Chem. Res. Toxicol. 1993,6, 548-553
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Selective Para Hydroxylation of Phenol and Aniline by Singlet Molecular Oxygen Karlis Briviba,t T h o m a s P. A. Devasagayam,? Helmut Sies,*Ft a n d Steen Steenken*J Institut fur Physiologische Chemie I, Universitiit Diisseldorf, Moorenstrasse 5, 0-4000 Diisseldorf, Germany, and Max-Planck-Institut fur Strahlenchemie, 0-4330 Mulheim, Germany Received February 22, 1993
Phenol reacts with singlet oxygen (l02) generated in aqueous solution (HzO or D2O)by (a) the exposure of methylene blue to light or (b) the thermal dissociation of the endoperoxide of 3,3’-( 1,4-naphthy1idene)dipropionateto lead selectively to hydroquinone as the primary product. The other isomers of phenol hydroxylation, catechol and resorcinol, were not observed. In agreement with the involvement of lo2 as the reactive species in the hydroxylation, in D20the yield of hydroquinone is 7 times that in H20, and the lo2 quenchers azide and the thiols, glutathione and dithiothreitol, suppress the production of hydroquinone. In contrast, the hydroxyl radical scavengers, tert-butyl alcohol, propanol, or sodium formate, are without effect. In a follow-up reaction, hydroquinone is converted into benzoquinone. Reaction of IO2 with aniline leads to the selective formation of 4-hydroxyaniline as the initial product. This is further converted to hydroquinone with formation of ammonia (deamination), and then to benzoquinone. On the basis of these results, the selective para hydroxylation of phenol or aniline may be used as an indicator for the involvement of singlet oxygen as compared to ‘OH radical- or cytochrome P450-mediated reactions.
Introduction Singlet molecular oxygen (‘0~)~ can be generated by photochemical reactions (photoexcitation)through transfer of excitation energy to ground-state oxygen (SO2)from a suitable excited triplet-state sensitizer (1, 2). It is produced also in biological systems by dark reactions (chemiexcitation), e.g., by lipid peroxidation, and by enzyme reactions such as those involving lactoperoxidase, lipoxygenase, and chloroperoxidase (3). Singlet oxygen is reactive with many biologically occurring compounds (2). In the case of DNA, ‘ 0 2 produces base modifications and leads to strand breaks. This process seems to be initiated by interaction with the most electron-rich base, guanine. In this reaction, guanine is hydroxylated at the 8-position (4,5). A similar reaction should, in principle, also be possible with other electronrich compounds such as phenols and aromatic amines. In fact, hydroxylation of salicylic acid by lo2 has recently been described (6). However, attempts to judge the generality of this reaction or to obtain information on the further reactions of the primary products have so far not been made. Due to the importance of phenols and related compounds such as aromatic amines in organic chemistry and in biology, the possible occurrence of hydroxylation by singlet oxygen of the parents of these families of compounds, Le., of phenol and aniline, was studied here. Surprisingly, there is very little information on the reactions of these simple aromatics, the emphasis so far
* Address correspondence to this author at the Max-Planck-Institut filr Strahlenchemie,D-4330MGlheim, Germany.Phone +49-208-3043535, fax +49-208-3043951. f UniversiW Dkseldorf. 2 Max-Planck-Institut filr Strahlenchemie. 1 Abbreviations: 1 0 2 = singlet molecular oxygen,sOz= triplet (groundstate)molecular oxygen, NDPOz = the endoperoxide of the disodium salt of 3,3’-(1,4naphthylidene)dipropionate,NDP = 3,3’-(1,4-naphthylidene)dipropionate. ., .
having been on more complicated, substituted systems (1, 2,7). From a toxicological point of view, it may be relevant that the singlet oxygen quenching ability of phenolic compoundscan be correlatedwith their capacity to protect against benzo[al pyrene-induced neoplasia (8).
Experimental Procedures Reagents. Deuterium oxide (99.8%), glutathione, phenol, hydroquinone, and aniline (distilled prior to use) were from Merck (Darmstadt, Germany) or Fluka (Basel, Switzerland) and of the highest purity available. DL-Dithiothreitol was from Sigma (St. Louis, MO). Water used in our studies was purified by Millipore filtration. The endoperoxide of the disodium salt of 3,3’-(1,4naphthy1idene)dipropionate(NDPOz) was prepared as described (9). Generation and Quenching of IO2 by Phenol, Hydroquinone, Aniline, and 4-Hydroxyaniline. (A) Photosensitization. Singlet oxygen was generated by exposure of methylene blue to visible light. The 3-mL samples contained 35 pM methylene blue, 1 mM of the aromatic, and 5 mM phosphate buffer in HzO or DzO at pH 6 (pD 6.4). They were illuminated on an optical bench with the focused light of a 220-W projector spotlamp at room temperature. A cutoff filter was used to eliminate light of X < 500 nm. The solutions were bubbled with oxygen throughout the illumination. Conversions were always < l o % , and in most cases < 1%. (B)Thermodissociationof NDPO,. Singlet oxygen was also generated by the thermal dissociation of NDP02, yielding 3,3’(1,4-naphthylidene)dipropionate(NDP) (9). The reactant was 1mM phenol in 50 mM phosphate buffer in H2O or DzO, pH 7.4 (pD7.4),at37OC. ThereactionwasstartedbyadditionofNDPOz to give a final concentration of 40 mM and stopped by adding 10 mM sodium azide. The rate of ‘ 0 2 generation was monitored by the formation of NDP (9). At 5 min after addition of 40 mM NDPOZ,the rate of 1 0 2 generation was 345 pM/min and decreased to about 315 pM/min after 30 min and to 25 pM/min after 60 min. Typical conversions were 1%based on phenol, or 0.02% based on NDP02. (C) HPLC Assays. Hydroquinone and benzoquinone were identified and quantified using HPLC on a 4.5 X 125 mm
0 1993 American Chemical Society
Chem. Res. Toricol., Vol. 6, No. 4,1993 549
Hydroxylation of Phenol by Singlet Oxygen
I
benzoquinone
I
Table I. Effect of Additives on Singlet Oxygen-Induced Formation of Hydroquinone and Benzoquinone from Phenol and of 4-Hydroxyaniline from Aniline hydroquinone benzoquinone 4-hydroxyaniline (pM) additive (NM) 74 (rM) % % none sodiumazide (10 mM)
4.3 100 0.6 100
