One-Electron Oxidation of Acetohydroxamic Acid: The Intermediacy of

Mar 22, 2011 - The pharmacological effects of hydroxamate derivatives have been attributed not only to metal chelation or enzyme inhibition but also t...
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One-Electron Oxidation of Acetohydroxamic Acid: The Intermediacy of Nitroxyl and Peroxynitrite Amram Samuni† and Sara Goldstein*,# †

Department of Molecular Biology, Medical School and #Chemistry Institute, The Accelerator Laboratory, The Hebrew University of Jerusalem, Jerusalem 91904, Israel ABSTRACT: The pharmacological effects of hydroxamate derivatives have been attributed not only to metal chelation or enzyme inhibition but also to their ability to serve as nitroxyl (HNO/NO) and nitric oxide (NO) donors. However, the mechanism underlying the formation of these reactive nitrogen species is not clear and requires further elucidation. In the present study, one-electron oxidation of acetohydroxamic acid (aceto-HX) by •OH, •N3, •NO2, CO3•, and O2• radicals was investigated using pulse radiolysis. It is demonstrated that only • OH, •N3, and CO3• radicals attack effectively and selectively the deprotonated form of the hydroxamate moiety, yielding the respective transient nitroxide radical. This nitroxide radical is a weak acid (CH3C(O)NHO•, pKa = 9.1), which decays via a pHdependent second-order reaction, 2k(2CH3C(O)NO•) = (5.6 ( 0.4)  107 M1 s1 (I = 0.002 M), 2k(CH3C(O)NO• þ CH3C(O)NHO•) = (8.3 ( 0.5)  108 M1 s1), and 2k(2CH3C(O)NHO•) = (8.7 ( 1.3)  107 M1 s1. The second-order decomposition of the nitroxide yields transient species, one of which decomposes via a first-order reaction whose rate increases linearly upon increasing [CH3C(O)NHO] or [OH]. One-electron oxidation of aceto-HX under anoxia does not give rise to nitrite even after exposure to O2, indicating that NO is not formed during the decomposition of the nitroxide radical. The presence of oxidants such as Tempol or O2 during CH3C(O)NO• decomposition had no effect on the reaction kinetics. Nevertheless, in the presence of Temopl, which does not react with NO but does with HNO, the formation of the hydroxylamine Tempol-H was observed. In the presence of O2, about 60% of CH3C(O)NO• yields ONOO, indicating that 30% NO is formed in this system. It is concluded that under pulse radiolysis conditions, the transient nitroxide radicals derived from one-electron oxidation of acetoHX decompose bimoleculary via a complex mechanism forming nitroxyl rather than NO.

’ INTRODUCTION Hydroxamate derivatives (RC(O)NR0 OH, HXs) form a class of compounds, which display interesting chemical and biological properties. The pharmacological effects of HXs have been attributed to metal chelation,1,2 enzyme inhibition,3 radical scavenging,4,5 formation of carcinogenic derivatives such as esters of hydroxamic acids,68 and formation of reactive nitrogen-derived species including NO916 and nitroxyl (HNO/NO).15,1720 Part of the toxicity of NO may be associated with its reaction with O2• to form peroxynitrite,13,2123 which has been implicated in various pathophysiological processes including acute and chronic inflammation, sepsis, ischemia-reperfusion, and neurodegenerative disorders.2430 HNO (pKa ≈ 11.4)31 is a unique species with novel and atypical chemistry, which exerts cytotoxicity in several cell types partially via oxidation of DNA and loss of thiols.3235 It has been reported that HNO and NO are formed upon oxidation of HXs either chemically,6,7,19,20,3640 enzymatically,12,38,41 or by the combination of heme proteins and H2O2.10,16,42,43 The formation of NO from hydroxyurea in reaction mixtures containing H2O2 and superoxide dismutase (SOD) or ceruloplasmin has been suggested to be mediated by •OH radicals derived from copper-catalyzed Fenton-like reaction.43 The r 2011 American Chemical Society

formation of HNO and NO from HXs involves the loss of two and three electrons, respectively. One-electron oxidation of HXs to their respective transient nitroxide radicals in aqueous solutions (reaction 1) has been previously shown by electron spin resonance (EPR) spectroscopy.79,19,20,42,4448

