Inhibitors for the Nitration of Phenol by Peroxynitrite

Nitration of phenol by PN has been reported to involve phenoxy radicals [6, 130]

and therefore can be considered as two one-electron oxidation steps mediated by peroxynitrous acid (ONOOH), or more precisely the radical cage form of decaying PN [72, 71]. Fig.34 shows the formation of nitro-phenols in the PN-phenol system at pH 6 in dependence of the concentration of some representative PN-scavengers. It is obvious, that two major groups of inhibitors for phenol nitration exist: The first one shows an exponential dependence (uric acids, dithio-purine and -pyrimidine), the second one a more linear dependence (thiols, ebselen, methionine, tyrosine and ascorbate) from scavenger concentration. This behavior is directly linked to the reactivity towards ONOOH or ONOO⁻ as will be shown later.

Tab.7 shows the IC₅₀-values of some natural antioxidants and synthetic compounds. It turned out, that among the best inhibitors of phenol nitration at pH 6 or scavengers for the radical cage form of PN are uric acid and its

Figure 34: Effect of scavengers on phe-nol nitration by PN in dependence of their concentration. The IC₅₀-values were deter-mined from these curves. Furthermore this graphic allowed to separate the scavengers into two major groups (linear and expo-nential dependence on concentration).

1.3- and 3.7-dimethyl analogues (DMUA), as well as 2-TBA, 2.6-DTPy and 2.6-DTPu, followed by cysteine, ascorbate, ebselen, 3.9-DMUA and GSH (left values). Se-met and methionine itself showed very poor protection in this system, whereas xanthine, allopurinol, caffeine, allantoin and alloxan, which are all structurally related to uric acid showed only poor protection (see Tab.8).

Exactly the same observations were made during phenol nitration at pH 7 (not shown). Very low IC₅₀-values have been reported for TEMPO/TEMPONE in phenol nitration reactions [106]. Therefore we also used this compound in our system and found IC₅₀-values of less than 2 µM for the nitration of 5 mM phenol by 800 µM PN (not shown). Additionally we found increased levels of 4-nitroso-phenol, when using TEMPO/TEMPONE, supporting the mechanism previously postulated for this reaction [106]. The results in the phenol system could be reproduced in a test system, where the same scavengers were employed to prevent BSA from nitration by PN (see Tab.9)[231].

Cysteine 64±14 / 425±125 Glutathione 181±20 / 380±57 Methionine 450±34 / 690±75 Se-methionine 250±13 / 170±28

Ascorbate 88±18 / 133±18 Uric acid 40±10 / 57±8

2-TBA 37±6 / 26±4 2.6-DTPu 35±13 / 36±14

2.6-DTPy 32±4 / 43.5±9.5 1.3-DMUA 36.5±1.5 / 25±12

3.7-DMUA 19±3 / - 3.9-DMUA 141±7 /

-Ebselen 127.5±4.5 / 190±11 Xanthine >1 mM />1 mM Allopurinol >1 mM />1 mM Caffeine >1 mM /

-Allantoin >1 mM / - Alloxan >1 mM /

-Table 7: IC₅₀-values [µM] of scavengers for phenol nitration at pH 6 or nitrosylation at pH 9 by peroxynitrite. First value for nitration (pH6), second value for nitrosylation (pH9). Mean-values of two independent series with at least four concentrations. 5mM phenol were reacted with 655µM PN at pH 6 and 400µM PN at pH 9.

At pH 9 the nitrosylation of phenol by PN was monitored and the effects of

4 RESULTS 60

Inhibitor [1 mM] % Nitration (pH6) % Nitrosation (pH9)

Adenosine 100 80

Table 8: Compounds with poor reactivity towards PN. Effect of 1 mM compound on nitration of 5 mM phenol by 655 µM PN at pH 6 and on nitrosation of 5 mM phenol by 400µM PN.

Values are the remaining percentage of nitration or nitrosation compared to the control without added compound.

Glutathione 150±26 Methionine 200±38

Ascorbate 170±17 Uric acid 75±13

2-TBA 45±12 1.3-DMUA 60±14

3.7-DMUA 185±37 3.9-DMUA > 200

Ebselen 180±12 Xanthine > 500

Allopurinol > 500

Table 9: IC₅₀-values [µM] for the inhibition of BSA nitration at pH 7 by peroxynitrite. Mean values of two independent series with at least four concentrations. Reaction of 15µM BSA with 1 mM PN. TBA, thiobarbituric acid; DMUA, dimethyluric acid.

the same scavengers as used above. These results are also shown in Tab.7(right values) and once more the uric acids and the thio-purines and -pyrimidines were best in preventing phenol from nitrosylation, suggesting that also this process involves radical species (also in the alkaline pH range high amounts of biphenols are formed, implicating the involvement of phenoxy radicals, see herefore Fig.23), for which the same group of scavengers is most effective.

In another experiment we used constant concentrations of PN, phenol and scavengers, but additionally measured the hydroxylation of phenol by PN (see Fig.35) and it turned out, that UA was most effective in suppressing hydroxy-products and benzoquinone, followed by 2-mercapto-benz-selenazol, 2-thio-barbituric acid and 2-mercapto-benz-thiazol. Ascorbate, GSH, cysteine,

ebselen and methionine showed only small effects on hydroxylation. In the case of 2,6-DTPu and -DTPy hydroxy-products and benzoquinone could not be determined because of interfering scavenger peaks (marked with ^∗). Additional experiments in this system showed no significant effect on neither PN-mediated hydroxylation nor nitration by PEP, oxalate, acetone, a-ketoglutarate, but a small decrease of nitration by acetaldehyde and a trialkyl-phosphine. Pyruvate showed a small increase of nitration (see Fig.37).

Figure 35: Effect of 200 µM scavenger on nitration (pH 7) and hydroxylation (pH 5) of 5 mM phenol by 1 mM PN at RT.

Products were determined by HPLC and error bars represent mean values out of 3 measurements.

Figure 36: Effect of 100µM scavenger on nitration of 5 mM phenol by 655 µM PN at pH 6 and RT, catalyzed by 5 µM MP-11. o- and p-nitro-phenol were quantified by HPLC, error bars indicate mean values out of 3 measurements.

Additionally a system containing phenol and MP-11 was used as a model for the metal-catalyzed tyrosine nitration in proteins. Nitration of 5 mM phenol

Figure 37: Effect of 200µM scav-enger on nitration of 5 mM phenol by 800 µM PN at pH 6 and RT.

Products were quantified by HPLC, error bars indicate mean values out of 3 measurements.

4 RESULTS 62 by 655 µM PN at pH 6 is increased by a factor of 4-5 by 5 µM MP-11. The same scavengers as tested above were employed in this system and it turned out that also in the metal-catalyzed nitration UA, 1.3-DMUA, 2.6-DTPu and -DTPy were much more effective in preventing phenol from nitration than were GSH, ascorbate, ebselen, methionine and Se-met (see Fig.36). 2-TBA showed an unusually small effect in this system.

4.2.3 Inhibitors for the Oxidation and Inactivation of ADH by PN