Reactions of Nitric Oxide, Peroxynitrite, and Carbonate Radicals with

Jan 23, 2004 - Department of Physical Chemistry, The Hebrew University of Jerusalem, ... Department of Molecular Biology, Hebrew UniversitysHadassah ...
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Chem. Res. Toxicol. 2004, 17, 250-257

Reactions of Nitric Oxide, Peroxynitrite, and Carbonate Radicals with Nitroxides and Their Corresponding Oxoammonium Cations Sara Goldstein,*,† Amram Samuni,‡ and Gabor Merenyi§ Department of Physical Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Department of Molecular Biology, Hebrew UniversitysHadassah Medical School, P.O. Box 12000, Jerusalem 91120, Israel, and Department of Chemistry, Nuclear Chemistry, The Royal Institute of Technology, S-10044 Stockholm 70, Sweden Received November 17, 2003

Cyclic nitroxides effectively protect biological systems against radical-induced damage. However, the mechanism of the reactions of nitroxides with nitrogen-derived reactive species and carbonate radicals is far from being elucidated. In the present study, the reactions of several representative piperidine- and pyrrolidine-based nitroxides with •NO, peroxynitrite, and CO3•were investigated, and the results are as follows: (i) There is no evidence for any direct reaction between the nitroxides and the •NO. In the presence of oxygen, the nitroxides are readily oxidized by •NO2, which is formed as an intermediate during autoxidation of •NO. (ii) •NO reacts with the oxoammonium cations to form nitrite and the corresponding nitroxides with k1 ) (9.8 ( 0.2) × 103 and (3.7 ( 0.1) × 105 M-1 s-1 for the oxoammonium cations derived from 2,2,6,6-tetramethylpiperidine-1-oxyl (TPO) and 3-carbamoyl-proxyl (3-CP), respectively. (iii) CO3•- oxidizes all nitroxides tested to their oxoammonium cations with similar rate constants of (4.0 ( 0.5) × 108 M-1 s-1, which are about 3-4 times higher than those determined for H-abstraction from the corresponding hydroxylamines TPO-H and 4-OH-TPO-H. (iv) Peroxynitrite ion does not react directly with the nitroxides but rather with their oxoammonium cations with k10 ) (6.0 ( 0.9) × 106 and (2.7 ( 0.9) × 106 M-1 s-1 for TPO+ and 3-CP+, respectively. These results provide a better insight into the complex mechanism of the reaction of peroxynitrite with nitroxides, which has been a controversial subject. The small effect of relatively low concentrations of nitroxides on the decomposition rate of peroxynitrite is attributed to their ability to scavenge efficiently •NO2 radicals, which are formed during the decomposition of peroxynitrite in the absence and in the presence of CO2. The oxoammonium cations, thus formed, are readily reduced back to the nitroxides by ONOO-, while forming •NO and O . Hence, nitroxides act as true catalysts in diverting peroxynitrite decomposition 2 from forming nitrating species to producing nitrosating ones.

Introduction Cyclic nitroxides have been used for decades as biophysical probes to monitor membrane dynamics, cellular pH, metabolism, and O2 levels (1-3). Later, their potential use as contrast agents for in vivo nuclear magnetic resonance imaging (MRI) (4-7) and electron paramagnetic resonance imaging (8, 9) was investigated. Their persistence in tissues and in the circulation (6, 10) and their susceptibility to enzymatic and nonenzymatic reduction (11) attracted much attention. Since their SOD mimetic activity (12) and antioxidative activity in general (13) as well as their catalytic effect in selective oxidation reactions (14, 15) have been recognized, research was directed at the mechanisms underlying their reaction with oxygen- and nitrogen-derived species. The kinetics and mechanisms of the reactions of nitroxides with nitrogen-derived reactive species are far * To whom correspondence should be addressed. Tel: 972-26586478. Fax: 972-2-6586925. E-mail: [email protected]. † The Hebrew University of Jerusalem. ‡ Hebrew UniversitysHadassah Medical School. § The Royal Institute of Technology.

from being elucidated. It has been shown that cyclic nitroxides, such as 3-carbamoyl-2,2,5,5-tetramethyl-3pyrroline-1-yloxy and 2,2,6,6-tetramethylpiperidine-1oxyl (TPO), do not react with •NO in deaerated solutions (16), and this could be the case for all simple nitroxides. In contrast, •NO reacts rapidly with nitronyl nitroxides, such as 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide, to form the corresponding imino nitroxides and • NO2 (17-19). Aromatic nitroxides were also reported to react with •NO, but in this case, the products and their yields under anoxia differed from those found in the presence of O2 (20, 21). There is also no agreement on the mechanism of the reaction of nitroxides with peroxynitrite (ONOO-/ONOOH), a reaction that diverts peroxynitrite decomposition from forming nitrating species to producing nitrosating ones (22-25). It has been shown that 4-OH-TPO does not react directly with peroxynitrite (24). However, 4-OH-TPO was shown to be a potent catalytic inhibitor of phenol nitration, while it stimulates phenol nitrosation by peroxynitrite in the presence of CO2 (23, 24). The reaction of ONOO- with CO2 forms about 33% •NO2 and CO3•- (26, 27), and nitration of phenols takes place via the reaction of CO3•- with phenols to form