The fate of the nitroxide radical is not clear, and several routes have been suggested for its decomposition, as described in Scheme 1, including (i) dismutation of the nitroxide radicals39,49 (reaction 2), forming the parent HX and RC(O)NdO, which hydrolyzes to RC(O)OH and HNO (reaction 3), (ii) unimolecular decomposition of the nitroxide radical to the respective aldehyde and NO (reaction 4),50,51 and (iii) unimolecular decomposition of the nitroxide radical via homolysis along the Received: February 23, 2011 Revised: March 9, 2011 Published: March 22, 2011 3022 | J. Phys. Chem. A 2011, 115, 3022–3028

The Journal of Physical Chemistry A Scheme 1. Possible Routes for the Decomposition of the Nitroxide Radical Formed via One-Electron Oxidation of RC(O)NHOH

CN bond, yielding the respective acyl radical (RC(O)•) and HNO (reaction 5).6,7,49 Mild oxidants like ferricyanide20 and compound II of heme proteins52 can oxidize the hydroxamate to the respective nitroxide radical and possibly the nitroxide radical to RC(O)NdO as well as HNO to NO. The mechanism is even more complex than described above. For example, the oxidation of C6H5C(O)NHOH yields N,O-dibenzoylhydroxylamine (C6H5C(O)NHOC(O) C6H5),6,37 which can be attributed to the recombination of the acyl radical with the nitroxide radical (reaction 6, shown below) or to the reaction of the acyl nitroso with the deprotonated form of HX (reaction 7, shown below).


AS was prepared in 10 mM NaOH, and the concentration was determined by the absorbance at 248 nm (ε = 8 mM1 cm1). N2O gas was passed through an oxygen trap (OXYTRAP, Alltech Associate Inc.). Analysis. Nitrite formation was assayed using the Griess reagent. Samples irradiated under anoxia require the addition of oxygen to enable nitrite formation via •NO autoxidation if the latter accumulates. Therefore, a relatively small volume of oxygenated water was gently mixed with the irradiated sample and let stand for several minutes before adding the Griess reagent. The absorption at 540 nm was read after 15 min against a nonirradiated sample. A calibration curve was prepared using known concentrations of nitrite. Hydroxylamine concentration was determined using the 8-HQ method.53 To a 1 mL sample was added 0.5 mL of 1% 8-HQ in methanol and 0.5 mL of 1 M Na2CO3. After 5 min of incubation at 95 °C and cooling, the absorption was read at 700 nm. A calibration curve was prepared using known concentrations of hydroxylamine. Hydroxylamine may be present in hydroxamic acids as a residue formed during synthesis or due to the hydrolysis of hydroxamic acid itself. Therefore, solutions of aceto-HX were prepared daily and analyzed for any hydroxylamine contaminants, which never exceeded 0.11%. The pH was adjusted using HClO4, phosphate buffer, borate buffer, or NaOH. EPR Measurements. EPR spectra were recorded using a JEOL X band JES-RE3X spectrometer operating at 9.5 GHz. Samples were injected into a flexible capillary, which was inserted into a quartz tube placed within the EPR spectrometer cavity. Radiolysis. Pulse radiolysis experiments were carried out using a 5-MeV Varian 7715 linear accelerator (0.11.5 μs electron pulses, 200 mA current). A 200 W Xe lamp produced the analyzing light. Appropriate cutoff filters were used to minimize photochemistry. Measurements were done using a 4 cm spectrosil cell with three light passes. All experiments were done at room temperature. Briefly, irradiation of aqueous solutions produced several species, as shown in eq 8. The numbers in parentheses are G values, which represent their respective yields (in 107 M Gy1), which are about 7% higher in the presence of high solutes concentrations. γ

H2 O f e aq ð2:6Þ, • OH ð2:7Þ, H• ð0:6Þ, H3 Oþ ð2:6Þ, H2 O2 ð0:72Þ

In the present study, acetohydroxamic acid (aceto-HX) has been selected as a simple hydroxamate derivative to be exposed to various one-electron oxidants in well-defined systems in order to elucidate the complex reaction mechanism(s) leading to the formation of HNO and/or NO.

