Effect of Nucleophilicity of the Aryl Thiol Cofactor on the Antioxidant Activity

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. 1Chemical 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. Periodically, aliquots were determined and the time required for 50% conversion of injected onto the reversed-phase column (Princeton thiols into disulfides (t1/2 values) was calculated from the C18 column, 4.6×150 mm, 5 µm) and eluted with peak areas at different time intervals using calibration methanol and water (85:15). The concentrations of the plots for the respective disulfides. 1022 INDIAN J CHEM, SEC A, AUG-SEPT 2013

As shown in Table 1, the t1/2 values for all the present study. The poor catalytic activities of ebselen selenium compounds (1), (5) and (16)-(24) were and its analogues in the presence of an aromatic thiol found to be lower than the control values in the such as PhSH has been reported earlier.16,17 Almost presence of selenium compounds (supplementary similar activities were also observed for the sec-amide- data). For a better and simplified comparison, relative based diselenides (18) and (19) corresponding to activities of all the compounds were calculated with ebselen analogues (16) and (17). The poor activities of respect to the control reactions (in the absence of any sec-amide-based compounds are mainly due to the selenium compound) as shown in Table 2. It is clear presence of strong Se···O non-bonded interactions that from Tables 1 and 2 that ebselen (1) and its analogues, lead to an extensive thiol exchange reaction at the (16) and (17), exhibited very poor catalytic activities in selenenyl sulfide intermediate as reported previously.21 the presence of all the aromatic thiols used in the Interestingly, the Se···O interactions could be reduced by the replacement of sec-amide group with the corresponding tert-amide counterparts having higher catalytic activities.21 The relatively higher activity of the tert-amide substituted diselenides (20) and (21) is clearly observed with all the thiols used in the present study (Tables 1 and 2). Compound (20) with methyl substituent on amide nitrogen group showed the highest activity among all the amide-based selenium compounds screened in the present study. The higher activity for the tert-amide-based diselenides as compared to the sec-amide-based compounds is mainly due to the reduction of thiol exchange reactions.21 The relative activities of different amide-based compounds were also found to be dependent on the nucleophilicity of thiols used in the present study. Aromatic thiols such as (26)-(29) having electron donating groups at the para-position are more nucleophilic than the thiols with electron withdrawing groups such as (30) and Fig. 2Chemical structures of the selenium compounds (1), (5) (31). As expected, all the amide-based compounds (1) and (16)-(24) used in the present study for the reduction of H2O2 in the presence of aromatic thiols (25)-(31). and (16-21) exhibited much higher relative activities in

Table 1Reduction of H2O2 by different selenium compounds (1), (5) and (16)-(24) in the presence of different thiols (25)-(31) a Comp. t1/2 (min) 25 26 27 28 29 30 31 Control 763.0±19.6 788.0±26.8 1016.0±31.2 756.0±22.7 730.0±45.9 415.0±9.1 571.0±3.5 1 664.0±4.9 509.0±9.8 641.0±7.7 542.0±24.0 589.0±9.8 323.0±4.9 536.0±36.0 16 663.0±10.6 572.0±9.9 660.0±11.8 642.0±17.3 542.0±2.8 308.0±22.6 483.0±13.5 17 633.0±21.2 634.0±2.8 662.0±19.7 778.0±19.7 672.0±16.2 338.0±8.4 544.0±15.9 18 466.0±2.1 303.0±0.7 363.0±2.1 399.0±11.3 377.0±8.5 269.0±2.8 541.0±9.5 19 454.0±3.5 333.0±19.6 381.0±3.2 438.0±26.1 389.0±14.1 313.0±14.8 576.0±13.4 20 160.0± 1.5 93.6±6.5 117.0±9.1 145.0±9.0 114.3±3.2 129.0±4.3 464.0±4.3 21 239.0±11.6 199.5±2.7 201.0±7.7 223.0±7.7 180.0±5.6 185.0±10.8 501.0±8.4 5 23.9±1.6 10.2±0.5 15.1±0.9 20.7±1.4 18.3±1.1 5.2±0.4 7.4±0.6 22 29.5±1.7 18.2±0.9 23.6±0.1 30.0±1.1 29.9±1.7 18.2±0.5 19.9±2.4 23 6.6±0.1 10.0±0.8 12.2±1.3 11.1±0.5 10.9±0.8 6.5±0.1 26.6±0.2 24 30.3±1.7 24.1±1.0 22.5±2.1 17.4±1.1 28.9±1.3 19.8±1.7 53.6±4.1 a The reactions were carried out in MeOH at 22 °C. Catalyst: 10.0 µM [except compounds (23)-(24)]; ArSH: 1.0 mM; H2O2: 2.0 mM. The control reactions were performed under identical conditions in the absence of any selenium compound. A lower concentration of compounds (23) and (24) (5.0 µM) was used as the conversion was too fast to be measured at 10.0 µM concentrations in presence of the various thiols except PhSH. [In presence of PhSH, 10.0 µM concentration of the catalysts (23) and (24) were used]. BHABAK et al.: EFFECT OF NUCLEOPHILICITY ON ANTIOXIDANT ACTIVITY OF GPx MIMICS 1023