10.1021/tx0342363 CCC: $27.50 © 2004 American Chemical Society Published on Web 01/23/2004

Reactions with Nitroxides and Their Oxoammonium Cations

the phenoxyl radicals, which competitively dimerize to biphenol or react with •NO2 to yield nitrophenol (28, 29). Carroll et al. (23) proposed that •NO2 reacts with 4-OHTPO to form an intermediate, which could react with the phenoxyl radical to form the nitrosophenol and regenerate the nitroxide. Bonini et al. (24) assumed that the reaction of •NO2 with 4-OH-TPO does not take place due to the relatively low reduction potential of the •NO2/NO2couple [1.04 V (30)]. They proposed that CO3•- is scavenged by 4-OH-TPO, rather than by the phenol, to form the oxoammonium cation. The oxoammonium cation is then reduced back to the nitroxide while oxidizing ONOO- to oxygen and •NO, the latter reacting with • NO2 to produce the nitrosating N2O3 species (24). Our recent pulse radiolysis study (31) demonstrates that • NO2 rapidly oxidizes 4-OH-TPO as well as other piperidine- and pyrrolidine-based nitroxides to the corresponding oxoammonium cations. The rate constants of these reactions are comparable to those of CO3•- reacting with 4-oxo-TPO (32) and 4-OH-TPO (33), i.e., (4-8) × 108 M-1 s-1. The products of the reactions of CO3•- with nitroxides have not yet been identified, although it is reasonable to assume that the radical oxidizes the nitroxides to the corresponding oxoammonium cations. In the present study, we investigated the reactions of •NO and peroxynitrite with piperidine- and pyrrolidine-based nitroxides and their corresponding oxoammonium cations. In addition, using the pulse radiolysis technique, we studied the reaction of CO3•- with the same nitroxides. Our results further elucidate the complex mechanism of the reaction of nitroxides with peroxynitrite and nitric oxide.

Experimental Section Materials. All chemicals were of analytical grade and were used as received. Water for solution preparations was distilled and purified using a Milli-Q purification system. The nitroxides TPO, 4-OH-TPO, 4-carboxy-TPO, and 3-carbamoyl-proxyl (3CP) were purchased from Aldrich, and 4-oxo-TPO was purchased from Alexis Biochemicals. The hydroxylamines 4-OH-TPO-H and TPO-H were prepared by catalytic reduction using H2 bubbled over Pt powder or by bubbling HCl gas through an ethanolic solution of the nitroxide followed by drying. Nitric oxide (C.P.) was purchased from Matheson Gas Products and was purified by passing it through a series of scrubbing bottles containing deaerated 50% NaOH and purified water in this order. Nitric oxide solutions were prepared in gastight syringes, and the solubility of •NO in water was taken to be 1.9 mM/atm (34). The oxoammonium cations were prepared using an electrochemical reactor as previously described (35), and their yields were determined using ferrocyanide, which was immediately oxidized to ferricyanide (420 ) 1000 M-1 cm-1). 3-CP+, TPO+, and 4-carboxy-TPO+ were prepared in aerated solutions containing 0.2-1 mM nitroxide in 4-10 mM PB at pH 6.8, whereas 4-OH-TPO+ and 4-oxo-TPO+ were unstable at neutral pH and therefore were prepared in 0.1 M HClO4. The yields of electrooxidation of the nitroxides to the corresponding oxoammonium cations exceeded 95%. These cations were stable for at least 2 h under the specified conditions except for 4-oxo-TPO+, whose half-life at pH 1 was ca. 10 min as compared to 0.3 s at pH 7.2. Peroxynitrite was synthesized through the reaction of nitrite with acidified H2O2 using a quenched flow with a computerized syringe pump (World Precision Instruments model SP 230IW), as described elsewhere (36). Briefly, 0.63 M H2O2 in 0.7 M HClO4 was mixed with 0.60 M nitrite and the mixture was quenched with 3 M NaOH at room temperature. The stock solutions contained about 0.13 M peroxynitrite, 4% residual nitrite, and 13% residual H2O2. The concentration of peroxynitrite was determined from its absorption at 302 nm using  ) 1670 M-1

