Indian Journal of Chemistry Vol. 52A, Aug-Sept 2013, pp. 1019-1025 Synthetic glutathione peroxidase mimics: Effect of nucleophilicity of the aryl thiol cofactor on the antioxidant activity Krishna Pada Bhabak, Debasish Bhowmick & Govindasamy Mugesh* Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore, 560 012, India Email: [email protected] Received 14 March 2013; accepted 30 April 2013 Catalytic activity of a series of potent amide- and amine-based organoselenium compounds are studied in the presence of various aromatic thiols having electron donating and electron withdrawing substituents on the phenyl ring. This study suggests that the antioxidant activities of the synthetic GPx mimics can be significantly increased by the incorporation of a suitable electron donating group on the phenyl ring of an aromatic thiol. Keywords: Glutathione peroxidase, Antioxidants, Organoselenium compounds, Aromatic thiols, Peroxide reduction, Thiols, Enzymatic activity, Enzyme mimics Glutathione peroxidase (GPx) is a selenocysteine with the reduction of peroxides. In presence of thiol containing mammalian antioxidant enzyme that cofactor such as GSH, the selenenic acid produces the catalyzes the reduction of harmful peroxides in the corresponding selenenyl sulfide intermediate presence of glutathione (GSH) as the thiol cofactor (E-SeSG). Nucleophilic attack of a second molecule and protects cells/biomolecules from oxidative of GSH at the Se-S bond regenerates the active site damage and inflammation.1 A catalytic mechanism with the release of GSH in its oxidized form (GSSG). has been proposed for the enzymatic activity that However, in the presence of a higher concentration of involves several redox reactions at the selenium peroxide such as in the condition of an ‘oxidative center of the active site selenocysteine residue. As stress’, the selenenic acid (E-SeOH) may undergo shown in Scheme 1, the selenol moiety (E-SeH) at the further oxidation to produce the corresponding active site reacts with peroxide (H2O2) and undergoes seleninic acid (E-SeO2H) or selenonic acid (E-SeO3H) oxidation to generate the selenenic acid (E-SeOH) intermediates that reduces the catalytic activities. (Scheme 1).1e-g Therefore, a rapid conversion of the selenenic acid to selenenyl sulfide and the regeneration play important roles for the higher catalytic activity. Owing to the high importance of GPx for the mammalian antioxidant defense, during the last few decades much research attention has been directed towards mimicing the active site structure of GPx. Several research groups have been working on the design and synthesis of small molecule organoselenium compounds that can mimic the GPx enzyme activity in the presence of thiol cofactor.2 The first successful synthetic GPx mimic reported in the literature was ebselen (1, 2-phenyl-1,2- benzisoselenazole-3-(2H)-one), which functions as an interesting anti-inflammatory drug.3 After the discovery of ebselen, different types of small- molecule selenium containing GPx mimics were discovered as shown in Fig. 1.4-14 These mimics 1020 INDIAN J CHEM, SEC A, AUG-SEPT 2013 Fig. 1Chemical structures of some representative synthetic GPx mimics reported in the literature. include heterocyclic compounds having Se-N selenol species for the catalysis. The undesired thiol covalent bond (1-4), various diselenides (5-9), exchange reaction is further enhanced by the presence monoselenides (10-12), cyclic selenenate ester (13) of Se···O/ Se···N nonbonded interactions. These thiol and spirodiazaselenurane (14). Based on their exchange reactions are more pronounced when reactivity towards thiols and peroxides, different aliphatic/aryl thiols are used as cofactor instead of catalytic mechanisms have been proposed for various natural cofactor GSH. For example, while ebselen has GPx mimics. Particularly, the diaryl diselenides been shown to exhibit reasonably good catalytic having basic amino group in the close proximity to activity with natural thiol GSH as the cofactor, it the selenium center such as (5-7) were found to mimic shows very poor antioxidant activity in the presence the GPx activity very effectively, mainly due to the of aryl/benzyl thiol for the reduction of peroxides.15-18 presence of the amino group, which plays crucial This was further supported by our studies on the roles in the catalytic mechanism.7b Notably, these detailed catalytic activities of ebselen and its diselenides follow the catalytic mechanism similar to analogues in the presence of different thiols and the native GPx enzyme involving selenol, selenenic peroxides.17 While the activities were much different acid and selenenyl sulfide as intermediates.1e-g in the presence of GSH and PhSH, the activities were In contrast to the amine-based mimics, ebselen and almost identical when different peroxides such as 17 sec-amide-based analogues exhibit relatively poor H2O2, Cum-OOH and t-BuOOH were used. A antioxidant activity due to the extensive thiol exchange number of different approaches were employed to