Structural requirements for the ferrihemoglobin-forming activity of glutathione S-conjugates of 4-dimethylaminophenol.

4-Dimethylaminophenol (DMAP) is a suitable cyanide antidote that rapidly forms ferrihemoglobin by catalytic transfer of electrons from ferrohemoglobin to oxygen. Deleterious methemoglobinemia, because of the catalytic cycling, is prevented by side reactions of oxidized DMAP with thiols, particularly with glutathione (GSH). In human red cells, both in vitro and in vivo, the formation of a transient bis-glutathione and a stable tris-glutathione adduct was observed. To investigate the reactivity of GSH adducts of DMAP, we synthesized various thioethers by oxidizing DMAP with PbO2 in 0.1 M sulfuric acid followed by reaction with GSH. The following compounds were isolated and characterized by 1H-NMR spectroscopy and determination of the pK values: 4-dimethylamino-2-(glutathione-S-yl)-phenol (2-GS-DMAP), 4-dimethylamino-3-(glutathione-S-yl)-phenol (3-GS-DMAP), 4-dimethylamino-2,5-bis(glutathione-S-yl)-phenol (2,5-bis GS-DMAP), 4-dimethylamino-2,6-bis(glutathione-S-yl)-phenol (2,6-bis GS-DMAP), and 4-dimethylamino-2,3,6-tris(glutathione-S-yl)-phenol (2,3,6-tris GS-DMAP). Ferrihemoglobin-forming activity was investigated with oxyhemoglobin, alkylated with N-ethylmaleimide (Hb-NES) to prevent binding of oxidized compounds to the protein SH groups. DMAP, 2,6-bis-GS-DMAP, and 2-GS-DMAP (0.1 mM each) completely oxidized Hb-NES (0.6 mM) in a decreasing order of activity (pH 7.4, 37 degrees C, air); the other derivatives were quite inactive. The same thioether reactivity was observed during autoxidation. Ferrihemoglobin formation by the reactive thioethers was greatly enhanced when the oxygen tension was increased from 2 to 100%. In contrast, variation of the oxygen tension had only marginal effects on the activity of DMAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Introduction 4-Dimethylaminophenol (DMAP) is a suitable cyanide antidote that rapidly forms ferrihemoglobin by catalytic transfer of electrons from ferrohemoglobin to oxygen. Deleterious methemoglobinemia is prevented by side reactions of oxidized DMAP with thiols, particularly with GSH. In human red cells, both in vitro and in vivo, the formation of a transient bis-glutathione and a stable tris-glutathione adduct was observed. The former still produced ferrihemoglobin, whereas the latter was inactive (1). This behavior contrasted with analogous thioethers from 1,4-hydroquinone and 4- aminophenol. Therefore, we decided to prepare a variety of glutathionyl derivatives of DMAP to investigate their reactivity. (DMAP) and the radioactive compound (U-4C-phenyl)-DMAP, specific activity 9 mCi/ mmoles, were synthesized by Farbwerke Hoechst (Frankfurt, Germany). Purified human hemoglobin, virtually free from catalase, superoxide dismutase, and glutathione peroxidase, was prepared by gel filtration and ion exchange chromatography (2). N-ethylmaleimide (Hb-NES) was prepared by incubating oxyhemoglobin with 1.1 equivalents Hb-NES (referred to SH groups) followed by dialysis against 0.2 M sodium phosphate buffer, containing 0.1 mM EDTA, pH 7.4, overnight at 40C. Hemoglobin and ferrihemoglobin were measured by the method reported by Kiese (3). HPLC was usually performed on i-Bondapak C18 (3.9 x 300 mm). The thioethers were separated with a formic acid (50 mM)/methanol (up to 20%) gradient, 1.5 ml/min. Radioactivity was measured in Bray's solution (4). Gas mixtures were prepared by a mixing pump from Wosthoff (Bochum, Germany). The pK values were determined spectroscopically in the presence of 2 mM sodium disulfite under argon to avoid autoxidation. The UV spectra showed isosbestic points that allowed estimation of the pK values according to the Henderson-Hasselbalch equation (5).

Results and Discussion
Fonnation of Glutathione S-Conjugates of DMAP To quantify the reaction products, U-14Cphenyl-DMAP was oxidized and reacted with GSH as described (smaller scale) and analyzed by HPLC. Besides DMAP, five compounds were formed. Figure 1 shows the product pattern after separation by HPLC. The distribution of radioactivity is shown in the lower part. The product pattern is indicative of sequential oxidation/ addition reactions. When isolated 2-GS-DMAP was oxidized and reacted with GSH, 2,6-bis GS-DMAP together with 2,3,6-tris GS-DMAP were formed. Such a reaction sequence was also formed when oxyhemoglobin served as oxidant (7). The pK values of the isolated derivatives as determined by spectroscopic titration (  (8). These data indicate that the electronic conjugation of the nitrogen lone-pair electrons with the aromatic ring may be impaired by the bulky glutathione substituent.

Autoxidaion ofDMAP and Its Glutathione S-Conjugates
In the absence of oxygen, DMAP in 0.2 M phosphate buffer, pH 7.4, at 37°C, was found to be stable for hours (9). Under air, half the DMAP had been autoxidized within 30 min. While 2-GS-DMAP and 2,6-bis GS-DMAP autoxidized more rapidly than DMAP, the other derivatives were quite stable ( Figure 2). These data suggest that derivatives with ortho substituents to the dimethylamino group are no longer susceptible to autoxidation. Interestingly, autoxidation kinetics showed a lag phase, indicating that reaction products may accelerate the autoxidation process (10).

Ferrihemoglobin Formation by DMAP and Its Glutathione S-Conjugates
The ferrihemoglobin-forming activity was investigated with Hb-NES to prevent binding of the compounds to the protein SH groups. DMAP, 2,6-bis GS-DMAP, and 2 order of activity; the other derivatives were rather inactive (Figure 3). This reactivity correlated with the proneness to autoxidation. In contrast to DMAP, ferrihemoglobin formation by the reactive thioethers exhibited an induction period.

Conclusion
Of the various glutathione S-conjugates of DMAP, either prepared chemically or formed within red cells, two were found to be rather reactive. 2-GS-DMAP and 2,6bis GS-DMAP autoxidized more rapidly than DMAP and formed ferrihemoglobin at an extent similar to DMAP. In contrast to the parent compound, the rate of ferrihemoglobin formation by the thioethers exhibited a lag phase and a marked influence on the oxygen tension, indicating that autoxidation products of the thioethers may be the ultimate oxidizing agents. The inactivity of glutathione S-conjugates substituted vicinal to the dimethylamino group points to steric effects of the bulky substituent leading to some distortion of the dimethylamino group, thereby inhibiting electronic conjugation of the system. The results illustrate once more that thioether formation of reactive arylamines should not be regarded as an exclusive detoxication reaction.