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Catalytic effects of glutathione peroxidase mimetics on the thiol reduction of cytochrome c
Chemico-Biological Interactions ELSEVIER
Chemico-Biological Interactions 93 (1994) 129-137
Catalytic effects of glutathione peroxidase mimetics on the thiol reduction of cytochrome c Lars Engman ~a , Anders Tunek b, Mathias Hallberg b, Anders Hallberg c aUppsala University, Institute of Chemistry, Department of Organic Chemistry, Box 531, S-751 21 Uppsala, Sweden bDepartment of Pharmacology, Astra Draco AB, P.O. Box 34, S-221 O0 Lund, Sweden CDepartment of Organic Pharmaceutical Chemistry, Uppsala University, P.O. Box 574, S-751 23 Uppsala, Sweden
Received 8 December 1993; revision received 28 January 1994; accepted I February 1994
Abstract
The reduction of ferric cytochrome c by various thiols was studied. It was found that Lcysteine, t-cysteine methyl ester and D-penicillamine were very efficient reductants for cytochrome c, whereas N-acetylated aminoacids (N-acetyl-L-cysteine and N-acetyl-Dcysteine) reacted considerably slower. A series of glutathione peroxidase mimetics and related compounds were studied as catalysts for the N-acetyl-t-cysteine reduction of ferric cytochrome c. Diphenyl diselenide, t-butylthio phenyl selenide, S-(phenylseleno)-glutathione, N-(phenylseleno)-phthalimide and a-(phenylselenenyl)-acetophenone were all efficient reduction catalysts. Diphenyl disulfide, Ebselen and several derivatives thereof were less potent catalysts whereas diaryl selenides and diphenyltelluride did not affect the rate of reduction when present in catalytic amounts. The catalysis of diphenyl diselenide, selenosulfides, a(phenylselenenyl)acetophenone, N-(phenylseleno)-phthalimide and Ebselen and derived compounds was suggested to involve the formation of areneselenolate ions as redox-active species capable of transferring one electron to the ferric cytochrome c. The resulting selenium centered arylseleno radicals would then dimerize to regenerate the catalyst in the diselenide form. In the presence of diaryl ditellurides and N-acetyl-L-cysteine, ferric cytochrome c was also rapidly reduced. This reaction was stoichiometric with respect to the ditelluride reagent. Keywords: Cytochrome c; Thiols; Diphenyl diselenide; Selenosulfides; Ebselen; a-(Phenyl-
selenenyl)-acetophenone; Diaryl ditellurides; Selenolate ions; Electron transfer; Catalytic mechanism * Corresponding author. 0009-2797/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009-2797(94)03277-F
130
L. Engman et al. ~Chem.-Biol. Interact. 93 (1994) 129-137
1. Introduction
Aerobic cells are susceptible to oxidative damage once their antioxidant capabilities are overwhelmed. Due to their high reactivity towards important biomolecules, oxygen species formed (superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen) are particularly harmful to the cell [1]. Another kind of oxidative stress damage is due to peroxidative degradation of unsaturated lipids in cell membranes [2]. Biological systems have developed several lines of protection against oxidants. These defence systems include antioxidants/radical scavengers like c~tocopherol and ascorbic acid, enzyme systems like glutathione peroxidases, superoxide dismutases and catalase, metal chelating proteins like ferritin, transferrin, lactoferrin and others [3]. The selenium containing glutathione peroxidases are believed to play a central role in the inactivation of peroxides, thus preventing the formation of alkoxyl, peroxyl and hydroxyl radicals. A few years ago, the organosetenium compound Ebselen (la) was described and shown to possess glutathione peroxidase-like activity [4,5]. Recently, a variety of organoselenium and organotellurium compounds were also shown to act as glutathione peroxidase mimetics. These compounds include diaryl diselenides [6], selenosubtilisin [7], c~-(phenylselenenyl)-acetophenone derivatives [8], diaryl ditellurides [9] and diaryl tellurides [10,11]. The highly reactive and biologically damaging free hydroxyl radical, or a reactive intermediate related to it [12], can be formed from hydrogen peroxide when suitable transition metals are present. The most abundant metal ion likely to catalyse this reaction is iron [13] (Fenton reaction; Eq. 1). There is also evidence that iron proteins act as Fenton catalysts, probably via induced release of ferrous iron from the proteins [14]. Thus, to minimize the toxic effects of hydroxyl radical formation in biological systems, it would seem desirable to keep the Fe(I0/Fe(lll) ratio low. It has been demonstrated [15,16] that certain selenium containing compounds (selenocysteine, selenite and glutathione peroxidase) catalyse the thiol mediated one-electron reduction of ferric cytochrome c [17a, 17b]. Since the ferric cytochrome c is probably a representative model for haeme proteins and its reduction can be monitored with great sensitivity and specificity by measuring the absorbance change at 550 nm, we decided to study the potentially toxic catalysis of its reduction by some recently developed glutathione peroxidase mimetics. 2. Materials and methods