Chem. Res. Toxicol., Vol. 17, No. 2, 2004 251 cm-1 (37) and that of H2O2 by its reaction with Fe2+ in 0.8 N H2SO4 to yield Fe3+ and using 302(Fe3+) ) 2197 M-1 cm-1. Methods. Stopped-flow kinetic measurements were carried out using the Bio SX-17MV Sequential Stopped-Flow from Applied Photophysics with a 1 cm optical path. Peroxynitrite in 0.01 M NaOH was mixed with the oxoammonium cation in PB at a 1:1 volume ratio. In another set of experiments, equal volumes of acidified nitroxide and nitrite were mixed. The reaction of •NO with the oxoammonium cations was studied by mixing equal volumes of the reactants in PB, whereas that of •NO with nitroxides was studied by mixing the reactants at a 1:5 volume ratio. Anaerobic experiments were carried out while flushing the syringe cups with inert gas. The formation and/or decay of the oxoammonium cations were followed at 280-295 nm, where the difference, ∆, between the absorptions of the oxoammonium cation and the corresponding nitroxide is relatively high, with ∆280 ) 800 M-1 cm-1 for 3-CP, ∆290 ) 510 M-1 cm-1 for TPO, ∆295 ) 250 M-1 cm-1 for 4-carboxy-TPO, ∆290 ) 235 M-1 cm-1 for 4-OH-TPO, and ∆290 ) 370 M-1 cm-1 for 4-oxo-TPO. The final pH in each experiment was measured at the outlet of the stopped-flow. All experiments were carried out at 25 °C. Each value given is an average of at least four measurements. When ∆OD was lower than 0.01, an average of five shots was taken for each measurement. Pulse radiolysis experiments were carried out with a Varian 7715 linear accelerator with 5-MeV electron pulses of 0.5 µs duration and a current of 200 mA. A 200 W Xe-Hg lamp produced the analyzing light. An appropriate cutoff filter was used to eliminate photochemistry and stray light. All measurements were made at room temperature in a 4 cm spectrosil cell using three light passes (apparent optical path length, 12.1 cm). The yield of CO3•- was determined from its absorption using 600 ) 1860 M-1 cm-1 (38). EPR spectra were recorded on a JEOL X band JES-RE3X operating at 9.5 GHz with the center field set at 3362 G (100 kHz modulation frequency, 1 G modulation amplitude, and 4 mW incident microwave power). Samples were drawn into a gas permeable Teflon capillary, with an inner diameter of 0.8 mm. The capillary was inserted into a quartz tube, open at both ends, and the tube was then placed within the EPR spectrometer cavity. The nitroxide concentration was calculated from the EPR signal intensity, using standard solutions of the nitroxide.

Results •

Reaction of NO with Oxoammonium Cation. The reaction of excess •NO with 3-CP+, TPO+, or 4-carboxyTPO+ was studied in the absence of oxygen and in the presence of 4 mM PB (pH 6.8 ( 0.1). No effect on the rate was observed upon increasing the concentration of PB to 50 mM. The decay of the oxoammonium cations was first-order, and k1 was determined from the dependence of kobs on [•NO] (Figure 1). We assume that the reaction of the oxoammonium cation with •NO forms the nitroxide and nitrite (equilibrium 1) k1

RN+dO + •NO + H2O y\ z RNO• + HNO2 + H+ k -1

K1 ) k1/k-1 (1) and that the rate of reaction -1 is extremely slow at pH 6.8. Hence, the values of k1 are obtained from the slopes of the lines in Figure 1 and are summarized in Table 1. Equilibrium 1 implies the formation of the oxoammonium cations through the reaction of the nitroxide with nitrous acid at low pH. Indeed, the formation of an absorption spectrum with a maximum around 290 nm was observed upon mixing equal volumes of deaerated solutions of 5 mM nitrite with the nitroxide in 0.02-0.2 M HClO4, which was attributed to the formation of the correspond-

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Table 1. Summary of Rate Constants Determined and Used in the Present Studya TPO (M-1 s-1)

k1 K1 (M) E° (V) (calcd)b E° (V) (literature) k2 (M-1 s-1) (31) k-2 (M-1 s-1) (31) k10 (M-1 s-1) k14 (M-1 s-1) a

4-OH-TPO

(9.8 ( 0.2) × 0.045 ( 0.005 0.74 0.75 (45), 0.744 (60) (7.1 ( 0.2) × 108 9 × 103 (6.0 ( 0.9) × 106 (4.0 ( 0.5) × 108 103

ND 1.8 ( 0.3 0.84 0.84 (31), 0.833 (60) (8.7 ( 0.2) × 108 (2.7 ( 0.4) × 105 ND (4.0 ( 0.5) × 108

4-carboxy-TPO (7.9 ( 0.3) × 0.8 ( 0.2 0.81 0.796 (60) ND ND ND ND

103

4-oxo-TPO

3-CP

ND 107 ( 13 0.94 0.93 (31), 0.917 (61) (7.1 ( 0.2) × 108 (1.1 ( 0.2) × 107 ND (4.8 ( 0.5) × 108 (32)

(3.7 ( 0.1) × 105 12.2 ( 1.4 0.88 0.89 (45) (7.1 ( 0.2) × 108 (1.5 ( 0.2) × 106 (2.7 ( 0.9) × 106 (4.0 ( 0.5) × 108

ND, not determined. b Calculated using K1 and E°(HNO2, H+/•NO, H2O) ) 0.82 V. Table 2. Yield of the Oxoammonium Cation Formed through the Reaction of 2.5 mM Nitrite with Various Concentrations of the Corresponding Nitroxide in Acidified Deaerated Solutions [H+]o [RNO•]o [R+NdO]o K-1 ) [RNO•][HNO2][H+]/ (M) (mM) (µM) [R+NdO][•NO] (M)a 3-CP

