In the H NMR spectrum two pairs of doublet resonances

In the 1H NMR spectrum two pairs of doublet resonances arising from the protons in the aromatic moiety of the two diastereoisomers can be readily observed around 7.5–7.6 ppm. These resonances can be used to detect and quantify the two diastereoisomers. From the results presented in Fig. 1A it can be derived that, upon chemical synthesis of EASG, equal amounts of A and B are formed, since the ratio of the peak areas is 48:52. From spectra obtained at increasing time intervals upon addition of EA to a 5 mM GSH solution the chemical formation of the glutathione conjugates could be followed in time. A spectrum of the incubation at one hour is shown in Fig. 1B. After 1 h, product A and product B were formed in a ratio of 46:54. About 24% of the parent compound ethacrynic Harringtonine is still present as well as 9% of an intermediate (peaks are observed around 7.13 and 6.77 ppm; Fig. 1B). A considerable amount of this intermediate is formed during the conjugation reaction, and it is most likely the enol tautomer of the glutathione conjugate, as presented in Fig. 2. However, the compound was not further identified in the present study. Fig. 1C presents the 1H NMR spectrum of an incubation of glutathione and ethacrynic acid in the presence of 47.6 μM GSTP1-1. The spectrum clearly shows that the GSTP1-1 catalyzed reaction between EA and GSH is stereoselective and results in formation of diastereoisomer A. At the highest concentration GSTP1-1 tested (47.6 μM), 87% of the formed EASG was diastereomer A, leaving 13% product B and about 7% of free ethacrynic acid after 40 min. The enzyme catalyzed conjugation reaction was corrected for the contribution of the chemical conjugation reaction, by subtracting the amount of diastereoisomers formed in the chemical reaction from the amount formed in the enzyme catalyzed reaction, in the linear part of the reaction. It appeared that no diastereoisomer B was formed by the enzyme catalyzed conjugation. Thus, GSTP1-1 is 100% stereoselective for formation of diastereoisomer A. In the enzyme catalyzed reaction only 18% of the EA added to the reaction mixture is present as the intermediate form after 1 min, rapidly decreasing to 14% after 2 min and to 0% after 12 min. When the formation of the intermediate conjugate is corrected for the chemical reaction, it appears that the amount of intermediate formed during the enzyme catalyzed reaction is essentially the result of the chemical reaction. No tautomerization of diastereoisomer A could be detected in the time course of the experiments (data not shown). Table 1 summarizes the stereoselectivity data for GSTP1-1 and other isoenzymes tested in various concentrations, corrected for the contribution of the chemical reaction. At low concentrations of enzyme the reactions were dominated by the chemical reaction; at higher concentrations of GST the enzyme catalyzed reaction became dominant and stereoselectivity could be registered. The GSTP1-1 mutant C47S was used to study the effect of the cysteine 47 residue on the stereoselectivity of the enzyme toward the reaction of EA and GSH. The reaction catalyzed by this mutant (C47S) showed equivalent stereochemistry as GSTP1-1. The isoenzymes of the α-class both catalyze the glutathione conjugation of ethacrynic acid. Only GSTA1-1 showed stereoselectivity, in favor of product A, but the formation of diastereoisomer B was catalyzed as well, although to a lesser extent. To investigate whether the stereoselectivity of EA-GSH conjugation observed in vitro with purified enzymes would also be relevant for EA-GSH conjugation in whole cells, where pH values are generally above 6.1, IGR-39 human melanoma cells were exposed to ethacrynic acid and the EASG, excreted into the medium, was analyzed for its diastereoisomeric content. Fig. 1D shows a typical 1H NMR spectrum of the medium after 2 h of exposure to EA. The chemical shift of the conjugates in the spectrum is slightly different from the spectrum of the purified EASG. This is the result of the high salt concentration, resulting from the HBSS. However, spiking the samples with purified EASG and EA showed that the proton resonances arise from the ethacrynic acid-glutathione conjugates. From Fig. 1D it appears that diastereoisomer A of EASG is the principal conjugate present in the medium of EA exposed IGR-39 cells.