 1 with heparin sodium
salt (HEP), chondroitin 4-sulfate sodium salt (ChS), hyaluronic acid
sodium salt (HA), and heparin disaccharide I-S was studied using
UV-vis spectroscopy in a 1:1 (v/v) water/methanol solution
buffered with 10 mM HEPES at pH 7.4. The binding of pyrocat-
echol violet with 1 causes a decrease around 430 nm and an increase
around 526 nm in its absorbance spectra with an isosbestic point
at 472 nm, and a color change from yellow to grayish purple.
Computer fitting of the titration data with a typical 1:1 binding
algorithm gave a binding constant of 7.1 × 103 M-1 11
. As shown
Heparin is a highly negatively charged oligosaccharide mainly
composed of repeating disaccharide units of 1f4 linked sulfated
iduronic acid and sulfated glucosamine.6 Anion-cation interaction
in Figure 1A, addition of heparin to a solution of 1 and the indicator
causes an absorbance increase around 430 nm and a decrease around
526 nm, with a color change back to yellow, revealing that the
indicator was displaced from the binding cavity of host 1 by the
analyte. A control experiment showed that direct addition of heparin
* To whom correspondence should be addressed. E-mail: anslyn@
ccwf.cc.utexas.edu.
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J. AM. CHEM. SOC. 2002, 124, 9014-9015
10.1021/ja020505k CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
was added to a solution of 2 and alizarin complexone. Therefore,
2 has far lower affinity than receptor 1 toward heparin.
In conclusion, a designed receptor possessing ammoniums and
a novel boronic acid-containing amino acid shows good affinity
and selectivity for heparin over similar polysaccharides possessing
lower anionic charge density. The affinity for heparin is similar to
that for a heparin disaccharide, indicating that disaccharidic units
are the likely sites for binding of 1. We are currently exploring the
use of the colorimetric indicator displacement method in serum.
Acknowledgment. We gratefully acknowledge support from the
NIH and the Beckman Center for Array Sensors. We also thank
Dr. Laura Keissling for first suggesting to us that heparin is an
attractive target for sensing applications.
Supporting Information Available: Experimental details for
syntheses and characterization of 1 and 2, absorbance spectral change
of PV upon addition of 1, and plots for determination of binding
constants (PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) (a) Despotis, G. J.; Gravlee, G.; Filos, K.; Levy, J. Anesthesiology 1999,
91, 1122-1151. (b) Hirsh, J.; Dalen, J.; Deykin, D.; Poller, L. Chest 1992,
102, 337S-351S.
(2) Gravlee, G. P.; Case, L. D.; Angert, K. C.; Rogers, A. T.; Miller, G. S.
Anesth. Analg. 1988, 67, 469-472.
(3) (a) Mitchell, G. A.; Gargiulo, R. J.; Husbeby, R. M.; Lawson, D. E.;
Pochron, S. P.; Schugner, J. A. Thromb. Res. 1978, 13, 47-52. (b) Umlas,
J.; Taff, R. H.; Gauvin, G.; Sweirk, P. Anesth. Analg. 1983, 62, 1095-
1099.
(4) (a) Mathison, S.; Bakker, E. Anal. Chem. 1999, 71, 4614-4621. (b) Ma,
S.-C.; Yang, V. C.; Fu, B.; Meyerhoff, M. E. Anal. Chem. 1993, 65, 2078-
2084.
(5) (a) Wiskur, S. L.; Ait-Haddou, H.; Lavigne, J. J.; Anslyn, E. V. Acc. Chem.
Res. 2001, 34, 963-972. (b) Ait-Haddou, H.; Wiskur, S. L.; Lynch, V.
M.; Anslyn, E. V. J. Am. Chem. Soc. 2001, 123, 11296-11297. (c) Wiskur,
S. L.; Anslyn, E. V. J. Am. Chem. Soc. 2001, 123, 10109-10110. (d)
Metzger, A.; Anslyn, E. V. Angew. Chem., Int. Ed. 1998, 37, 649-652.
(e) Niikura, K.; Metzger, A.; Anslyn, E. V. J. Am. Chem. Soc. 1998, 120,
8533-8534.
(6) Casu, B. In AdVances in Carbohydrate Chemistry and Biochemistry;
Tipson, R. S., Horton, D., Eds.; Academic Press: Orlando, 1985; Vol. 43,
pp 51-134.
(7) (a) Mulloy, B.; Linhard, R. J. Curr. Opin. Struct. Biol. 2001, 11, 623-
628. (b) Hileman, R. E.; Fromm, J. R.; Weiler, J. M.; Linhardt, R. J.
Bioessays 1998, 20, 156-167. (c) Sasisekharan, R.; Venkataraman, G.
Curr. Opin. Chem. Biol. 2000, 4, 626-631.
Figure 1. (A) Absorbance spectra of a 0.054 mM PV and 0.16 mM 1
solution in 1:1 H2O/MeOH buffered with 10 mM HEPES at pH 7.4 in the
presence of 0-1.75 mM HEP. (B) Absorbance change at 526 nm of the
PV and 1 solution upon addition of different analytes.