Table 2Relative activities of compounds (1), (5) and (16)-(24) in the presence of thiols (25)-(31) as compared to the control reactions Comp. Relative activity 25 26 27 28 29 30 31 Control 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1 1.14 1.54 1.58 1.39 1.23 1.28 1.06 16 1.15 1.37 1.54 1.17 1.35 1.34 1.18 17 1.20 1.24 1.53 0.97 1.08 1.22 1.05 18 1.63 2.60 2.79 1.89 1.94 1.54 1.05 19 1.68 2.36 2.66 1.73 1.87 1.32 0.99 20 4.76 8.42 8.68 5.21 6.40 3.21 1.23 21 3.19 3.94 5.05 3.39 4.05 2.24 1.14 5 31.92 77.25 67.28 36.52 39.89 79.81 77.16 22 25.86 43.29 43.05 25.2 24.41 22.80 28.6 23 115.6 157.6 166.5 136.2 135.18 127.69 42.93 24 25.18 65.66 88.34 86.89 50.34 41.92 21.46

the presence of thiols (26) and (29) than their activities corresponding amino group. As shown in Table 2, in the presence of (30) and (31) (Table 2). While a tert-amine-based diselenide (5) exhibited 6.7 times maximum rate for all the amide-based compounds was higher activity than the corresponding tert-amide observed in the presence of thiol (27) with 4-hydroxy analogue (20) and the sec-amine substituted diselenide group, the lowest activity was observed when thiol (31) (23) showed almost 70 times higher activity as with electron withdrawing 4-nitro group was compared to the corresponding sec-amide-based employed. diselenide (18). A similar increase in the activity is also

