Nitroso compounds and their use as spin traps

Details Nitroso compounds and their use as spin traps Nitroso compounds and their use as spin traps

2001-12-11

2004-07-27

Ceperley, Mary E. (Department: 1641)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C436S116000, C436S119000, C436S127000, C436S173000, C562S058000

Reexamination Certificate

active

06767715

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to novel nitroso compounds and to their use as spin traps.
BACKGROUND OF THE INVENTION
It is now widely accepted that Reactive Oxygen (RO) and Reactive Nitrogen (RN) species (including free radicals) are involved in the pathogenesis of several disease states. The only technique which can detect low concentrations of radicals directly is electron spin resonance (ESR) spectroscopy. Although this technique is highly sensitive (thresholds of 10
−7
-10
−6
M spins), it is not directly applicable to the study of biological oxidations. A more useful method, permitting ESR investigation of short-lived reactive free radicals by transforming them into more persistent species, is the so-called “spin trapping” method. The technique of spin trapping makes use of a diamagnetic compound in which all the electrons are in pairs. The diamagnetic compound, known as the spin trap, reacts with a free radical (R′) which contains an unpaired electron with spin. Reaction of the spin trap with a reactive free radical, results in the formation of a relatively stable, ESR-observable spin adduct. In favourable cases, the free radical, R′, can be identified from the ESR parameters of the spin adduct (e.g. hyperfine coupling constants, g-factor).
This technique was established by Jansen et al, JACS (1968) 90:5909-10. Since then, there has been much research on the synthesis of suitable spin traps. The spin traps that have been most commonly employed are those designed so that, on reaction with a free radical, a nitroxide is formed.
A commercially available spin trap is 3,5-dibromo-4-nitrosobenzenesulphonate, sodium salt (DBNBS). See Kaur et al, JCS Chem. Comm. (1981) 142-3. DBNBS is a water-soluble aromatic C-nitroso spin trap which has been reported to trap the sulphite radical anion (SO
3
−
), superoxide, alkyl, nitric oxide and selenite anion radicals. DBNBS has also been reported to detect an oxidising species in uremic plasma, as it is oxidized to its radical cation DBNBS
+
which is then detected by ESR spectroscopy. See Roselaar et al, Kidney International (1995) 48:199-206, and WO-A-92/18874.
Reist et al, FEBS Lett. (1998) 423:231-4, indicates that sulphite is toxic to the lung and can cause allergic reactions such as bronchoconstriction in asthmatics. The toxic effects of sulphite in combination with peroxynitrite neuronal cells are reported by Reist et al, J. Nephrology (1998) 71:2431-8. An effective spin trap for sulphite is therefore of great potential value.
Matsuo et al, Free Radical Biology and Medicine (1998) 25:929-35, reports a very sensitive ‘ELISA-ESR’ method for Hepatitis B surface (HBs) antigen detection, using 4-hydrazonomethyl-1-hydroxy-2,2,5,5-tetramethyl-3-imidazoline-3-oxide (HHTIO) as the spin trap. In this method, beads are coated with the first HBs-antibody, and a second HBs-antibody is labelled with horseradish peroxidase (HRP). In the presence of HBs antigen, the HRP-labelled antibody will be linked to the antigen and the antigen linked to the beads. After washing, the antigen-antibody complex is added to a solution containing p-acetamidophenol (p-AP), hydrogen peroxide and HHTIO. The p-AP is converted into phenoxy radicals by the action of HRP in the presence of hydrogen peroxide, and the phenoxy radical is trapped by HHTIO to form a stable nitroxide radical which can be detected by ESR spectroscopy.
SUMMARY OF THE INVENTION
Novel compounds according to this invention are 3,5-dichloro-4-nitrosobenzenesulphonate (DCNBS) and 3,5-dimethyl-4-nitrosobenzenesulphonate (DMNBS) and salts thereof.
DCNBS at least has several advantages over DBNBS. These include improved solubility of the spin trap in aqueous systems and narrower ESR signals (which result in a greater signal
oise ratio).
DCNBS has been used successfully to detect an oxidant in the dialysate of patients with renal failure (the oxidant oxidized DCNBS to its radical cation DCNBS
+
which was subsequently detected by ESR spectroscopy).
DCNBS will trap nitric oxide. It is also a potential spin trap for alkyl free radicals, superoxide and the selenite radical anion (SeO
3
−
).
DCNBS has been found to be a more sensitive spin trap than DBNBS for the sulphite radical anion (SO
3
−
). DMNBS is also superior to DBNBS or its isotopic analogues (
15
N and d
2
), or DCNBS. DMNBS was shown to give an ESR signal for the SO
3
−
adduct of more than 20 times that obtained with DBNBS.
Accordingly, DMNBS as a spin trap for the sulphite radical anion has enormous potential. In addition, DMNBS may be used as a detector molecule for the presence of antibody-peroxidase complexes in ELISAs with ESR detection.


REFERENCES:
patent: WO 92/18874 (1992-10-01), None
patent: WO 02 31495 (2002-04-01), None
Roselaar et al., “Detection of oxidants in uremic plasma by electron spin resonance spectroscopy”, Kidney International, vol. 48, 1995, pp. 199-206.
Ozawa et al. “Spin-Trapping Of Sulfite Radical Anion, SO3., By A Water-Soluble, Nitroso-Aromatic Spin-Trap”, Biochemical and Biophysical Research Communication, vol. 142, No. 2, 1987, pp. 410-416.
Matsuo et al., “Highly Sensitive Hepatitis B Surface Antigen Detection By Measuring Stable Nitroxide Radical Formation With ESR Spectroscopy”, Free Radical Biology & Medicine, vol. 25, No. 8. pp. 929-935, 1998.
Reist et al., “Sulphite enhances peroxynitrite-dependent œ1-antiproteinase inactivation. A mechanism of lung injury by sulphur dioxide?”, FEBS Letters, 423, 1998, pp. 231-234.
Reist et al., “Toxic Effects of Sulphite in Combination with Peroxynitrite on Neuronal Cells”, Journal of Neurochemistry, vol. 71, No. 6, 1998, pp 2431-2438.
Orrell et al., “Monomer-Dimer Solution Equilibria of 2,4,6-Trialkylnitrosobenzenes and 2,4,6-Trialkylnitrosobenzene/Nitrosobenzene Mixtures. A Study Using One-and Two-dimensional NMR Techniques”, J. Chem. Soc. Perkin Trans., 1990, pp. 1297-1303.
Azoulay et al., “Aromatic C-Nitroso Compounds. Thermodynamics and Kinetics of the Equilibrium between 2,6-Dimethylnitrosobenzene and its trans-Dimer”, J.C.S. Perkin II, pp. 256-259.
Janzen et al., “Two Decades of Spin Trapping”, Advances in Free Radical Chemistry, vol. 1, pp. 253-295.

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