When solutions saturated with N2O ([N2O] = 24 mM) are irradiated at pH > 3, eaq is converted into •OH (reaction 9), and in cases where H• does not react sufficiently fast with any of the added substrates, it produces •OH via reaction 10. e aq þ N2 O þ H2 O f • OH þ N2 þ OH k9 ¼ 9:7  109 M1 s1

Milli-Q system. All chemicals were of the highest available grade and were used as received. Aceto-HX, 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (Tempol), 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS2), 8-hydroxyquinoline (8-HQ) and modified Griess reagent were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.); Angeli’s salt (Na2N2O3, AS) was a product of Cayman Chemical Company; hydroxylamine-hydrochloride was a product of Hopkin & Williams. Stock solution of


H• þ N2 O f • OH þ N2

’ MATERIALS AND METHODS Reagents. Water for solution preparation was purified using a


k10 ¼ 2:1  106 M1 s1


Secondary radicals like •N3, •NO2, or CO3• are formed upon irradiation of N2O-saturated solutions containing a solute (S) such as N3, NO2, or CO32, respectively (reaction 11). •

OH þ S f S• þ OH


When the solutions are saturated with mixtures of N2O/O2 (ratio g 0.8), only H• reacts with O2, forming superoxide radicals. In oxygenated or aerated solutions containing formate 3023 |J. Phys. Chem. A 2011, 115, 3022–3028

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Table 1. Rate Constants (M1 s1) of •OH, •N3, and CO3• Reactions with Aceto-HX at Various pHs pH




(2.7 ( 0.2)  108


(3.4 ( 0.1)  107

7.0 7.8


(6.4 ( 0.2)

(4.5 ( 0.3)  108



(1.8 ( 0.1)  109


(1.5 ( 0.1)  108

9.3 (3.4 ( 0.2)  10

9.5 11 11.2 •

Figure 1. The reaction of aceto-HX with OH. Competition kinetic plots demonstrating the effect of added aceto-HX on the yield of ABTS• in N2O-saturated solutions containing 20 μM ABTS2 at pH 6.7 (8 mM phosphate buffer) and 40 μM ABTS2 at pH 11 followed at 416 nm. The optical path length was 12.1 cm, and the dose was 2.5 Gy. (Inset) The dependence of k12 on pH. The sigmoidal fit resulted in an upper value of (4.0 ( 0.1)  109 M1 s1, a lower value of (1.7 ( 0.1)  108 M1 s1, and pKa = 8.9 ( 0.07.

ions or methanol, all of the primary water-derived radicals are converted into superoxide radicals.54 The knowledge of the rate constants of aceto-HX with eaq and H• formed by radiolysis (eq 8) is essential for designing appropriate experimental conditions. The reaction of eaq with acetoHX was studied directly in a deoxygenated solution containing 0.050.2 M t-butanol by following the decay of eaq at 700 nm yielding ke = (6.4 ( 0.2)  108 and (1.6 ( 0.4)  108 M1 s1 at pH 7.9 and 10.9, respectively. An upper value of 1  107 M1 s1 was estimated using phenol as a competing agent for H• reaction with aceto-HX, that is, up to 10 mM aceto-HX had no effect on the product yield of H• reaction with 0.5 mM phenol (k = 1.7  109 M1 s1)55 at pH 3 in a N2O-saturated solution containing 0.2 M t-butanol.


(4.0 ( 0.1)  109

(2.8 ( 0.1)  108 (3.9 ( 0.1)  109

Figure 2. The absorption spectrum of the transient species observed 10 μs after the pulse in N2O-saturated solutions containing 0.4 mM aceto-HX without (O) and with (9) 10 mM N3 at pH 11. (Inset) The dependence of the initial absorption at 270 and 290 nm on the pH, reflecting a pKa = 9.1. The dose was 18.9 Gy and the optical path 12.1 cm.

ABTS• formation in the absence of aceto-HX.

Oxidation of Aceto-HX by One-Electron Oxidants. •OH

radicals were generated upon irradiation of N2O-saturated solutions at pH 6.713. The rate constant of •OH reaction with aceto-HX (reaction 12) was determined using competition kinetics against 20 or 40 μM ABTS2 (reaction 13). •

