History and Properties of PN

PN (oxoperoxonitrate (1-)) was first observed in 1901 by Bayer and Villiger in the reaction of nitrous acid and hydrogen peroxide, but was ill-defined as pernitric acid (HNO₄). In 1935 Gleu and Hubold introduced the name peroxynitrite for this compound. In 1954 Halfpenny and Robinson observed

1 INTRODUCTION 12 hydroxylation and nitration reactions of aromatics by PN. At the same time Anbar and Taube postulated the mechanism for PN formation by nitrosation of H₂O₂. In 1964 Papee and Petriconi proved the formation of PN in the photolysis of nitrate. In 1976 traces of PN were found in mars minerals by the NASA Viking-sonde. In the 80’s, the role of PN in smog chemistry and in the destruction of ozone in the stratosphere was discussed and first indictions were found for the formation of PN in vivo.

Today it is known, that PN can be formed in vivo by the nearly diffusion controlled reaction of ·NO and O^·−₂ :

·NO + O^·−₂ −→ ONOO⁻ (30)

The velocity of this reaction (k=4.3-10 x 10⁹ M⁻¹s⁻¹ [7, 8, 9]) is a factor of 2-10 faster compared to the velocities of the reactions of Mn- and Cu,Zn-SOD with superoxide (see also equation (21) and k-values given in 1.2.3). For the suppression of PN formation in vivo the ·NO concentration is essential. If the nitric oxide concentration gets too high, SOD’s cannot compete with it for the superoxide anion and formation of PN will be favored [5].

PN is a structural isomer of nitrate, but lies 150 KJ·mol⁻¹ higher in energy [56]. Kept at -20 to -80 ^◦C in alkaline solutions PN can be stored for 4 weeks up to months without serious losses. The intense yellow color of the PN-anion can be used for its quantification as proposed by Hughes and Nicklin in 1968, by the use of ε302 = 1670 M⁻¹cm⁻¹ [57]. In alkaline solutions PN is present in its cis-conformation (as shown previously by ¹⁵N-NMR and Raman measurements [58, 59] and x-ray structure analysis [60]) and undergoes a very slow decomposition to form oxygen and nitrite [61]. The cis-form of ONOO⁻ is 14.6 KJ·mol⁻¹ more stable than the trans-form of PN-anion [56]. In neutral solutions PN-anion undergoes protonation to peroxynitrous acid with a pK_a-value of 6.8 [56], which is much more reactive and isomerizes to nitrate by a yet not completely understood mechnism [56]. ε302 of ONOOH is a factor of 100 lower compared to that of ONOO⁻ (unpublished observation of Kissner and Koppenol). ONOOH is thought to be present in the trans-conformation, because trans-ONOOH is 4.18 KJ·mol⁻¹ more stable than cis-ONOOH [56, 62].

The halflife of ONOOH in aqueous phosphate buffer at pH 7.4 is 2.7 s at 25 ^◦C and 0.8 s at 37 ^◦C [61, 63]. Fig.4 shows a compilation of the protonation and conformation equilibria and pathways for the decomposition of PN, as well as

isomerization energies and pK_a-values [56, 61, 62].

Figure 4: Isomerization and decomposition of PN.

Concerning the postulated activated intermediate of PN, ONOOH^∗, there are two theories. Older studies about ONOOH-isomerization proposed a vibronical excitation in the trans-form by a combined deforming vibration along the N-O-O angle and a stretching vibration along the O-OH bond (see also Fig.5) [56].

During these vibrations the O-O bond is weakened and the endbonded O-atom approaches the N-atom. The intermediate can be regarded as a three-membered ring, which has either singlet or triplet character. In cis-ONOOH these vibrations are hindered. On an orbital basis, Fig.6shows that in the trans-ONOOH HOMO and 2^nd HOMO a bonding overlapping of the N- and O-orbitals becomes possible, whereas in the cis-form it is anti-bonding [59]. The radical cage mechanism of PN-isomerization and 1e-oxidative reactivity was discussed very controversly in literature [64, 65, 66, 67, 68, 69, 70].

The current opinion about PN-isomerization and 1e-oxidative reactivity has changed. Detailed kinetic measurements [71, 72], ESR measurements [73, 74, 75] and CIDNP experiments [6] favor the radical cage as reactive intermediate during PN-isomerization and also the observed 1e-oxidations are explainable this way. Equation (31) shows the formation of the radical pair in the solvent cage and the subsequent recombination to nitrate [71, 72]. If this cage encounters a 1e-donor (D-H), then a cage will be formed with a ·NO₂ and donor radical pair and a molecule of water (equation (32)) [6]. This cage can either collapse by recombination of the donor and nitrogen dioxide radical, which would be a nitration or form nitrite and D⁺, which would be hydrolyzed by water and form a

1 INTRODUCTION 14

Figure 5: Formation of an excited in-termediate during isomerization of PN to nitrate by vibronical activation.

Figure 6: Explanation of PN-isomerization on the basis of ab initio calculated molecule orbitals of PN. The MO’s were calculated by using the 6-311+G^∗ basis-set.

hydroxy product. Of course it may also happen, that some of the radical species escape from the cage and react in a different way. The yield of these radical species is still under discussion and varies from 5 [76] -40 % with respect to ONOOH concentration [71, 72].

ONOOH [ONO· ·OH]_cage −→ NO⁻₃ (31)

[ONO· ·OH]_cage + D−H −→ [D· ·ONO]_cage + H₂O (32) Also in the decomposition of PN at alkaline pH there are new findings. The formation of nitrite and oxygen is more complicated than shown in Fig.4. The following equations will introduce the major reactions taking place in an alkaline solution of PN [24, 61, 77].:

ONOOH ·NO₂ + ·OH k = 1.2−1.3s⁻¹ (33) Reaction (33) contributes most to the decay of PN in the physiological pH range (by formation of hydroxyl radicals which react with PN by equation(37)), above pH 8 reaction(34)causes PN-decomposition. Reaction(35)and (36)are

too slow to play a role, especially since the reaction of ·NO with ·NO₂ (mainly produced in the autoxidation of ·NO in oxygenated solutions) is much faster (see (38)). N₂O₃ is the intermediate that accelerates PN-decomposition at alkaline pH [77]. Some other reactions may play a role in this decomposition, so the formation of ONOO· and O₂NOO⁻ and their further reactions [77].