Nitrogen Dioxide Reaction with Nitroxide Radical Derived from

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A: Kinetics and Dynamics

Nitrogen Dioxide Reaction with Nitroxide Radical Derived from Hydroxamic Acids: The Intermediacy of Acyl Nitroso and Nitroxyl (HNO) Eric Maimon, Amram Samuni, and Sara Goldstein J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.8b02300 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018

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The Journal of Physical Chemistry

Nitrogen Dioxide Reaction with Nitroxide Radical Derived from Hydroxamic Acids: The Intermediacy of Acyl Nitroso and Nitroxyl (HNO)

Eric Maimona, Amram Samunib, Sara Goldsteinc*

a

Nuclear Research Centre Negev, Beer Sheva, Israel, bInstitute of Medical Research Israel-Canada,

Medical School, The Hebrew University of Jerusalem, Jerusalem 91120, Israel, cInstitute of Chemistry, The Accelerator Laboratory, the Hebrew University of Jerusalem, Jerusalem 91904, Israel

*Corresponding Author S. Goldstein. E-mail: [email protected]; Tel: 972 54 7659996

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ABSTRACT

Hydroxamic acids (RC(O)NHOH) form a class of compounds, which display interesting chemical and biological properties The chemistry of RC(O)NHOH) is associated with one- and twoelectron oxidations forming the respective nitroxide radical (RC(O)NHO•) and acyl nitroso (RC(O)N=O), respectively, which are relatively unstable species. In the present study the kinetics and mechanism of •NO2 reaction with nitroxide radicals derived from acetohydroxamic acid, suberohydroxamic acid, benzohydroxamic acid and suberoylanilide hydroxamic acid have been studied in alkaline solutions. Ionizing radiation was used to generate about equal yields of these radicals demonstrating that the oxidation of the transient nitroxide radical by •NO2 produces HNO and nitrite at about equal yields. The rate constant of •NO2 reaction with the nitroxide radical derived from acetohydroxamic acid has been determined to be (2.5 ± 0.5) x 109 M-1s-1. This reaction forms a transient intermediate absorbing at 314 nm, which decays via a first-order reaction whose rate increases upon increasing the pH or the hydroxamic acid concentration. Transient intermediates absorbing around 314 nm are also formed during the oxidation of hydroxamic acids by H2O2 catalyzed by horseradish peroxidase. It is shown that HNO is formed during the decomposition of these intermediates, and therefore they are assigned to acyl nitroso compounds. This study provides for the first time a direct spectrophotometric detection of acyl nitroso compounds in aqueous solutions allowing the study of their chemistry and reaction kinetics.


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1. INTRODUCTION Hydroxamic acids (RC(O)NHOH, HXs) form a class of compounds, which display interesting chemical and biological properties.1,2 Recent studies have highlighted HXs biological activities, which some of them are associated with their ability under oxidative stress to generate nitroxyl (HNO) and nitric oxide (NO).2-8 The chemistry of HXs is associated with one- and two-electron oxidations forming the respective nitroxide radical (RC(O)NHO•) and acyl nitroso (RC(O)N=O), respectively, which are relatively unstable species. Hydrolysis of acyl nitroso (denoted also nitrosocarbonyl) and its reaction with nucleophiles generate HNO,6,9-11 which readily undergoes dimerization followed by dehydration to give N2O12. The presumed generation of acyl nitroso has been generally evidenced by trapping it with conjugated 1,3-dienes yielding stable cycloadducts10,13 or by detection of N2O7,10,11,14-17. So far, the only direct evidence for acyl nitroso was provided by time-resolved infrared spectroscopy in nonaqueous solution.11,18 The kinetics and mechanism of cyclic stable nitroxide radicals reactions with nitrogen dioxide (•NO2) have been previously studied.19,20 It has been demonstrated that this reaction proceeds via an inner-sphere electron transfer mechanism forming the respective oxoammonium cations. The bimolecular rate constants have been determined to be (7 – 8) x 108 M-1s-1 implying that cyclic nitroxides are among the most efficient •NO2 scavengers. Similarly, •NO2 reaction with nitroxide radicals derived from HXs might generate the respective acyl nitroso compounds. In the present study we used ionizing radiation for studying •NO2 reaction with nitroxide radicals derived from various HXs demonstrating that the oxidation of the nitroxide radical by •NO2 produces HNO and nitrite at about equal yields. In addition, the transient intermediate formed in the case of acetohydroxamic acid and also during the enzymatic oxidation of HXs by horseradish peroxidase and H2O2. It is shown that HNO is formed during the decomposition of these intermediates, which are assigned to acyl nitroso compounds. 3 ACS Paragon Plus Environment