Figure 1. Reaction of oxoammonium cation with •NO. The decay of the oxoammonium cation was followed at 281-295 nm upon mixing equal volumes of 0.22-1.7 mM •NO in 4 mM PB with deaerated solutions containing 38-130 µM nitroxide and 4 mM PB. The final pH was 6.8 ( 0.1.

ing oxoammonium cation. The yields of the oxoammonium cations as a function of [nitroxide]o or [H+]o and the experimental values for K1 are given in Table 2. Hence, ∆E1° ) E°(RN+dO/RNO•) - E°(HNO2, H+/•NO, H2O) ) (RT/F)lnK1, and E°(RN+dO/RNO•) was calculated using K1 and E°(HNO2, H+/•NO, H2O) ) 0.82 V, which was obtained from the known accurate values of ∆fG° ) (•NO) ) 24.4 kcal/mol, ∆fG°(H2O) ) -56.7 kcal/mol, and ∆fG°(HNO2) ) -13.3 kcal/mol (39). The calculated values of E°(RN+dO/RNO•) are summarized in Table 1. The agreement of these values with the literature reduction potentials (Table 1) supports the assumption of the establishment of equilibrium 1, irrespective of the detailed mechanism of the oxidation of the nitroxide by nitrous acid. Reaction of •NO with Nitroxides. It was previously found that TPO does not react with •NO in deaerated solutions (16), but its reaction with •NO in the presence of oxygen has not yet been studied. The oxidation of •NO by oxygen produces •NO2 and N2O3 as oxidizing and nitrosating intermediates (40-42). We have recently shown that •NO2 rapidly oxidizes various nitroxides to the corresponding oxoammonium cations (eq 2; Table 1) (31). k2

RNO• + •NO2 y\ z RN+dO + NO2- K2 ) k2/k-2 k -2

(2)

Therefore, the reaction of •NO with O2 in the presence of nitroxides should form the corresponding oxoammonium cations, which are readily reduced by •NO and by nitrite. Indeed, a transient absorption at 290 nm was observed when oxygen-saturated solutions of 3-CP or TPO were mixed with 0.57 mM •NO at a 5:1 volume ratio to yield 1 mM O2 reacting with 95 µM •NO in the presence of 0.22-2 mM nitroxide at pH 6.8 (10 mM PB). A typical

0.01 0.01 0.01 0.01 0.1 0.1 0.1 0.1 0.1 TPO 0.01 0.01 0.01 0.01 0.01 0.1 0.1 4-carboxy-TPO 0.01 0.01 0.01 0.01 0.01 0.1 0.1 4-OH-TPO 0.05 0.05 0.05 0.05 4-oxo-TPO 0.05 0.05 0.05 0.05

4.8 2.4 1.2 0.6 3.7 1.85 0.92 0.46 0.23 2.22 1.11 0.56 0.28 0.17 1.33 0.67 5.07 2.54 1.27 0.634 0.317 1.27 0.634 5.0 2.5 2.5 2.5 2.5 2.5 2.5 2.5

77 55 40 28 250 180 130 89 61 637 471 318 196 126 1033 582 286 208 150 107 74 484 323 485 382 255 162 73 53 36 28

13.6 13.4 12.6 12.8 12.1 11.6 10.8 11.0 10.8 5.0 × 10-2 4.1 × 10-2 3.7 × 10-2 3.6 × 10-2 4.2 × 10-2 4.0 × 10-2 4.9 × 10-2 0.91 0.87 0.82 0.96 0.76 0.66 0.63 1.93 1.54 1.72 2.06 112 107 115 94

a The concentrations of [H+] and [HNO ] were calculated using 2 pKa(HNO2) ) 3.15, [R+NdO] ) [•NO], and [RNO•] ) [RNO•]o [R+NdO].

kinetic trace monitored in the presence of 2 mM 3-CP is shown in Figure 2. Ferrocyanide reduces oxoammonium cations with a rate constant approaching the diffusioncontrolled limit (31), whereas the rate constant for the reaction of •NO2 with ferrocyanide is merely 3 × 106 M-1 s-1 (43); i.e., lower than that for •NO2 reacting with TPO or 3-CP by more than 2 orders of magnitude (31). Therefore, we added 0.15 mM ferrocyanide to the reaction mixture in order to scavenge all of the oxoammonium cations formed. The formation of ferricyanide was followed at 420 nm and was found to be second-order with kobs ) (5.3 ( 0.5) × 103 M-1 s-1. The formation rate was independent of the type of nitroxide or its concentration. A typical kinetic trace recorded in the presence of 2 mM 3-CP and 0.15 mM ferrocyanide is shown in the inset of Figure 2. The yield of ferricyanide in the presence of 0.22-2 mM 3-CP or TPO approached 90% (∆[Fe(CN)63-]/ [•NO]o ≈ 0.9). The reaction was also studied under limiting concentrations of O2. Aerated solutions containing 1.2 mM

