OCTOBER/NOVEMBER 1996 VOLUME 9, NUMBER 7 © Copyright 1996 by the American Chemical Society
Articles The Reaction of 2-(Methylseleno)benzanilide with Peroxynitrite Hiroshi Masumoto and Helmut Sies* Institut fu¨ r Physiologische Chemie I, Heinrich-Heine-Universita¨ t Du¨ sseldorf, Postfach 101007, D-40001 Du¨ sseldorf, Germany Received March 27, 1996X
2-(Methylseleno)benzanilide (MeSe), a main metabolite of ebselen in vivo, rapidly reacts with peroxynitrite, yielding 2-(methylseleninyl)benzanilide (MeSedO) as the sole selenium-containing product. The reaction proceeds efficiently at acidic pH where peroxynitrous acid (ONOOH) is predominant, suggesting that MeSe reacts faster with ONOOH than with the peroxynitrite anion (ONOO-). MeSe is much less reactive with hydrogen peroxide. The second-order rate constant for the reaction at 22 °C was estimated from the relationship between substrate consumption and peroxynitrite concentration as 2.2 × 104 M-1 s-1 and 2.7 × 103 M-1 s-1 at pH 3.9 and 7.0, respectively, and from intermolecular competition with ascorbic acid as 3.4 × 103 M-1 s-1 at pH 7.1. The rate constant is about 2 orders of magnitude lower than that for the reaction of ebselen with peroxynitrite, but about 1-2 orders of magnitude higher than that reported in the literature for methionine or ascorbate. The selenoxide can be reduced back to MeSe by thiols. MeSe has previously been considered to be an inert metabolite of ebselen, but may show antioxidant activity against peroxynitrite.
Introduction Peroxynitrite (ONOO-/ONOOH)1 is a reactive species generated from superoxide anion radical and nitrogen oxide (1-3). Peroxynitrite may be formed in vivo (4-7). The peroxynitrite anion (ONOO-) is relatively stable in * Address for correspondence: Institut fu¨r Physiologische Chemie I, Heinrich-Heine-Universita¨t Du¨sseldorf, Postfach 101007, D-40001 Du¨sseldorf, Germany. Phone, +49-211-81-12707; fax, +49-211-8113029; e-mail,
[email protected]. X Abstract published in Advance ACS Abstracts, August 15, 1996. 1 Note that the recommended IUPAC nomenclature for peroxynitrite is oxoperoxonitrate(1-); for peroxynitrous acid, hydrogen oxoperoxonitrate. The term peroxynitrite is used in the text to refer generically to both oxoperoxonitrate(1-) (ONOO-) and its conjugate acid, hydrogen oxoperoxonitrate (ONOOH). When the discussion is limited to either the anion or the acid, its specific name is used.
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alkaline solution, and its conjugate acid, peroxynitrous acid (ONOOH), with pKa ) 6.8 (8) isomerizes to nitrate with the decay constant of 1.3 s-1 at 25 °C (8). Peroxynitrous acid oxidizes compounds via a metal-independent hydroxyl radical-like mechanism (1, 9-12). Peroxynitrite can undergo a number of reactions of pharmacological and toxicological interest including nitration of phenolics and aromatic amino acids (12-16) and the generation of DNA base damage (17) and is thought to play an important role in inflammatory tissue damage. The recent description of high reactivity of the selenoorganic compound, ebselen2 (2-phenyl-1,2-benzisoselenazol-3(2H)-one), with peroxynitrite (18) has sparked interest in selenium-containing compounds as to their © 1996 American Chemical Society
1058 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
potential function in scavenging peroxynitrite. We have recently determined the second-order rate constant for ebselen in the reaction with peroxynitrite to be 2.0 × 106 M-1 s-1 at pH g8 and 25 °C3 and also have examined the kinetics of the seleno-methylated derivative of ebselen, 2-(methylseleno)benzanilide (MeSe). This methylated derivative reacts less avidly with peroxynitrite, but it reacts still faster than the rate constants reported for small biologically occurring compounds, e.g., ascorbate (19), cysteine (20), methionine (21), or tryptophan (16, 22). As one important aspect for biological efficacy is the maintenance of a steady state, the reducibility of the oxidation product to revert to the parent substrate is an essential feature. Ebselen Se-oxide can be reduced back to ebselen by thiols such as GSH (23, 24). It was of interest to examine the reaction product(s) for the methyl derivative, as also there the potential for a regeneration reaction exists, based on the reduction by GSH (25). In the present work, we address the chemistry in the reaction of MeSe with peroxynitrite to produce 2-(methylseleninyl)benzanilide (MeSedO), and also some competition experiments with ascorbate are presented. MeSe is a main metabolite of ebselen in vivo (26, 27). MeSe is oxidized by NADPH/O2-dependent oxidations mediated by cytochrome P450 (28) and flavin monooxygenase (29), giving MeSedO as a product.
Materials and Methods Reagents. 2-(Methylseleno)benzanilide was a kind gift from Rhoˆne-Poulenc-Nattermann (Cologne, FRG). Desferrioxamine mesylate and DTPA were from Sigma Chemicals (St. Louis, MO). Other chemicals and solvents were from E. Merck (Darmstadt, FRG). Peroxynitrite was synthesized from sodium nitrite and hydrogen peroxide using a quenched-flow reactor as previously described with minor modifications (1, 18). The peroxynitrite solution was treated with MnO2 powder to eliminate residual hydrogen peroxide, and the solution was filtered. Peroxynitrite was concentrated by freeze fractionation at -20 °C. Peroxynitrite concentration was determined with a Perkin Elmer Lambda 2 spectrophotometer (U ¨ berlingen, FRG) at 302 nm (302 nm ) 1670 M-1 cm-1) (30). 2-(Methylseleninyl)benzanilide was synthesized from MeSe as previously described (28). The structure was confirmed by IR, MS, and 1H NMR (500 MHz) spectra and elemental analysis. The iron complex, Fe(III)EDTA, was prepared by mixing solutions of iron sulfate and sodium EDTA in the ratio 1:1. General. Prior to experiments, the concentration of the stock peroxynitrite solution was determined by measuring absorbance at 302 nm of an aliquot of the stock peroxynitrite solution diluted in 0.05 M aqueous NaOH. A small correction was made for the absorbance due to the presence of a contaminant, most likely nitrite, that absorbs at that wavelength. Phosphate buffer is used as a least reactive buffer in terms of accelerating peroxynitrite decomposition (31). Reaction System. Typically, peroxynitrite diluted in distilled water was added to a solution of MeSe in 50 mM sodium phosphate buffer (1.0 mL) while vigorously vortexing to start the reaction at room temperature (22 °C). An incubation time of 5 min assured total peroxynitrite decomposition at neutral and acidic pH, as t1/2 of peroxynitrite is