2.1. Compounds All thiols used were commercially available except for Noacetyl-D-cysteine which was obtained from Astra Draco AB (Lund, Sweden). Cytochrome c from horse heart (type III) was obtained from Sigma. Catalysts tested were obtained from commercial sources or prepared according to literature methods Scheme 1 (1-15): Ebselen (la) [ 18], 2-phenyl- 1,2-benzisothiazol-3(2H)-one (1 b) [ 18], 2,2' -diselenobis(benzanilide) (2a) [18], diphenyl selenide (3a) [19], bis(4-aminophenyl) selenide (3b) [20], bis(4(dimethylamino)phenyl) selenide (3c) [21], bis(4-acetamidophenyl) selenide (3d) [20],
L. Engman et al./Chem.-Biol. Interact. 93 (1994) 129-137
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132
L. Engman et aL ~Chem.-Biol. Interact. 93 (1994) 129-137
bis(4-nitrophenyl) selenide (3e) [22], diphenyl telluride (4) [23], diphenyl ditelluride (5a) [24], bis(4-methoxyphenyl) diteiluride (5b) [24], bis(4-aminophenyl) ditelluride (5e) [10], bis(4-(dimethylamino)phenyl) ditelluride (5d) [24], t-butylthio phenyl selenide (8a) [9], S-(phenylselenenyl)-glutathione (8b) [25], ~-(phenylselenenyl)acetophenone (9) [8], 2,2'-diselenobiphenyl (10) [26], S-(N-phenyl(2-carboxamido)phenylselenenyl)-glutathione (llb) [27], l-octylthio-N-phenyl(2-carboxamido)phenyl selenide (lle) [9], t-butylthio phenyl telluride (14) [9].
2.2. t-Butylthio-N-phenyl ( 2-carboxamido ) phenyl telluride (15a ) To a stirred solution of t-butyl mercaptan (0.020 g, 0.22 mmol) in CH2C12 (5 ml) was added N-phenyl(2-carboxamido)phenyltellurenyl iodide [18] (0.080 g, 0.18 mmol) and dry pyridine (30/~1, 0.18 mmol). After 16 h at ambient temperature, the reaction mixture was evaporated under reduced pressure in the presence of some silica gel and submitted to flash chromatography (SiO2/CH2C12:hexanes = 1:1) to afford the title compound, 0.066 g (90%), m.p. 134-135°C. Analysis calculated for C17H19NOSTe: C, 49.44; H, 4.64. Found: C, 49.42; H, 4.72. JH NMR 6(CDC13): 1.43 (s, 9H), 7.20 (t, 1H), 7.33-7.53 (several peaks, 4H), 7.61-7.65 (several peaks, 2H), 7.74 (d, 1H), 8.07 (s, 1H), 8.58 (d, 1H). 2.3. S-[N-phenyl(2-carboxamido)phenyltellurenyl] glutathione (15b) To a stirred solution of N-phenyl(2-carboxamido)phenyltellurenyl iodide (0.10 g, 0.22 mmol) in DMF (1.0 ml) was added glutathione (0.068 g, 0.22 mmol) and dry pyridine (18 #1, 0.22 mmol). Addition of water (1 ml) caused an exothermic reaction. Further addition of water (5 ml) to the resulting clear yellowish solution caused separation of an oil. This crystallized upon trituration with ethanol to give 0.106 g (72%) of yellowish crystals of the title compound, m.p. 205-208°C. The material could not be further purified by recrystallization. Analysis calculated for C23H26N4OySTe: C, 43.84; H, 4.16. Found: C, 42.77; H, 3.96. ~H NMR 6(DMSO-d6): 1.80-2.05 (several peaks, 2H), 2.29-2.48 (several peaks, 2H), 3.18 (d, 2H), 3.36 (t, IH), 3.67 (d, 2H), 4.40 (m, 1H), 7.20 (t, 1H), 7.38-7.74 (several peaks, 6H), 8.33-8.51 (several peaks, 3H), 8.67 (t, IH), 10.82 (s, 1H). 2.4. Cytochrome c reduction assay Instrumentation. A Varian DMS 100 double beam spectrophotometer was used. The absorbance at 550 nm was continuously recorded. Solutions. The assay buffer was 0.1 M KH2PO4, pH 7.5, containing 0.05% EDTA. Cytochrome c was dissolved in the assay buffer (18 mg/ml; 1.5 mM) and this stock solution kept one ice. The catalysts were dissolved in CH3CN (3 mM) and kept at ambient temperature. Stock solutions of thiols in the assay buffer were normally 240 mM. These were freshly prepared (on the day) and kept on ice. Assay. The cuvette holders and the assay buffer were maintained at 37°C. The reference cuvette contained only the assay buffer. To the sample cyvette were added assay buffer (2.75 ml) and cytochrome c solution (100 tA). After 3 rain, thiol solution (100 tA) appropriately diluted was added, followed after another 7-10 min by catalyst solution (50 ~1). Acetonitrile in the concentrations used did not influence the reduction rate which was expressed as absorbance increase/min.