Reactions with Nitroxides and Their Oxoammonium Cations

Figure 2. Autoxidation of •NO in the presence of 3-CP. Oxygenated solution of 2.4 mM 3-CP in 10 mM PB was mixed with 0.57 mM •NO at a 5:1 volume ratio to yield final concentrations of 1 mM O2, 95 µM •NO, and 2 mM 3-CP at pH 6.8. The formation and decay of the absorption were followed at 290 nm. Inset: The kinetic trace obtained at 420 nm upon the addition of 0.15 mM ferrocyanide (∆OD420 ) 0.083; kf ) 4.9 × 103 M-1 s-1).

ferrocyanide and 3-CP or TPO were mixed at a 1:5 volume ratio with •NO-saturated solutions to yield 1.43 mM •NO reacting with 40 µM O2 in the presence of 0.14.8 mM nitroxide and 0.2 mM ferrocyanide at pH 6.7 (10 mM PB). No spectral changes in the absence of oxygen or ferrocyanide were observed at 290 nm. The yield of ferricyanide measured in the presence of 4.5 mM 3-CP or TPO was 72 ( 5 µM, i.e., ∆[Fe(CN)63-]/[O2]o ) 1.8 ( 0.1. The rate of formation of ferricyanide was first-order with kobs ) 5.1 ( 0.4 s-1, independent of the type of nitroxide added or its concentration. The rate-determining step of the oxidation of •NO by oxygen is the formation of •NO2, which can be described by reactions 3-5 and by rate eq 6 (40-42):

2•NO + O2 f 2•NO2 k3 ) (2-2.9) × 106 M-2 s-1 (40-42) (3) •

NO + •NO2 h N2O3

(4)

N2O3 + H2O f 2NO2- + 2H

(5)

•

-

d[O2] 1 d[ NO] )) k3 [•NO]2[O2] 4 dt dt

(6)

The oxidation of nitroxides by •NO2 forms the corresponding oxoammonium cations (reaction 2), which decay via reactions 1 and -2. However, in the presence of ferrocyanide, which scavenges efficiently the oxoammonium cations, the whole process is described by reactions 7 and 8 and rate eq 9.

2•NO + O2 + 2RNO• f 2RN+dO + 2NO2-

(7)

RN+dO + Fe(CN)64- f RNO• + Fe(CN)63- fast (8) •

-

+

d[O2] 1 d[RN dO] 1 d[ NO] )) ) k3[•NO]2[O2] 2 dt dt 2 dt (9)

Under limiting concentrations of •NO, each mole of •NO yields 1 mol of ferricyanide, the formation of ferricyanide is a second-order process, and kobs ) 2k3[O2]. Indeed, the

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measured yield of ferricyanide approached 90%, and from the value of kobs ) (5.3 ( 0.4) × 103 M-1 s-1 in the presence of 1 mM O2, we calculated k3 ) (2.7 ( 0.2) × 106 M-2 s-1. Under limiting concentration of O2, each mole of O2 yields 2 mol of ferricyanide at infinite concentrations of the nitroxide, the rate of formation of ferricyanide being first-order and kobs ) k3[•NO]2. We measured kobs ) 5.1 ( 0.4 s-1 in the presence of 1.43 mM • NO and hence calculated k3 ) (2.5 ( 0.2) × 106 M-2 s-1. These results demonstrate that during autoxidation of • NO the nitroxides are oxidized to the corresponding oxoammonium cations, being unstable in the presence of nitrite and •NO. They can, however, be trapped by appropriate reductants such as ferrocyanide. In addition, the formation of •NO2 was rate limiting under limiting concentrations of •NO and in the presence of 2 mM nitroxide, which implies that this rate is at least 10 times faster than the rate of •NO reacting with the nitroxide. It then follows that the rate constant for a direct reaction between •NO and TPO or 3-CP cannot exceed ca. 10 M-1 s-1. Reaction of Peroxynitrite with Nitroxides. The reaction of peroxynitrite with TPO, 4-OH-TPO, 4-oxoTPO, and 3-CP was studied at pH 6.9 using the stoppedflow. Deaerated solutions of 0.02-1 mM nitroxide in 0.2 M PB were mixed at a 1:1 volume ratio with 0.36-0.41 mM peroxynitrite in 10 mM NaOH. The decay of peroxynitrite, which was followed at 302 nm, was first-order with kd ) 0.49 ( 0.01 s-1. This rate constant increased in the presence of each of the nitroxides tested to 0.65 ( 0.02 s-1, independent of the nitroxide concentration when in the range of 10-500 µM. The addition of 7.5 mM nitrite had no effect on the observed rate constant in the presence of the nitroxides, whereas in their absence it decreased by ca. 10%, i.e. to kd ) 0.44 ( 0.01 s-1. These results demonstrate that nitroxides do not react directly with peroxynitrite at neutral pH. Reaction of Peroxynitrite with the Oxoammonium Cation. The reaction of peroxynitrite with excess 3-CP+ or TPO+ was studied using the stopped-flow. Equal volumes of 15-40 µM peroxynitrite in 10 mM NaOH were mixed with 150-700 µM 3-CP+ or TPO+ in 0.5 M phosphate or 50 mM acetate buffer, and the decay of the absorption at 290 nm was followed. The reaction at pH > 5.2 was too fast for a kinetic study using this technique, and at pH < 4.1, it became too slow to compete efficiently with the self-decomposition of ONOOH, the latter reaction having kd ) 1.2 ( 0.1 s-1 (44). The absorption decayed via a fast first-order reaction, and kobs increased linearly with [RN+dO] at a constant pH (Figure 3) and decreased as pH decreased at a constant [RN+dO]. The second-order rate constant at each pH was obtained from the dependence of kobs on [RN+dO]. The results, which are summarized in Table 3, demonstrate that ONOO-, rather than its conjugate acid ONOOH, reduced the oxoammonium cation (reaction 10).

