270
A.L. Kleschyov et al. / European Journal of Medicinal Chemistry 58 (2012) 265e271
5
0 mU/ml) system (pH 7.4). The chemiluminescence signals were
References
recorded in the presence of 50 mM lucigenin, 1 mM DTPA using
[
1] B.P. Soule, F. Hyodo, K. Matsumoto, N.L. Simone, J.A. Cook, M.C. Krishna,
J.B. Mitchell, The chemistry and biology of nitroxide compounds, Free Radic.
Biol. Med. 42 (2007) 1632e1650.
chemiluminometer Lumat 9507. The chemiluminescence signals
were recorded in the absence or in the presence of increasing
concentrations of 1a18 or 4-hydroxy-TEMPO [16]. The concentra-
tion of the heparin-bound TEMPO groups was calculated from the
[2] K. Matsumoto, H. Yakumaru, M. Narazaki, H. Nakagawa, K. Anzai, H. Ikehira,
N. Ikota, Modification of nitroxyl contrast agents with multiple spins and their
proton T(1) relaxivity, Magn. Reson. Imaging 26 (2008) 117e121.
known content of the TEMPO-modified disaccharides in 1a18
.
[
3] D.I. Potapenko, M.A. Foster, D.J. Lurie, I.A. Kirilyuk, J.M. Hutchison,
I.A. Grigor’ev, E.G. Bagryanskaya, V.V. Khramtsov, Real-time monitoring of
drug-induced changes in the stomach acidity of living rats using improved
pH-sensitive nitroxides and low-field EPR techniques, J. Magn. Reson. 182
5.5. Binding to vascular tissue
(2006) 1e11.
[
[
4] A.A. Bobko, I. Dhimitruka, J.L. Zweier, V.V. Khramtsov, Trityl radicals as
persistent dual function pH and oxygen probes for in vivo electron para-
magnetic resonance spectroscopy and imaging: concept and experiment,
J. Am. Chem. Soc. 129 (2007) 7240e7241.
Rat aortic rings (3 mm long) were incubated in solutions of
different HPNs (10 M or 100 M) for 30, 60, 90, 120 or 180 min
m
m
with following washed out (3 times) with Krebs buffer. EPR spectra
of the rings were recorded at room temperature using an X-band
radiospectrometer MS 200 (Magnettech GmbH, Berlin). Instrument
parameters were 10 mW microwave power, 0.1 mT amplitude
modulation, 100 kHz modulation frequency, sweep field 5 mT and
5] J. Shen, S. Liu, M. Miyake, W. Liu, A. Pritchard, J.P. Kao, G.M. Rosen, Y. Tong,
K.J.
Liu,
Use
of
3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-
pyrrolidinyloxyl as an EPR oximetry probe: potential for in vivo measure-
ment of tissue oxygenation in mouse brain, Magn. Reson. Med. 55 (2006)
1433e1440.
[
[
[
[
6] C.S. Winalski, S. Shortkroff, R.V. Mulkern, E. Schneider, G.M. Rosen, Magnetic
resonance relaxivity of dendrimer-linked nitroxides, Magn. Reson. Med. 48
6
0 s sweep time.
(2002) 965e972.
7] S. Goldstein, A. Samuni, K. Hideg, G. Merenyi, Structure-activity relationship of
cyclic nitroxides as SOD mimics and scavengers of nitrogen dioxide and
carbonate radicals, J. Phys. Chem. A 110 (2006) 3679e3685.
8] V.D. Sen’, V.A. Golubev, Kinetics and mechanism for acid-catalized dispro-
portionation of 2,2,6,6-tetramethylpiperidine-l-oxyl, J. Phys. Org. Chem. 22
(2009) 138e143.
9] G.G. Borisenko, I. Martin, Q. Zhao, A.A. Amoscato, V.E. Kagan, Nitroxides
scavenge myeloperoxidase-catalyzed thiyl radicals in model systems and in
cells, J. Am. Chem. Soc. 126 (2004) 9221e9232.
5.6. MRI studies
1 2
NMR measurements of T and T relaxation time in the heparine
polynitroxide phantom probes were performed using Siemens Tim
Trio 3T scanner (Erlangen, Germany). T measurements were per-
1
formed with the saturation recovery using Spoiled Gradient
Recalled Echo sequence (SGRE). The sequence parameters were:
[10] M.D. Rees, E.C. Kennett, J.M. Whitelock, M.J. Davies, Oxidative damage to
extracellular matrix and its role in human pathologies, Free Radic. Biol. Med.
ꢀ
2
TE
¼
4
ms, flip angle
¼
90 , FOV
¼
120 mm ,
44 (2008) 1973e2001.