290 nm (e.g., Figure 4) indicate that the second-order decay of the nitroxide radical leads to a formation of an intermediate, which subsequently decomposes via a pH-independent first-order reaction (k = 67 ( 14 s1) into another transient absorbing species having a maximum optical absorption at 315 nm (Figure 4, inset). The latter species decayed at 270360 nm via a first-order reaction into nonabsorbing species. The observed first-order rate constant increased upon increasing [OH] or [CH3C(O)NHO] (Figure 5), resulting in kobs = 4.2  103  [CH3C(O)NHO] þ 126  [OH] s1. Nitrite was not detected before and after exposing the irradiated solutions to oxygen, indicating that NO is not formed during the self-decomposition of the nitroxide radical under anoxic conditions. Effect of Tempol. Tempol does not react with NO but does with HNO.59 However, the product of the latter reaction has not been previously identified, and we studied this reaction using the 3025 |J. Phys. Chem. A 2011, 115, 3022–3028

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azide radical was used as an oxidant to avoid any interference from O3• potentially produced at high pH via the reaction of O2 with O• (pKa = 11.7).

’ DISCUSSION One-electron oxidation of aceto-HX leads to the formation of the respective transient nitroxide CH3C(O)NHO• (pKa = 9.1). Under all experimental conditions, the nitroxide radicals decompose bimolecularly. Under anoxic conditions NO2 is not produced even after exposure of the pulsed solution to O2, indicating that NO is not formed during this oxidation process. The lack of nitrite precludes any reaction of NO with HNO (pKa = 11.4),31 which readily yields nitrite via reactions 1619.62 HNO þ • NO f N2 O2  þ Hþ Figure 7. Difference spectra obtained following pulse irradiation of solutions saturated with a mixture of 90% N2O and 10% O2 containing 0.5 mM aceto-HX at pH 11.3. The residual spectra were measured 45 s after delivering one, three, and five pulses, and the latter was also measured 30 min later (red trace). The dose was 20.5 Gy/pulse and the optical path 4 cm.

HNOdonor AS coupled with EPR. We found that the EPR signal of 100 μM Tempol decreased with time upon exposure to 220 or 480 μM AS in aerated solutions containing 40 mM PB (pH 7.1). The EPR signal was fully restored upon the addition of 1 mM ferricyanide, indicating that Tempol is reduced by HNO to its respective EPR-silent hydroxylamine, Tempol-H (reaction 15).59 Tempol þ HNO f Tempol-H þ NO k15 ¼ 8  104 M1 s1


Up to 150 μM, Tempol, which has no appreciable absorption at λ > 270 nm, had no effect on the kinetics of the decay of CH3C(O)NO• formed upon pulse irradiation of N2O-saturated solutions containing 2 mM aceto-HX at pH 11, where •OH reacts exclusively with CH3C(O)NHO. However, the EPR signal of Tempol decreased linearly with the number of pulses delivered into the solution (Figure 6) and was almost fully restored upon the addition of 1 mM ferricyanide. The radiolytically produced H• radicals readily reduce Tempol to Tempol-H,60 and the decrease of the EPR signal of Tempol should be corrected for this process. Thus, one calculates from the slope of the line in Figure 6 that [Tempol-H]/[CH3C(O)NO•]o = 0.13 ( 0.01. In this system, nitrite is accumulated, though at very low yield, that is, [NO2]/[CH3C(O)NO•]o = 0.037 ( 0.009 (Figure 6). Effect of O2. The kinetics were unaffected upon the addition of up to 20% O2, where 90% of the primary radicals are •OH, • N3, or CO3• and the rest is O2•, which does not react with the hydroxamate. However, O2 did affect the residual absorption at alkali pH. The irradiated cell was removed from the pulse radiolysis setup to the diode array spectrophotometer, where the spectral changes at 267 and 302 nm demonstrate the formation of two species (Figure 7), where the half-life of the former absorbing at 267 nm is significantly shorter than that of the species absorbing at 302 nm (Figure 7). The half-life of the species absorbing at 302 nm increased with the pH. Its optical absorption disappeared upon acidification and could not be restored upon alkalization. These results demonstrate that in alkaline solutions and in the presence of O2, one of the final decomposition products of CH3C(O)NO• is the peroxynitrite ion.61 The yield of peroxynitrite at pH 1213 was determined to be [ONOO]/[CH3C(O)NO•] = 0.30 ( 0.03. At pH 13,