RN+dO + ONOO- f RNO• + •NO + O2

(10)

This finding is in keeping with results previously reported, where the reactivity of H2O2 toward oxoammonium cations was scrutinized (45). Similarly, HO2- was the reactive species, while the conjugate acid, H2O2, proved inert. Using pKa(ONOOH) ) 6.6 (44), we calculated k10 ) (6.0 ( 0.9) × 106 and (2.7 ( 0.9) × 106 M-1 s-1 for TPO+ and 3-CP+, respectively (Table 3). Using

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Figure 3. Observed first-order rate constant of the reaction of peroxynitrite with TPO+ at pH 5.0 or with 3-CP+ at pH 5.2. Peroxynitrite (22-34 µM) in 10 mM NaOH was mixed with either 3-CP+ (136-634 µM) or TPO+ (78-603 µM) in 0.5 M PB at a 1:1 volume ratio, and the absorption was followed at 282 or 290 nm, respectively. Table 3. Reaction of Peroxynitrite with Excess of TPO+ or 3-CP+ in the Presence of 0.25 M Phosphate (a) or 50 mM Acetate (b) Buffers pH

kapp (M-1 s-1)

k(ONOO- + RN+dO) (M-1 s-1)

4.2 (a) 4.6 (a) 5.0 (a) 5.2 (a) 5.0 (b) 4.7 (a) 4.3 (b) 4.2 (a)

(2.5 ( 0.1) × 104 (5.0 ( 0.4) × 104 (1.6 ( 0.1) × 105 (8.8 ( 0.2) × 104 (7.3 ( 0.2) × 104 (4.2 ( 0.3) × 104 (1.8 ( 0.1) × 104 (1.0 ( 0.1) × 104

(6.3 ( 0.3) × 106 (5.1 ( 0.3) × 106 (6.3 ( 0.7) × 106 (2.2 ( 0.1) × 106 (3.0 ( 0.1) × 106 (3.3 ( 0.3) × 106 (3.6 ( 0.1) × 106 (2.5 ( 0.3) × 106

RN+dO TPO+ TPO+ TPO+ 3-CP+ 3-CP+ 3-CP+ 3-CP+ 3-CP+

290(ONOOH) ) 250 M-1 cm-1 (46), ∆290(3-CP+ - 3-CP) ) 720 M-1 cm-1, and ∆290(TPO+ - TPO) ) 510 M-1 cm-1, we determined ∆[3-CP+]/[peroxynitrite]o ) 1.7-1.8 and ∆[TPO+]/[peroxynitrite]o ) 0.9-1.0. These results imply that •NO, which is formed via reaction 10, is readily oxidized by 3-CP+. On the other hand, oxidation of •NO by TPO+ is relatively slow and, therefore, does not compete efficiently with other reactions of •NO. Reaction of CO3•- with Nitroxides. Upon irradiating N2O-saturated aqueous solutions containing 0.1-0.5 M sodium carbonate (pH 9-10.7), the carbonate radical was generated via the following reactions (the radiation chemical yields of the species are given in parentheses):

Figure 4. Reaction of CO3•- with nitroxides and hydroxylamines. The decay of CO3•- was followed at 600 nm upon pulse irradiation of N2O-saturated solutions containing 0.1-0.5 M carbonate at pH 10.5-10.7 in the presence of various concentrations of TPO (O), 4-OH-TPO (0), 3-CP (2), 4-oxo-TPO (X), TPO-H (b), or 4-OH-TPO-H (9).