Matrix ¼ 128 ꢃ 128 pixel, number of averages ¼ 2. The repetition
[
11] C.S. Wilcox, A. Pearlman, Chemistry and antihypertensive effects of tempol
time TR was varied from 20 to 3000 ms to acquire the relaxation
and other nitroxides, Pharmacol. Rev. 60 (2008) 418e469.
recovery curve. T
2
measurements were performed with the
[12] C. Aliaga, E.A. Lissi, O. Augusto, E. Linares, Kinetics and mechanism of the
reaction of a nitroxide radical (tempol) with a phenolic antioxidant, Free
Radic. Res. 37 (2003) 225e230.
multiple spin-echo (CPMG) sequence using the same spatial reso-
lution parameter settings. The echo train consisted of 15 echoes
with the echo time TE varied from 45 ms with the step 22.5 ms. The
repetition time was TR ¼ 2000 ms and number of averages ¼ 2. MRI
measurements of heparinepolynitroxide pre-treated isolated
porcine blood vessels were performed on Siemens Tim Trio 3T
[13] R.A. Smith, C.M. Porteous, A.M. Gane, M.P. Murphy, Delivery of bioactive
molecules to mitochondria in vivo, Proc. Natl. Acad. Sci. U. S. A. 100 (2003)
5407e5412.
[
14] P. Wipf, J. Xiao, J. Jiang, N.A. Belikova, V.A. Tyurin, M.P. Fink, V.E. Kagan,
Mitochondrial targeting of selective electron scavengers: synthesis and bio-
logical analysis of hemigramicidin-TEMPO conjugates, J. Am. Chem. Soc. 127
(2005) 12460e12461.
1
scanner using a standard extremity (knee) coil. The T -weighted
images were acquired using the spin-echo sequence with TR varied
[
[
15] J.C. Stoclet, E. Troncy, B. Muller, C. Brua, A.L. Kleschyov, Molecular mechanisms
underlying the role of nitric oxide in the cardiovascular system, Expert Opin.
Investig. Drugs 7 (1998) 1769e1779.
16] T. Munzel, I.B. Afanas’ev, A.L. Kleschyov, D.G. Harrison, Detection of super-
oxide in vascular tissue, Arterioscler. Thromb. Vasc. Biol. 22 (2002) 1761e
from 30 to 500 ms. Other imaging parameters were TE ¼ 16 ms,
2
matrix size 256 ꢃ 256, slice thickness 1 mm, FOV ¼ 58 mm . The
MRI measurements have demonstrated that the increased relax-
ivity could be used for the signal enhancement of the blood vessel
1768.
[17] D.D. Heistad, Endothelial function in the time of the giants, J. Cardiovasc.
wall (T
1
-weighting effect). In these experiments, the maximal
Pharmacol. 52 (2008) 385e392.
[
[
[
18] P. Kuppusamy, P. Wang, R.A. Shankar, L. Ma, C.E. Trimble, C.J. Hsia, J.L. Zweier,
In vivo topical EPR spectroscopy and imaging of nitroxide free radicals and
polynitroxylealbumin, Magn. Reson. Med. 40 (1998) 806e811.
19] P.W. Buehler, C.R. Haney, A. Gulati, L. Ma, C.J. Hsia, Polynitroxyl hemoglobin:
a pharmacokinetic study of covalently bound nitroxides to hemoglobin plat-
forms, Free Radic. Biol. Med. 37 (2004) 124e135.
contrast-to-noise enhancement factor approached 3.5.
5.7. In vivo EPR
20] R.J. Linhardt, 2003 Claude S. Hudson award address in carbohydrate
chemistry. Heparin: structure and activity, J. Med. Chem. 46 (2003) 2551e
Mice were anesthetized with ketamine/xylazyne and heparine
polynitroxide 1b45 (1.5 mMe0.5 ml) were injected intraperitone-
ally; the mouse tail was fixed in the resonator of an X-band EPR
spectrometer (MS 200 Magnettech) and the spectra EPR were
sequentially recorded as described previously [26].
2
564.
[21] K. Na, S. Kim, K. Park, K. Kim, D.G. Woo, I.C. Kwon, H.M. Chung, K.H. Park,
Heparin/poly(l-lysine) nanoparticle-coated polymeric microspheres for stem-
cell therapy, J. Am. Chem. Soc. 129 (2007) 5788e5789.
[
22] O. Gemma, D. Meyer, A.N. Uhrin, Hulme, enabling methodology for the end
functionalization of glycosaminoglycan oligosaccharides, Mol. Biosyst.
2008) 481e495.
4
(
Acknowledgments
[23] T.D. Bjornsson, D.E. Schneider, A.R. Hecht, Effects of N-deacetylation and N-
desulfation of heparin on its anticoagulant activity and in vivo disposition,
J. Pharmacol. Exp. Ther. 245 (1988) 804e808.
This work was partially supported by grants from BMWi
[
24] U. Landmesser, S. Spiekermann, S. Dikalov, H. Tatge, R. Wilke, C. Kohler,
D.G. Harrison, B. Hornig, H. Drexler, Vascular oxidative stress and endo-
thelial dysfunction in patients with chronic heart failure: role of xanthine-
oxidase and extracellular superoxide dismutase, Circulation 106 (2002)
3073e3078.
(
KF2445701AK9) and DFG (SCHR 687/2).
Appendix A. Supplementary data
[
25] Y. Chu, R. Piper, S. Richardson, Y. Watanabe, P. Patel, D.D. Heistad, Endocytosis
of extracellular superoxide dismutase into endothelial cells: role of the
heparin-binding domain, Arterioscler. Thromb. Vasc. Biol. 26 (2006) 1985e
1990.