reaction at selenenyl sulfide intermediate.15-17 Due to overcome this complication with thiol exchange the higher electrophilicity of selenium than sulfur, the reaction with various modifications in the synthetic incoming thiol preferably attacks at the selenium mimics to efficiently reduce peroxides.16,19,20 In center at Se-S bond in the selenenyl sulfide addition to this, it was thought worthwhile to study intermediate, preventing the regeneration of active the influence of the electronic effects of various BHABAK et al.: EFFECT OF NUCLEOPHILICITY ON ANTIOXIDANT ACTIVITY OF GPx MIMICS 1021 functional groups at the aromatic ring of PhSH on the disulfide (RSSR) produced in the reaction were antioxidant activities of well-known GPx mimics. determined at 254 nm (315 nm for p-NO2C6H4SH) Therefore, in the present study, we report the with the aid of calibration plots of pure disulfides influence of different electron donating and (RSSR) as an external standard. The plots for t1/2 were withdrawing substituents on the aromatic ring in obtained by sigmoidal curve fitting. The concentration PhSH towards its nucleophilic reactivity. of thiol and H2O2 used in the assay were 1.0 mM and 2.0 mM, respectively. The catalyst concentration used Materials and Methods was 10 µM (except compounds 23 and 24. 10 µM and General procedure 5 µM concentrations were used for compounds n-Butyllithium (nBuLi) was purchased from Acros 23 and 24 with different PhSH and thiols, Chemical Co. (Belgium). Methanol was obtained respectively). from Merck and dried before use. All other chemicals were of the highest purity available. All the reactions Results and Discussion were carried out under nitrogen with use of standard Considering the presence of glutamine (Gln) and vacuum-line techniques. Because of the unpleasant aspartate (Asp) residues in the close proximity of 23 odors and toxic nature of several of the reaction selenocysteine (Sec) center at the active site of GPx, mixtures involved, most manipulations were carried most of the synthetic mimics were designed having an out in a well-ventilated fume hood. Et2O and THF amino or amide group close to the selenium center. A was dried over sodium metal with benzophenone. number of compounds having much improved Thin layer chromatography analyses were carried out antioxidant activities as compared to the existing amine on pre-coated silica gel plates (Merck), and the spots and amide-based synthetic GPx mimics have been were visualized with UV radiation. Column reported in last few years from our group. For example, chromatography was performed on glass columns the antioxidant potency of the amide-based mimics loaded with silica gel or on automated flash could be improved by replacing the sec-amide group 21 chromatography systems (Biotage) with use of with the corresponding tert-amide counterpart, preloaded silica cartridges. 1H (400 MHz), whereas the activity of the benzylamine-based mimics 13C (100.56 MHz), and 77Se (76.29 MHz) NMR could be increased further either by the incorporation spectra were obtained on a Bruker 400 MHz NMR of a methoxy group on the phenyl ring or by replacing 8,22 spectrometer. Chemical shifts are cited with respect to the tert-amino group with a sec-amino group. 1 13 SiMe4 as internal ( H and C) and Me2Se as external Keeping all these developments in mind, we have (77Se) standard. Mass spectral studies were carried out chosen some mimics such as (1), (5) and (16-24) as on a Bruker Daltonics Esquire 6000 plus mass model GPx mimics (Fig. 2) to study the electronic spectrometer with ESI-MS mode analysis. effect of different substituents on the phenyl ring of Compounds (1), (5), (15)-(23) were synthesized PhSH towards GPx activities in the presence of H2O2 3,7a,8,17,21,22 following the literature methods.3,7a, 8,17,21,22 as substrate. Several electron donating groups (-OMe, -OH, -Me and -tBu) and electron withdrawing Determination of GPx-like activity by HPLC method groups (-Cl and -NO2) are used at the para-position of GPx-like activity was carried out by high the phenyl ring of PhSH. The disulfides corresponding performance liquid chromatography (HPLC) to the thiols (25)-(31) (Fig. 2) were synthesized consisting of a 2695 separation module and a 2996 following the literature method.24 The pure disulfides of photodiode-array detector and a fraction collector. known concentrations were used to make independent The assays were performed in 1.8 mL sample vials calibration plots to determine the antioxidant activities and a built-in autosampler was used for sample of the mimics. During the assay, the formation of injection. In this assay, mixtures containing a 1:2 disulfide from the corresponding thiol in the presence of molar ratio of thiol and peroxide in methanol at room different selenium compounds was monitored by temperature (22 oC) were used as model systems. reverse-phase HPLC method. The amount of Runs with and without catalyst were carried out under corresponding disulfide produced in each reaction was identical conditions.