similar to that of 3-CP+, which at neutral pH was stable for at least 2 h. To verify the formation of 3-CP+ in the reaction of CO3•- with 3-CP, we used the stopped-flow technique to study the decay of 90 µM 3-CP+ in 0.1 M carbonate buffer at pH 9.7-10.9. The decay was firstorder, and kobs was linearly dependent on [OH-] resulting in kOH ) (7.0 ( 0.3) × 105 M-1 s-1 (results not shown), which at pH 10.5 corresponds to kobs ) 221 ( 10 s-1. This value is in agreement with kobs ) 193 ( 12 s-1 that was measured for the decay of a transient formed via the reaction of CO3•- with 3-CP at pH 10.5. In the case of TPO, both the spectrum and the reactivity of TPO+ remained unaffected for at least 20 min upon increasing the pH from 6.8 to 10.2. Hence, a sample of N2Osaturated solution containing 0.5 mM TPO and 0.1 M carbonate at pH 10.6 was repetitively pulsed (total dose of 680 Gy) and scanned for the absorption change with the diode array spectrophotometer. The spectrum of the accumulated product corresponded to that of TPO+, and its yield was within experimental error identical to that of CO3•- formed by the radiation. Therefore, we conclude that CO3•- oxidized the nitroxide to the corresponding oxoammonium cation, and from the slope of the lines in Figure 4, we determined k14 ) (4.0 ( 0.5) × 108 M-1 s-1 for the four nitroxides tested.

RNO• + CO3•- f RN+dO + CO32-

γ

H2O 98 eaq-(2.6), •OH (2.7), H• (0.6),

(14)

+

H3O (2.6), H2O2 (0.72) (11) eaq- + N2O f N2 + OH- + •OH

(12)

OH + CO32- f CO3•- + OH-

(13)

•

The decay of 8-9 µM CO3•- in 0.1 M carbonate at pH 10.6 was followed at 600 nm (600 ) 1860 M-1 cm-1) and was second-order with 2k ) (2.6 ( 0.3) × 107 M-1 s-1. In the presence of excess nitroxide, this decay turned from second-order into a fast first-order reaction, and kobs increased linearly with increasing [nitroxide] (Figure 4). The four nitroxides tested showed similar reactivity toward CO3•- (Figure 4). In the case of 3-CP, the formation of a transient absorption with a maximum at 280 nm was observed, ∆280 ) 740 ( 20 M-1 cm-1, which at pH 10.5 decayed in a first-order reaction with kobs ) 193 ( 12 s-1. The spectrum of the transient species was

The rate constants for the reaction of CO3•- with TPO-H and 4-OH-TPO-H were also determined. The EPR spectrum and its intensity verified the formation of the nitroxides via these reactions, and from the slopes of the lines in Figure 4, we determined k14a ) (1.2 ( 0.1) × 108 and (1.7 ( 0.1) × 108 M-1 s-1 for TPO-H and 4-OH-TPO, respectively.

CO3•- + RNO-H f HCO3- + RNO•

(14a)

These results demonstrate that both cyclic nitroxides and their respective hydroxylamines are efficient scavengers of carbonate radicals.

Discussion The present results demonstrate that pyrrolidine- and piperidine-based nitroxides do not react directly with

Reactions with Nitroxides and Their Oxoammonium Cations

Chem. Res. Toxicol., Vol. 17, No. 2, 2004 255

•

NO or peroxynitrite at physiological pH. The small effect of the addition of low concentrations of nitroxides on the decomposition rate of peroxynitrite is attributable to their ability to scavenge efficiently •NO2 radicals that are formed during the homolysis of ONOOH. While •OH reacts rapidly and at comparable rates with ONOO-, NO2-, and each of the nitroxides tested, •NO2 radicals are efficiently scavenged by the nitroxides forming the corresponding oxoammonium cations. The latter are reduced back to the nitroxides by ONOO- (reaction 10), whereas •NO mainly reacts with •NO2 to form N2O3, which hydrolyzes fast into nitrite. Hence, the nitroxides act as true catalysts (24), as is illustrated in Scheme 1. The effect of the nitroxides on the decay rate of peroxynitrite is minor, but in their presence, more nitrite is formed at the expense of nitrate, as was previously observed at pH e 7.4 (24). We showed that cyclic nitroxides are oxidized by CO3•and •NO2 to the corresponding oxoammonium cations with similar rate constants (Table 1). This finding provides a better explanation for the inhibitory effect of nitroxides on the nitration of phenols (PhOH) by peroxynitrite (22-24). The reaction of peroxynitrite with excess CO2 in the presence of phenols at pH 7.5 proceeds via reactions 15-20, where the rate constant for the reaction of ONOO- with CO2 has been previously determined to be 3 × 104 M-1 s-1 and the radical yield is ca. 33% (26, 27, 47): -

•

•-

ONOO + CO2 f NO2 + CO3

k15 ) 1 × 104 M-1 s-1 (15) ONOO- + CO2 f NO3- + CO2 4

k16 ) 2 × 10 M

-1 -1

s

(16)

PhOH + CO3•- f PhO• + HCO3k17 ) (2-5) × 107 M-1 s-1 (17) PhOH + •NO2 f PhO• + NO2- + H+ k18 ) 3 × 105 M-1 s-1 (18) 2PhO• f diphenol 2k19 ) 5 × 108 M-1 s-1 (19) •

NO2 + PhO• f nitrophenol k20 ) (1-3) × 109 M-1 s-1 (20)