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2. MATERIALS AND METHODS Water for preparation of the solutions was purified using a Milli-Q purification system. All chemicals from Sigma-Aldrich were of the highest available grade and were used as received: acetohydroxamic acid (aceto-HX), suberohydroxamic acid (subero-HX), benzohydroxamic acid (benzo-HX), horseradish peroxidase (HRP, Type VI) and Griess reagent. Suberoylanilide hydroxamic acid (SAHA) was purchased from LC Laboratories and stock solution of 0.25 M was prepared in dimethylsulfoxide (DMSO). The structures of HXs are given in Fig. 1.

aceto-HX

benzo-HX

SAHA

subero-HX

Figure 1. Structures of aceto-HX, benzo-HX, SAHA and subero-HX

H2O2 was obtained as a 30% solution from Merck and its concentration was determined by the molybdate-activated iodine assay (ε352 = 25800 M-1cm-1).21 The concentration of HRP was determined spectrophotometrically using ε403 = 100 mM-1cm-1.22 Nitrite was assayed using the Griess reagent. The absorption at 540 nm was read 15 min after mixing the reagent with the sample. Calibration curves were prepared using known concentrations of nitrite. Radiolysis. Pulse radiolysis experiments were carried out using a 5-MeV Varian 7715 linear accelerator (1.5 µs electron pulses, 200 mA current). A 200 W Xe lamp produced the analyzing light. Measurements were carried out in a 4 cm spectrosil cell with three passes of the analyzing light. The ionizing radiation dose was determined using N2O-saturated solution containing 5 mM ferrocyanide where ε420(Fe(CN)63–) = 1000 M-1cm-1 and G(Fe(CN)63–) = 6.7 µM Gy-1 or Fricke dosimeter where ε302(FeIII) = 2200 M-1cm-1 and G(FeIII) = 15.6 µM Gy-1.23 Steady-state radiolysis was carried out with a 4 ACS Paragon Plus Environment

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137


Cs source. The dose rate was determined using the Fricke dosimeter. All experiments were done at

room temperature. Gas chromatography (GC). Sample solutions (7 mL) were placed in a glass vial (12 mL) sealed with a rubber septum and were deaerated by He. A gas aliquot of the reaction headspace (2 mL) was taken and 1 mL at room pressure and temperature was injected onto a 5890 Hewlett-Packard gas chromatograph equipped with a thermal conductivity detector, a 10 ft - 1/8 inch Porapak Q column at an operating oven temperature of 70 °C (injector and detector 150 °C) and a flow rate of 20 mL min-1 (He, carrier gas). The yields of N2O were calculated on the basis of a standard curve prepared by injecting known amounts of N2O gas (Maxima, Israel).

3. RESULTS 3.1. Radiolysis. Irradiation of aqueous solutions produces several species as shown in eq. 1. The numbers in parenthesis are G-values representing the respective yields (in 10-7 M Gy-1), which are higher in the presence of high concentrations of solutes. γ H2O → e–aq(2.6), •OH (2.7), H• (0.6), H3O+ (2.6), H2O2 (0.72)

(1)

In deaerated solutions containing NO3– at pH > 10, e–aq and H• are scavenged by NO3– forming NO3•2– and HNO3•– (pKa = 7.524) radicals, which undergo hydrolysis yielding •NO2 (reactions 2 – 4).

e–aq + NO3– → NO3•2–

k2 = 9.7 x 109 M-1s-1 25

(2)

H• + NO3– → HNO3•–

k3 = 1 x 107 M-1s-1 26

(3)

NO3•2– + H2O → •NO2 + 2OH–

k4 = 5.5 x 104 s-1 26

(4)



In the presence of excees concentrtions of NO3– over CH3C(O)NHOH (pKa = 9.0 ± 0.16,27) at pH > 10, •

OH is scavenged by CH3C(O)NHO– forming CH3C(O)NO•– (pKa = 9.1 ± 0.16) (reaction 5) while NO3–

reduction by eaq- and H• generates NO3•2–.