Table 1. Binding Constants between 1 and Anionic Analytesa,b
-1
analyte (as sodium salts)
binding constant (M )
heparin
chondroitin 4-sulfate
hyaluronic acid
3.8 × 104
6.4 × 103
c
heparin disaccharide I-S
6.2 × 103
a Measured by competitive spectrophotometry11 in 1:1 (v/v) water/
methanol buffered with 10 mM HEPES at pH 7.4. b Concentration of
glycosaminoglycans refers to their disaccharide units. c Spectral change was
too small to estimate a binding constant.
(8) (a) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 1910-1922. (b) Sugasaki, A.; Sugiyasu, K.; Ikeda,
M.; Takeuchi, M.; Shinkai, S. J. Am. Chem. Soc. 2001, 123, 10239-
10244. (c) Cabell, L. A.; Monahan, M.-K.; Anslyn, E. V. Tetrahedron
Lett. 1999, 40, 7753-7756. (d) Takeuchi, M.; Ikeda, M.; Sugasaki, A.;
Shinkai, S. Acc. Chem. Res. 2001, 34, 865-873. (e) Mizuno, T.;
Fukumatsu, T.; Takeuchi, M.; Shinkai, S. J. Chem. Soc., Perkin Trans. 1
2000, 407-413. (f) Kobayashi, H.; Nakashima, K.; Ohshima, E.; Hisaeda,
Y.; Hamachi, I.; Shinkai, S. J. Chem. Soc., Perkin Trans. 2 2000, 997-
1002. (g) Ikeda, M.; Shinkai, S.; Osuka, A. Chem. Commun. 2000, 1047-
1048. (h) James, T. D.; Shinkai, S. In Topics in Current Chemistry;
Penade´s, S., Ed.; Springer-Verlag: Berlin, Heidelberg, New York, 2002;
Vol. 218, pp 159-200.
(9) (a) Iverson, D. J.; Hunter, G.; Blount, J. F.; Damewood, J. R.; Mislow,
K. J. Am. Chem. Soc. 1981, 103, 6073-6083. (b) Kilway, K. V.; Siegel,
J. S. J. Am. Chem. Soc. 1992, 114, 255-261. (c) Gottlieb, H. E.; Ben-
Ary, C.; Hassner, A.; Marks, V. Tetrahedron 1999, 55, 4003-4014.
(10) (a) Jon, S. Y.; Kim, J.; Kim, M.; Park, S.-H.; Jeon, W. S.; Heo, J.; Kim,
K. Angew. Chem., Int. Ed. 2001, 40, 2116-2119. (b) Kim, S.-G.; Ahn,
K. H. Chem. Eur. J. 2000, 6, 3399-3403. (c) Chin, J.; Walsdorff, C.;
Stranix, B.; Oh, J.; Chung, H. J.; Park, S.-M.; Kim, K. Angew. Chem.,
Int. Ed. 1999, 38, 2756-2758. (d) Tam-Chang, S.-W.; Stehouwer, J. S.;
Hao, J. J. Org. Chem. 1999, 64, 334-335. (e) Hartshorn, C. M.; Steel, P.
J. Chem. Commun. 1997, 541-542. (f) Stack, T. D. P.; Hou, Z.; Raymond,
K. N. J. Am. Chem. Soc. 1993, 115, 6466-6467. (g) Weizman, H.;
Libman, J.; Shanzer, A. J. Am. Chem. Soc. 1998, 120, 2188-2189. (h)
Rathore, R.; Lineman, S. V.; Kochi, J. K. J. Am. Chem. Soc. 1997, 119,
9393-9404. (i) Walsdorff, C.; Saak, W.; Pohl, S. J. Chem. Soc., Dalton
Trans. 1997, 1857-1861. (j) Szabo, T.; O’Leary, B. M.; Rebek, J., Jr.
Angew. Chem., Int. Ed. 1998, 37, 3410-3413. (k) Hoskins, B. F.; Robson,
R.; Slizys, D. A. Angew. Chem., Int. Ed. Engl. 1997, 36, 2752-2755.
(11) Conners, K. A. Binding Constants, The Measurement of Molecular
Complex Stability; John Wiley and Sons: New York, 1987.
to the indicator does not change the absorbance spectrum at all.
The binding constant (3.8 × 104 M-1) between 1 and heparin was
measured by competitive spectrophotometric method.11 Addition
of chondroitin or hyaluronic acid also decreases the absorbance
around 526 nm, but the saturated absorbance changes are only about
60 and 10%, respectively, of that in the case of heparin (Figure
1B), and the binding constants are lower (Table 1). These binding
constants are not for a discrete 1:1 stoichiometry, since undoubtedly
more than 1 equiv of 1 can associate with each heparin strand. To
measure a binding constant relevant to a 1:1 stoichiometry, we
examined heparin disaccharide I-S. The disaccharide has a high
affinity also, indicating that there is only a modest enhancement
of the affinity due to polymerization.
The selectivity shown in Table 1 is related to the anionic charge
density of the glycosaminoglycan analytes, that is, HEP > ChS >
HA, suggesting that electrostatic interactions play a dominant role
in the binding. To prove that the boronic acid groups in receptor 1
do play a role in the binding, compound 2 lacking the boronic acid
groups was synthesized and used as a control. No significant change
was observed on the spectrum of pyrocatechol violet upon the
addition of 2. Instead, alizarin complexone was used as an indicator,
which gave a small change on the absorbance spectrum upon
binding with 2. However, no displacement took place when heparin
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