The rate constants for reactions 17-20 are estimations based on literature data available for different phenols (29, 43, 48-51). According to this mechanism, the nitration yield of phenols is expected to approach the radical yield, i.e., ca. 33%, provided that the nitration efficiency of reaction 20 is 100% and that the concentration of the phenol is sufficiently low for it not to compete efficiently with PhO• for •NO2. In the case of tyrosine, the yield of 3-nitrotyrosine formed in reaction 20 is about 45%; therefore, the nitration yield was found to be ca. 15% (29). Nitroxides inhibit the formation of nitrophenol because they rapidly react with •NO2 (Table 1) and, therefore, efficiently compete with PhO• for •NO2. The oxoammonium cation, thus formed (reaction 2), oxidizes ONOO- to •NO (reaction 10), and the latter reacts rapidly

Scheme 1

with the phenoxyl radical to form nitrosophenol (reaction 21). •

NO + PhO• f nitrosophenol k21 ) (1-2) × 109 M-1 s-1 (52) (21)

However, the oxoammonium cations are also expected to oxidize phenolate ions (reaction 22), as has been shown in the case of tyrosine (53):

RN+dO + PhO- f RNO• + PhO•

(22)

Judging by our preliminary results on tyrosine, k22 is expected to be close to the diffusion-controlled limit. Hence, even if the neutral phenol were to be unreactive, and in view of the pKa of most phenols being close to 10, the effective rate constant of RN+dO for the oxidation of phenols at pH 7.5 should be above 106 M-1 s-1. Consequently, nitroxides act as true catalysts in diverting peroxynitrite decomposition from forming nitrating species to producing nitrosating ones, all in agreement with literature results (23, 24). However, the oxidation of phenol to phenoxyl radical by the oxoammonium cation suggests that the inhibition of the formation of nitrophenol is accompanied not only by the formation of nitrosophenol but also by the formation of biphenol. The maximum nitrosation yield is, therefore, highly dependent on the experimental conditions, namely, the formation of biphenol increases at the expense of nitrosophenol upon increasing the ratio [PhO-]/[ONOO-]. We note that when the 4-position of the phenol is blocked, as is the case for tyrosine, the addition of •NO to the 2-position of the phenoxyl radical does not take place (54, 55). In this case, •NO is expected to react with • NO2 to produce N2O3, which can nitrosate a nucleophile (reaction 23) in a direct competition with its hydrolysis to nitrite (reaction 5).

N2O3 + PhO- f nitrosophenol + NO2-

(23)

The nitrosation yield through this pathway (reactions 4, 5, and 23) is expected to be relatively low at physiological pH, because the pKa of the phenolic OH group is around 10 and because of the fast hydrolysis of N2O3, which is catalyzed by HPO42- and HCO3- (k5 ) 2 × 103 + 1.9 ×

256

Chem. Res. Toxicol., Vol. 17, No. 2, 2004

106[HCO3-] + 8 × 105[HPO42-] s-1) (41, 56, 57). Hence, under physiological conditions, the addition of nitroxides inhibits the formation of 3-nitrotyrosine, whereas it increases the formation of dityrosine by peroxynitrite. It has already been shown that the yield of nitration of tyrosine by authentic peroxynitrite in the presence of CO2 is remarkably reduced when peroxynitrite is formed via continuous and equal generation of •NO and O2•- (29, 58). In addition, the yield of dityrosine is enhanced and approaches ca. 33% (29, 58). This was shown mainly to be due to efficient scavenging of •NO2 by tyrosine, and reaction 24 was shown to affect the product yields only when O2•- was generated in excess of •NO (29).

O2•- + TyrO• f products k24 ) 1.5 × 109 M-1 s-1 (50, 59) (24) Hence, under continuous and equal generation of •NO and O2•-, the addition of even 1 µM nitroxide is expected to inhibit the formation of 3-nitrotyrosine, simply because even such low concentrations of nitroxides are sufficient to compete efficiently with TyrO• for •NO2. In the case where the phenoxyl radical reacts with •NO to form nitrosophenol (reaction 21), reaction 25 becomes important upon continuous and equal generation of •NO and O2•-.

O2•- + PhO• f products k25 ) (0.5 - 3.0) × 109 M-1 s-1 (50, 59) (25) Consequently, the main product is expected to be nitrosophenol, and its yield will not be affected by the addition of nitroxides.

Conclusions Several representative piperidine- and pyrrolidinebased nitroxides do not react directly with •NO or peroxynitrite at neutral pH, but they are oxidized by • NO2, the latter being formed as an intermediate during autoxidation of •NO or during the decomposition of peroxynitrite in the absence and presence of CO2. The oxoammonium cations thus formed are reduced back to the nitroxides by •NO or ONOO- with the simultaneous formation of nitrite or •NO and O2. These results rationalize the mechanism underlying the catalytic effect of nitroxides in diverting peroxynitrite decomposition from forming nitrating species to producing nitrosating ones.

Acknowledgment. This research was supported by a grant from the Israel Science Foundation of the Israel Academy of Sciences.

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