k16 ¼ 5:8  106 M1 s1


NO þ • NO f N2 O2 

k17 ¼ 3  109 M1 s1 ð17Þ

N2 O2  þ • NO f N3 O3 

k18 ¼ 5:4  109 M1 s1 ð18Þ

N3 O3  f N2 O þ NO2 


The kinetic traces (e.g., Figure 4) indicate that the bimolecular decomposition of CH3C(O)NO• does not proceed via an outersphere electron-transfer mechanism (e.g., Figure 3). This process yields an absorbing species with a maximum at 315 nm that decomposes via its reaction with CH3C(O)NHO and OH. When the oxidation proceeded in the presence of Tempol or O2, which had no effect on the kinetics, Tempol-H and ONOO were formed, indicating that HNO/NO is produced during the bimolecular decomposition of CH3C(O)NO•. The yield of the nitroxyl was determined to be ∼30% of CH3C(O)NO• formed via the reaction of •OH/•N3 with CH3C(O)NHO at pH 1213 in the presence of O2, that is, about 60% of the nitroxide radicals yield nitroxyl. Under such conditions, the dimerization of HNO (reaction 20) is negligible, and HNO readily deprotonates to NO (reaction 21), which reacts with O2 to form ONOO (reaction 22).31 HNO þ HNO f N2 O þ H2 O


k20 ¼ 8  106 M1 s1


HNO þ OH h NO þ H2 O ¼ 4:9  104 M1 s1 , k-21 ¼ 120 s1


NO þ O2 f ONOO k22 ¼ 2:7  109 M1 s1


In deaerated solutions, the yield of Tempol-H was ∼13% of CH3C(O)NO• formed via the reaction of •OH with CH3C(O)NHO at pH 10.6. Tempol is reduced by HNO, forming Tempol-H and NO (reaction 15), where NO reacts at a diffusioncontrolled rate with NO (reaction 17), leading to the formation of N2O and NO2 (reactions 18 and 19). Assuming that 60% of the nitroxide radicals yield nitroxyl, simulation of reactions 1521 yields 13% Tempol-H using the literature values of k16k21 and k15 = 4.5  104 M1 s1, which is in good agreement with the reported value.59 The spin density on the CH3C(O)NO• is evenly distributed over the OCNO group, and therefore, the recombination of the nitroxide radicals could produce various adducts, where 3026 |J. Phys. Chem. A 2011, 115, 3022–3028

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Scheme 2. Proposed Mechanism for the Decomposition of CH3C(O)NO• Derived from One-Electron Oxidation of CH3C(O)NHO

the OO ones are expected to be very unstable.63 We, therefore, suggest that the bimolecular recombination of CH3C(O)NO• yields about 60% OO adducts, which dismutate to yield acetoHX and CH3C(O)NdO. The latter is known to decompose via hydrolysis (catalyzed by OH)18,64,65 or via reaction 737 to form nitroxyl. The other 40% recombination may be attributed to CC, CN, CO, and NN adducts, of which one absorbs at 267 nm (Figure 7) or decomposes to yield a product absorbing at this wavelength. The proposed mechanism is described in Scheme 2, where adduct 1 is attributable to the OO adducts and adduct 2 is attributable to the other recombinations. The reaction kinetics could not be studied under physiological pH because of the relatively low absorption of the nitroxide radical. However, product analysis using •OH as an oxidant at pH 7.0 demonstrates that nitrite does not accumulate under anoxic conditions but does in the presence of O2, though at extremely low yield of a few percent in the presence of 10% O2. This can be rationalized by assuming a competition between dimerization of HNO and its relatively slow reaction with O2 (reaction 23),59 which eventually forms peroxynitrite. HNO þ O2 f NO þ HO2 • k23 ¼ ð1  3Þ  103 M1 s1


Consequently, nitrite is formed during peroxynitrite decomposition, which yields about 28% •NO2 and •OH.61,66 In our system, • OH is scavenged by aceto-HX, and because the radiation gives rise to about 10% O2•, nitrite is mainly formed via its reaction with •NO2.61,66 We note that the mechanism described in the present study does not necessarily apply under steady-state radiolysis, where first-order decomposition of the nitroxide radical better competes with second-order decomposition. The steady-state radiolysis is currently under investigation using additional derivatives of hydroxamates. Our preliminary results demonstrate that nitrite is not accumulated under anoxic conditions, implying that NO is not formed under these conditions as well.

’ AUTHOR INFORMATION Corresponding Author

*Phone 972-2-6586478. Fax: 972-2-6586925. E-mail: [email protected]

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