CH3C(O)NHO– + •OH → CH3C(O)NHO•– + OH–

k5 = 4 x 109 M-1s-1 6

(5)

Several subsequent kinetic processes were monitored at 270 - 360 nm as demonstrated in Fig. 2 (275 nm) and in Fig. 3 (320 nm). In Fig. 2A the initial absorption formed at the end of the pulse due to the formation of NO3•2– 24,28 and CH3C(O)NO•– 6 decays via a first-order reaction resulting in kobs = (4.7 ± 0.3) x 104 s-1, which is attributed to NO3•2– hydrolysis (reaction 4).

0.3

0.3

B

A 0.2

0.2

40 ms

1 ms

A275

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.1

0.1 20 µs

1 ms

0.0

0.0

Time

Time

Figure 2. Kinetic traces observed at 275 nm upon pulse-irradiation of deaerated solutions containing 3 mM NO3– and 0.4 mM aceto-HX acid at pH 10.7. The dose was 23.2 Gy/pulse and the optical path 12.1 cm.

The following process obeys second-order kinetics (Figs 2A and 2B) and is attributed to •NO2 reaction with CH3C(O)NO•–. The rate constant has been calculated using ε(CH3C(O)NO•–)6 and ε(•NO2) ≤ 200 M-1cm-1 at 270 – 290 nm28,29 to be (2.5 ± 0.5) x 109 M-1s-1. The dimerization of CH3C(O)NO•– (2k = 5.6 x 107 M-1s-1)6 was ignored since its rate is far lower. The second-order reaction is followed by a first6 ACS Paragon Plus Environment

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order one, which at λ < 290 nm is well separated in time from the preceding reaction, resulting in kobs = 31 ± 3 s-1 (e.g., Fig. 2B). This process generates a transient intermediate with maximum absorption at 314 nm (Fig. 3, inset), which decays via a first-order reaction to non-absorbing products at 270 - 360 nm (Fig. 3). The observed-first order rate constant increases upon increasing the pH or [CH3C(O)NHO–], e.g., kobs = 0.62 ± 0.02 and 1.15 ± 0.05 s-1 in the presence of 0.2 and 0.4 mM hydroxamate, respectively, at pH 10.7.

0.06 0.06 0.04

0.04

0.02

A320

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.00 270

300

330

360

0.02 2s

10 ms

0.00

Time

Figure 3. Kinetic traces observed at 320 nm upon pulse-irradiation of deaerated solution containing 3 mM NO3– and 0.4 mM CH3C(O)NHO at pH 10.7. Inset: the absorption of the transient species formed 40 ms after the pulse. The dose was 23.2 Gy/pulse and the optical path 12.1 cm.

Similar kinetics profile has been reported for RC(O)NHO– oxidation by •OH or •N3 where the secondorder kinetics were attributed to the dimerization of RC(O)NO•– generating the same transient species absorbing at 314 nm.6,7 Accumulation of N2O and nitrite was determined following both pulse- and γ-irradiation of deaerated solutions containing 2 - 20 mM NO3– and 0.5 mM aceto-HX at pH 10.9 - 11.7. Fig. 4 demonstrates that accumulation of N2O and NO2– increases linearly with the irradiation dose resulting



in G(NO2–) = 3.1 ± 0.1 and G(N2O) = 1.5 ± 0.1. Similar yields were obtained when aceto-HX was replaced by subero-HX (Fig. 4B).

200

B

200

A

-

NO2

NO2

-

150

µM

150

µM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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100

50

100

N2O

50

N2O

0

0 0

10

20

0

30

150

300

450

600

750

Dose (Gy)

pulses

Figure 4. Accumulation of N2O and NO2– obtained upon (A) pulse-irradiation of deaerated solutions containing 0.5 mM aceto-HX and 20 mM NO3– at pH 10.9 (21 Gy/pulse); (B) γ-irradiation of deaerated solutions containing 0.5 mM aceto-HX (circle symbols) or subero-HX (square symbols) and 20 mM NO3– at pH 10.9.

