4-Substituted 1-Chloro-2-nitrobenzenes:  Structure−Activity Relationships and Extension of the Substrate Model of Rat Glutathione S-Transferase 4-4

Abstract

In the present study, eleven 4-substituted 1-chloro-2-nitrobenzenes were tested for their GSH conjugation capacity when catalyzed by base or rat glutathione S-transferase (GST) 4-4. Kinetic parameters (ks and K(m), kcat, and kcat/K(m)) were determined and subsequently used for the description of structure-activity relationships (SAR's). For this purpose, eight physicochemical parameters (electronic, steric, and lipophilic) of the substituents and five computer-calculated parameters of the substrates (charge distributions and several energy values) were used in regression analyses with the kinetic parameters. The obtained SAR's are compared with corresponding SAR's for the GSH conjugation of 2-substituted 1-chloro-4-nitrobenzenes, previously determined [Van der Aar et al. (1996) Chem. Res. Toxicol. 9, 527-534]. The kinetic parameters of the 4-substituted 1-chloro-2-nitrobenzenes correlated well with the Hammett sigma p- constant; the Hammett sigma p constant corrected for "through resonance", while the corresponding kinetic parameters of the 2-substituted 1-chloro-4-nitrobenzenes did not. The base- and GST 4-4-catalyzed GSH conjugation reactions of 2-substituted 1-chloro-4-nitrobenzenes depend to a different extent on the electronic properties of the ortho substituents, suggesting the involvement of different rate-limiting transition states. The base- and GST 4-4-catalyzed conjugation of 4-substituted 1-chloro-2-nitrobenzenes, however, showed a similar dependence on the electronic properties of the para substituents, indicating that these substrates are conjugated to GSH via a similar transition state. Multiple regression analyses revealed that, besides electronic interactions, also steric and lipophilic restrictions appeared to play an important role in the GST 4-4-catalyzed GSH conjugation of 4-substituted 1-chloro-2-nitrobenzenes. Finally, the 4-substituted 1-chloro-2-nitrobenzenes were also used to extend the previously described substrate model for GST 4-4 [De Groot et al. (1995) Chem. Res. Toxicol. 8, 649-658], by which a specific steric restriction of substrates for GST 4-4 became clear.

Online Biochemical Detection of Glutathione-S-Transferase P1-Specific Inhibitors in Complex Mixtures

“…Acrolein was obtained from Acros (Den Bosch, the Netherlands). 4-Chloro-3-nitrobenzoic acid butyl ester 26 and n-butyl-2-chloro-5-nitrobenzoate 27 were synthesized as described by Van der Aar et al 27 Sodium cholate, cinnamaldehyde, 4-chloro-3-nitrobenzaldehyde, and 2-chloro-5-nitrobenzonitrile were obtained from Aldrich (Zwijndrecht, the Netherlands). Enzyme-linked immunosorbent assay (ELISA) blocking reagent was purchased from Roche (Mannheim, Germany), and bovine serum albumin (BSA) was from Life Technologies (Paisley, UK).…”

“…Table 1 lists all IC 50 values obtained. CDNB, a commonly used substrate for GSTs, 26 was also tested. CDNB, however, gave significant quenching of fluorescence at high concentrations (> 0.5 mM) and hence prevented the construction of accurate IC 50 curves.…”

Fluorogenic probes using 4-substituted-2-nitrobenzenesulfonyl derivatives as caging groups for the analysis of human glutathione transferase catalyzed reactions

“…To date, the effects of electron-withdrawing groups on S N Ar reactions catalyzed by rat GSTs have been examined using several 2-or 4-substituted CDNB analogs, and a linear correlation was reported between the reactions catalyzed by the rat GSTs and the Hammett substituent constant (s À ). [21][22][23][24][25] For the 2-substituted CDNB analogs, 23,24 the catalysis of the reaction by GST1-1 and GST7-7 was found to be dependent on the s value in the same way as the non-enzymatic reaction. In contrast, the reactions catalyzed by GST3-3 and GST4-4 were independent of the s value.…”

Development of Specific Electrochemical Biosensor Based on Chitosan Modified Screen Printed Carbon Electrode for the Monitoring of Captan Fungicide

“…5. In the sole substrate buffer, almost obvious one oxidation and one reduction charging current peaks appeared at Epa of 0.27 V and Epc of 0.16 V owing to the traditional GSH-CDNB conjugation reaction by GST catalysis [13] that can be summarized as CDNB + GSH �⎯� GS-DNB + HCl . The peak separation between them was about 0.1 V indicating profuse diffusion behaviour of the analytic substrate toward the electrode surfaces ( Fig.…”