It is known that the amine-based diselenides exhibit observed for the isopropyl substituted amide and amine compounds. much higher antioxidant activities than the corresponding amide substituted GPx mimics.7a,7b,8 A As observed for the amide-based compounds, the number of tert-amine based diselenides with very high enhancement of the catalytic activity was found to be antioxidant activities have been reported in the dependent on the nucleophilicity of aromatic thiols. literature having very weak or no Se···N non-bonded However, the nucleophilicity of aromatic thiol has a interactions.7c,8 For example, the tert-amine-based more pronounced impact on the catalytic activity of diaryl diselenide (5) that possesses reasonably weak amine-based GPx mimics than that of amide substituted Se···N interaction exhibits moderate antioxidant compounds. In general, all the amine-based diselenides activity.7a,8 A dramatic enhancement in the antioxidant exhibited higher activities in the presence of thiols with activity was observed for compounds such as (7), (8) electron donating substituents on the phenyl ring. For and (23) having different substitutions. It should be example, while the relative activity of compound (5) in noted that all these modifications led to a weaker or no the presence of PhSH was ~32 times, almost 77, 67, 36 Se···N non-bonded interaction resulting in much less and 40 times higher activity was observed in the or no thiol exchange reactions.7c,8,22 As expected, the presence of thiols with -OMe, -OH, -Me, and -tBu tert-amine substituted compounds (5) and (22) substitutions, respectively, at the para-position of the exhibited significantly high activity in the presence of phenyl ring (Table 2). In addition to the electron all the aryl thiols, (25)−(31), used in the present study. donating substituents, a much higher relative activity of As shown previously, an enhancement in the catalytic (5) such as 80- and 77-fold respectively was observed in activity was observed for the corresponding sec-amine- the presence of the thiols (30) and (31) having electron based compounds (23) and (24). For example, in the withdrawing (–Cl and –NO2) groups on the phenyl ring. presence of thiophenol as cofactor, the Me-substituted While the tert-amine-based diselenides (5) and (22) amide-based compounds (16), (18) and (20) exhibited showed higher activities in the presence of substituted poor catalytic activities and only a slight increase in the thiols (26-31) than in presence of PhSH, the sec-amine activity (3-fold) is observed upon the conversion of substituted diselenides (23) and (24) exhibited lower sec-amide group (18) to the corresponding tert-amide relative activities in the presence of thiols (30) and (31) analogue (20). However, a dramatic enhancement is having electron withdrawing –Cl and –NO2 groups, observed upon the conversion of amide group to the respectively. These discrepancies are probably due to the 1024 INDIAN J CHEM, SEC A, AUG-SEPT 2013

higher basicity of the sec-amino group than that of the synthetic mimics, the present study suggests tert-amine analogues. Owing to the lower pKa of the 4-hydroxythiophenol (27) to be a better co-substrate for thiol group in (30) and (31), the incoming thiol future studies. Furthermore, the catalytic activities of approaching the sec-amine-based compounds may the known active GPx mimics that are screened in the undergo deprotonation in the presence of sec-amine- presence of PhSH may exhibit much higher rate in the based diselenides to the corresponding thiolates presence of 4-hydroxythiophenol, making them highly that are stabilized by the electron withdrawing 4-Cl potent GPx mimics. and 4-NO2 substituents, decreasing the nucleophilicity of the thiolate for catalysis. As the –NO2 group Conclusions is much more electron withdrawing than the In the present study, the GPx mimetic activity of a –Cl group, the relative activity of sec-amine-based series of amide- and amine-based organoselenium compounds (23) and (24) was the lowest in the compounds has been investigated in the presence of present of thiol (31). various aromatic thiols with electron donating and To understand the effect of different aromatic electron withdrawing substituents on the phenyl ring. thiols on the catalytic activity of GPx mimics, we This is the first study that describes the importance of have chosen the most active mimic (23) in the present electronic contribution of various substituents on study as a representative molecule. The formation of phenyl ring of aromatic thiols towards GPx activity of disulfides from different thiols in the presence of synthetic mimics. This study reveals that the compound (23) was plotted as a function of time as antioxidant activities can be significantly increased by shown in Fig. 3. It is clear from the plot that the the incorporation of a suitable electron donating group compound exhibited much higher activities in the such as 4-hydroxyl moiety on the phenyl ring of an presence of thiols with electron donating groups. The aromatic thiol. The higher GPx activity is probably maximum catalytic activity was observed in the due to the increase in the nucleophilic character of the presence of 4-hydroxy substituted thiophenol (27). thiol in the presence of an electron donating Similar to (23), all other mimics in the present study substituent. The electronic effect of para-substituents exhibited maximum activity in the presence of thiol is more pronounced for the GPx activity of amine- (27). While PhSH is commonly used as an aromatic based mimics than for that of the corresponding thiol cofactor for the determination of GPx activity of amide-based compounds. This study further indicates that 4-hydroxythiophenol acts as a better thiol cofactor than PhSH for screening new synthetic mimics in future.

Supplementary Data Supplementary data associated with this article, i.e., Tables S1-S84, are available in the electronic form at http://www.niscair.res.in/jinfo/ijca/IJCA_52A(8- 9)1019-1025_SupplData.pdf.

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