Secondary radicals like •N3 or Br2•– are formed when the irradiated solution contains also N3– or Br–, respectively 25. Under limiting concentrations of RC(O)NHO– both radicals oxidize RC(O)NHO– where the rate constant in the case of •N3 is much higher, e.g., k(Br2•– + SAHA) = (4.9 ± 0.1) x 107 M-1s-1 and k(•N3 + SAHA) = (5.1 ± 0.1) x 109 M-1s-1 at pH > 11.7 Accumulation of N2O following pulse-irradiation of deaerated solutions containing 0.5 mM aceto-HX, 2 mM nitrate and 20 mM azide ion at pH 11.5 increased linearly with the number of pulses resulting in G(N2O) = 1.4 ± 0.1. The accumulation of NO2– could not be determined due to interference of N3– with the Griess assay. Since both SAHA and benzo-HX contain an aromatic ring (Fig. 1), Br2•– was used as the oxidizing radical, which contrary to •

OH, reacts extremely slowly, if at all, with aromatic moiety and with DMSO used to solubilize

SAHA.25 Br2•– was preferred over •N3 since azide ions interfers with the Griess assay. However, due to the relatively low reaction rate constant of HXs oxidation by Br2•–, accumulation of N2O and NO2– was 8 ACS Paragon Plus Environment

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determined following steady-state irradiation since under pulse radiolysis conditions the selfdecomposition of Br2•– (2 x 109 M-1s-1)25 cannot be ignored. Relatively high [Br–] were required to compete efficiently with DMSO for •OH, and therefore the experiments with all HXs were carried out in the presence of 0.5 M NaBr. The results presented in Fig. 5 demonstrate that accumulation of N2O and NO2– following γ-irradiation of deaerated solutions containing 0.5 mM HX (SAHA, benzo-HX, aceto-HX), 20 mM NO3– and 0.5 M NaBr at pH 10.9 increases linearly with the irradiation dose resulting in G(NO2–) = 3.7 ± 0.1 and G(N2O) = 1.9 ± 0.2 for the three HXs tested. These yields are higher than those obtained in the absence of Br– since the G-values increase substantially in the presence of 0.5 M Br–.30,31

300

benzo-HX aceto-HX SAHA

NO2

-

200

µM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100

N2O 0 0

30

60

90

120

Dose (Gy)

Figure 5. Accumulation of N2O and NO2– obtained upon γ-irradiation of deaerated solutions containing 0.5 mM aceto-HX (circle symbols), benzo-HX (square symbols) or SAHA (triangle symbols, 0.5 M NaBr and 20 mM NO3– at pH 10.9.

Pulse-irradiation of a solution containing 3 mM NO3–, 0.4 mM CH3C(O)NHOH and 2.4% O2 (29 µM) at pH 10.7 resulted in a residual persistent absorbance with a maximum around 300 nm, which disappeared upon acidification and could not be restored upon alkalization. It is assumed that this absorbing product is peroxynitrite since NO– (pKa = 11.4)12 readily reacts with O2 forming ONOO– (k = 9 ACS Paragon Plus Environment


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2.7 x 109 M-1s-1)12. Hence, the formation of ONOO– provides further evidence for HNO generation via •

NO2 reaction with CH3C(O)NO•–. G(ONOO–) = 1.47 ± 0.09 has been determined using ε302(ONOO–) =

1670 M-1cm-1,32 which is expected to be lower than G(•NO2) since O2 is readily reduced by H• (reaction 6), and to some extent also by NO3•2– (reaction 7) followed by O2•– reaction with •NO2 (reaction 8), i.e., G(•NO2) = Ge - GH - 2 x Ge x k8[O2]/(k8[O2] + k4) ≈ 1.42, which is in excellent agreement with the experimentally observed yield of ONOO–.

H• + O2 → HO2• NO3•2– + O2 → NO3– + O2•– O2•– + •NO2 → NO2– + O2

k6 = 1.9 x 1010 M-1s-1 25 k7 = 2.4 x 108 M-1s-1 25 k8 = 4.5 x 109 M-1s-1 29,33

(6) (7) (8)

3.2. Enzymatic oxidation of HXs by HRP/H2O2. Peroxide reduction by electron donors like HXs catalyzed by HRP (R-PorFeIII) proceeds via reactions 9 – 11 where k10 > k11.34-37

R-PorFeIII + H2O2 → R-+•PorFeIV=O + H2O

k9 = 1.7 x 107 M-1s-1

(9)

R-+•PorFeIV=O + X− → R-PorFeIV=O + X•

(10)

R-PorFeIV=O + X− + 2H+ → R-PorFeIII + X• + H2O

(11)

In the present study X• = RC(O)NHO•, which under anoxic conditions mainly decomposes bimolecularly yielding, among other products, CH3C(O)N=O.6,7 HRP forms spectroscopically distinct, reversible complexes with HXs where the greatest affinity has been shown by HXs having an aromatic group such as benzo-HX.38 Upon the addition of H2O2 to a solution containing HRP and HXs (excluding benzo-HX) at pH 7.0, HRP is immediately converted into R-PorFeIV=O (compound II) as reflected by the absorption of the Soret peak at 420 nm and two absorption peaks at 527 and 556 nm 10 ACS Paragon Plus Environment

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(Figure 6B).39 Upon complete consumption of H2O2, compound II is reduced back to HRP. During the catalytic process a transient species with maximum absorbance at 314 nm is accumulated as demonstrated for aceto-HX in Fig. 6A.

0.25

0.25

30 s

A

314 nm

Absoebance

420 nm 0.15

0.10

403 nm

80 s 10 s

O2

0.20

0.20

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.15

B

x10

0.10

0.05

0.05 0.00

0

20

40

100

200

300

300

Time (s)

400

500

600

700

λ (nm)

Figure 6. Oxidation of 1 mM aceto-HX by 150 µM H2O2 catalyzed by 1.7 µM HRP at pH 7.0 (20 mM phosphate buffer) under anoxia. (A) Spectral changes monitored at 314, 403 and 420 nm; (B) Spectra measured 10, 30 and 80 s after the addition of HRP, and the latter after exposure to air.

When H2O2 is fully consumed, A314 decays via a first-order reaction (Fig. 6A) and the observed rate constant increases as [HX] or pH increases. As demonstrated in Fig. 6, HRP is recovered after H2O2 is fully consumed, and then it is converted to other heme species via its reaction with one of the products formed during A314 decay, presumably HNO, forming R-PorFeIINO (Fig. 6B). The latter slowly decomposes while recovering HRP, a process which is accelerated upon the addition of O2 (Fig. 6B), –

i.e., R-PorFeIINO undergoes auto-oxidation in the presence of O2 yielding NO3 and regenerating HRP.40 The reaction mixture was sampled for N2O determination at the end of the catalytic process, i.e., upon full consumption of H2O2, and when the decay of A314 was completed where the yield of N2O is about half that of ∆[H2O2] = [H2O2]o (Table 1). The results demonstrate that N2O yield at the end of 11 ACS Paragon Plus Environment


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the catalyic process is significantly lower than that measured at the end of A314 decay (Table 1). This implies that N2O formation, i.e., HNO formation, is associated with the decomposition of the transient species absorbing at 314, which decomposes to some extent also during the catalytic process.

Table 1. Yields of N2O accumulated during HXs oxidation by H2O2/HRP at pH 7.0 (20 mM PB) under anoxia sampled both at the end of the catalytic process and when the decay of A314 was completed. The error is ±10%. HX

[HX], mM

[HRP], µM

[H2O2], µM

aceto-HX

0.5

1.6

280

[N2O], µM End of A314 decay 132

aceto-HX

1

1.6

400

230

aceto-HX

1

3

420

39

236

subero-HX

1

3

400

37

190

SAHA

0.4

3

250

7

98

SAHA

0.4

1.6

280

[N2O], µM End of catalysis

152

4. DISCUSSION Transient nitroxide radicals derived from one-electron oxidation of HXs readily react with •NO2 and the proposed mechanism in alkaline solutions is described by equations 12 - 15.

RC(O)NO•– + •NO2 → adduct

(12)

adduct → CH3C(O)N=O + NO2–

(13)

RC(O)N=O + RC(O)NHO– → RC(O)NHOC(O)R + NO–

(14)

RC(O)N=O + OH– → RC(O)O– + HNO

(15)

As demonstrated for aceto-HX, the reaction proceeds via an inner-sphere electron transfer mechanism where initially a transient adduct is formed (k12 = (2.5 ± 0.5) x 109 M-1s-1), which decomposes to yield nitrite and acyl nitroso (k13 = 31 ± 3 s-1). The decomposition of the acyl nitroso yields HNO, RC(O)O– 12 ACS Paragon Plus Environment

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and RC(O)NHOC(O)R (reactions 14 and 15) where the latter has been identified in the case of benzohydroxamic acid.41,42 Hence, under anoxic conditions the yield of nitrite should be similar to that of HNO and about twice that of N2O in agreement with the experimentally obtained yields for all HXs tested. The detection of ONOO– as an end-product in the presence of O2 provides further evidence for HNO generation via •NO2 reaction with CH3C(O)NO•–. Our assignment of the transient intermediate absorbing at 314 nm to acyl nitroso is supported by the results obtained while studying the catalytic oxidation of HXs by HRP/H2O2. It is demonstrated that the chemical, spectral, and reaction kinetics properties of the transient intermediate absorbing at 314 nm are identical to those of the intermediate observed upon •NO2 reaction with CH3C(O)NO•–, i.e., an electrophile that readily reacts with nucleophiles such as RC(O)NHO– and OH–. In addition, it is demonstrated that the decomposition of this transient intermediate yields HNO (Table 1). The chemistry of RC(O)NHO• resembles that of cyclic stable nitroxide radicals, which is associated with their one-electron oxidation forming acyl nitroso and oxoammomium cation, respectively, and their one-electron reduction yielding RC(O)NHOH and the hydroxylamine, respectively (Fig. 7). Unlike RC(O)NHO•, cyclic nitroxides are stable radicals and their chemistry is primarily associated with a one-electron exchange among their reduced and oxidized states (Fig. 7B).

O R

(B)

acyl nitroso

nitroxide

O NHO

+

.

ee-

R

H+

HNO + R

Nu

2e-, 2H+

H

N O.

+

H

O R

oxoammonium cation

O

Nu: N O

nitroxide

e-

e-

+ N=O 2e-

N OH NHOH hydroxylamine

(A) hydroxamic acid

Figure 7. Three oxidation states of (A) hydroxamic acid and (B) 2,2,6,6-tetramethyl-piperidine-N-oxyl (TEMPO). 13 ACS Paragon Plus Environment


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Contrary to the relatively unstable acyl nitroso compounds, some oxoammonium cations such as those derived from TEMPO and 3-carbamoyl proxyl are relatively stable,43 and their salts can be synthesized and used for the oxidation of alcohols to aldehydes and aldehydes to carboxylic acids.44,45 The oxoammonium cations are readily reduced back to their respective nitroxides or hydroxylamines, and thus cyclic stable nitroxides can protect against oxidative damage acting both as reducing and oxidizing species while being continuously recycled.46-48

5. CONCLUSIONS This study provides for the first time a direct spectrophotometric detection of acyl nitroso compounds in aqueous solutions allowing the study of their chemistry and reaction kinetics. The rate constant of •NO2 reaction with transient nitroxide radicals derived from HXs is somewhat higher than that of its reaction with cyclic nitroxides forming acyl nitroso compounds and oxoammonium cations, respectively. While the oxoammomium cation is readily reduced back to the parent nitroxide, the acyl nitroso decomposes to yield HNO. Therefore, cyclic nitroxides are highly effective antioxidants while hydroxamic acids serve under oxidative stress as HNO-donors.

ACKNOWLEDGEMENT This work has been supported by the Pazy Foundation Grant number 276/17.


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Goldstein, S.; Samuni, A. One-Electron Oxidation of Acetohydroxamic Acid: The Intermediacy of Nitroxyl and Peroxynitrite. J. Phys. Chem. A 2011, 115, 3022-3028.

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Samuni, Y.; Wink, D. A.; Krishna, M. C.; Mitchell, J. B.; Goldstein, S. Suberoylanilide Hydroxamic Acid Radiosensitizes Tumor Hypoxic Cells in Vitro Through the Oxidation of Nitroxyl to Nitric Oxide. Free Radic. Biol. Med. 2014, 73, 291-298.

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Gutierrez, M. M.; Almaraz, A. E.; Bari, S. E.; Olabe, J. A.; Amorebieta, V. T. The HNO Donor Ability of Hydroxamic Acids Upon Oxidation with Cyanoferrates(III). J. Coord. Chem. 2015, 68, 3236-3246.



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Evans, A. S.; Cohen, A. D.; Gurard-Levin, Z. A.; Kebede, N.; Celius, T. C.; Kebede, N.; Celius, T. C. ; Miceli, A. P.; Toscano, J. P. Photogeneration and Reactivity of Acyl Nitroso Compounds. Can. J. Chem. 2011, 89, 130-138.

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Shafirovich, V.; Lymar, S. V. Nitroxyl and Its Anion in Aqueous Solutions: Spin States, Protic Equilibria, and Reactivities Toward Oxygen and Nitric Oxide. Proc. Natl. Acad. Sci. USA 2002, 99, 7340-7345.

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Memeo, M. G.; Quadrelli, P. Generation and Trapping of Nitrosocarbonyl Intermediates. Chem. Rev. 2017, 117, 2108-2200.

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King, S. B.; Nagasawa, H. T. Chemical Approaches Toward Generation of Nitroxyl. Methods Enzymol. 1999, 301, 211-220.

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Xu, Y.; Alavanja, M.-M.; Johnson, V. L.; Yasaki, G.; King, S. B. Production of Nitroxyl (HNO) at Biologically Relevant Temperatures from the Retro-Diels-Alder Reaction of NHydroxyurea-Derived Acyl Nitroso-9,10-Dimethylanthracene Cycloadducts. Tetrahedron Lett. 2000, 41, 4265-4269.

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Samuni, Y.; Samuni, U.; Goldstein, S. The Mechanism Underlying Nitroxyl and Nitric Oxide Formation from Hydroxamic Acids. Biochim. Biophys. Acta 2012, 1820, 1560-1566.


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Goldstein, S.; Samuni, A. Biologically Relevant Chemistry of Nitroxides. Stable Radicals: Fundamental and Applied Aspects of Odd-Electron Compounds; Wiley & Sons, Ltd, 2010; pp 433-460.


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TOC Graphic

acyl nitroso

nitroxide O R

O NHO

+

.

ee-

R

O

Nu: N O

H

+

HNO + R

Nu

2e-, 2H+

H

O R

NHOH

hydroxamic acid



Structures of aceto-HX, benzo-HX, SAHA and subero-HX 254x190mm (96 x 96 DPI)

ACS Paragon Plus Environment

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Kinetic traces observed at 275 nm upon pulse-irradiation of deaerated solutions containing 3 mM NO3– and 0.4 mM aceto-HX acid at pH 10.7. The dose was 23.2 Gy/pulse and the optical path 12.1 cm. 254x190mm (96 x 96 DPI)



Kinetic traces observed at 320 nm upon pulse-irradiation of deaerated solution containing 3 mM NO3– and 0.4 mM CH3C(O)NHO at pH 10.7. Inset: the absorption of the transient species formed 40 ms after the pulse. The dose was 23.2 Gy/pulse and the optical path 12.1 cm. 254x190mm (96 x 96 DPI)


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Accumulation of N2O and NO2– obtained upon (A) pulse-irradiation of deaerated solutions containing 0.5 mM aceto-HX and 20 mM NO3– at pH 10.9 (21 Gy/pulse); (B) γ-irradiation of deaerated solutions containing 0.5 mM aceto-HX (circle symbols) or subero-HX (square symbols) and 20 mM NO3– at pH 10.9. 254x190mm (96 x 96 DPI)



Accumulation of N2O and NO2– obtained upon γ-irradiation of deaerated solutions containing 0.5 mM acetoHX (circle symbols), benzo-HX (square symbols) or SAHA (triangle symbols, 0.5 M NaBr and 20 mM NO3– at pH 10.9. 254x190mm (96 x 96 DPI)


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Oxidation of 1 mM aceto-HX by 150 µM H2O2 catalyzed by 1.7 µM HRP at pH 7.0 (20 mM phosphate buffer) under anoxia. (A) Spectral changes monitored at 314, 403 and 420 nm; (B) Spectra measured 10, 30 and 80 s after the addition of HRP, and the latter after exposure to air. 254x190mm (96 x 96 DPI)



Three oxidation states of (A) hydroxamic acid and (B) 2,2,6,6-tetramethyl-piperidine-N-oxyl (TEMPO). 254x190mm (96 x 96 DPI)


Page 28 of 28

Nitrogen Dioxide Reaction with Nitroxide Radical Derived from

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