L. Engrnan et aL / Chern.-BioL Interact. 93 (1994) 129-137
133
3. Results and discussion
A series of thiols were tested with respect to their ability to reduce ferric cytochrome c in the absence of added catalysts but in the presence of EDTA as a chelating agent. Without this chelator, the reduction of cytochrome c by any thiol was considerably faster and variable due to transition metal catalysis of the reaction. It was found that L-cysteine, L-cysteine methyl ester and D-penicillamine were very efficient reducing agents for cytochrome c (at 550 nm the absorbance increased > 0.2 absorbance units/min in 1.0 mM solutions of the thiols containing 50 #mol/l of ferric cytochrome c) whereas the corresponding N-acetylated aminoacids reacted considerably slower (the absorbance increased ---0.002 absorbance units/min for Nacetyl-L-cysteine and N-acetyl-D-cysteine). Since the D and L forms of Nacetylcysteine were equally reactive, the steric requirements in this electron transfer process do not seem essential. This is in contrast to the powerful chiral recognition observed with these compounds towards deacetylases [28]. The lower reactivity of N-acylated amino acids towards cytochrome c may be due to the higher pKa value of the thiol group, resulting in a lower concentration of the redox-active thiolate form of the compounds (pKa = 8.5 for the sulfhydryl group of cysteine and 9.5 for N-acetylcysteine [29]). By using N-acetyl-L-cysteine as a thiol reducing agent in the ferric cytochrome c system, we then investigated the catalytic activity of a series of glutathione peroxidase mimetics and related compounds with the potential of being good one-electron donors. It turned out that diphenyl selenide (3a) and a variety of 4-substituted derivatives 3b-3e thereof, as well as diphenyl telluride (4), had little, if any, catalytic activity when present at the 10 moi% level with respect to cytochrome c. However, diphenyl diselenide (2b) and diaryl ditellurides (5) were found to actively promote the reaction. Interestingly, the mode of action for the two classes of compounds is quite different, as shown in Fig. 1 for diphenyl diselenide and diphenyl ditelluride. Whereas the organoselenium compound acts in a truly catalytic fashion to reduce cytochrome c (initially very rapidly, then more slowly), all four organotellurium compounds seem to act in a stoichiometric fashion as judged by the amount of ferrous cytochrome c formed in the rapid initial reaction. Fig. 2 shows the increase of the absorption of ferrous cytochrome c at 550 nm with time as the concentration of diphenyl diselenide is varied. In the absence of thiols, diphenyl diselenide and other catalysts tested did not reduce ferric cytochrome c. As shown in Fig. 1, diphenyl disulfide (6) is a poorer catalyst than its corresponding selenium and tellurium analogues. Ebselen (la) catalysed the reduction of cytochrome c very poorly (Fig. 1). The sulfur analogue lb of Ebselen was completely void of any catalytic effect. It is well known that an equilibrium is rapidly established when diselenides and thiols are brought together [30,31] (Eqs. 2-3; diphenyl diselenide used as an example). It is likely that the benzeneselenolate ion 7 formed acts as a one-electron reductant towards cytochrome c as shown in Eq. 4. The resulting selenium centered radical could then dimerise to regenerate the catalyst in the diselenide form [15]. The spontaneous reduction of cytochrome c by N-acetyl-L-cysteine and other thiols probably also involves electron transfer from thiolate ions to iron. However, since
134
L. Engman et al. ~Chem.-Biol. Interact. 93 (1994) 129-137
Absorbance (550 nm) 1.11.00.9 0.8
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Absorbance (550 nm) 1.1 7
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Fig. 2. Absorbance as a function of time for the N-acetyl-L-cysteine (1 mM) reduction of cytochrome c (50/~M) in the presence ofdiphenyl diselenide (0.625 #M, 1.25 #M, 2.5 ~M, 5/zM and 10 #M final concentration). The catalyst was added at ,' = 1.5 rain to an aqueous solution (pH = 7.5) of the other reactants containing 0,05% EDTA.
L. Engman et al. ~Chem.-Biol. Interact. 93 (1994) 129-137
135
selenolate ions are stronger reductants than thiolate ions (Se is less electronegative than S) and selenols are more acidic than thiols (benzeneselenol is essentially dissociated at physiological pH, whereas, for example, N-acetylcysteine contains only a minute fraction of the thiolate form) the uncatalysed reduction of cytochrome c by thiols is relatively slow. In accord with the proposed mechanism, alkyl aryl selenosulfides 8a and 8b, selenenamide 8¢ and a-(phenylselenenyl)acetophenone (9), which all could be expected [25] to readily generate benzeneselenolate ion in the presence of thiol, showed the similar catalytic activity as diphenyl diselenide (not shown). Diselenide 10 turned out to be inactive as a catalyst. Due to the intramolecular nature of the backward reaction, the equilibrium with thiol is probably shifted strongly to the diselenide side with this compound. The high oxidation potential of Ebselen (1.59 V versus Ag/AgCI [32]) suggests that the compound does not exert its catalytic effect by direct one-electron transfer from selenium to iron. Since Ebselen is known to be rapidly ring-opened by any thiol present [33], it is likely to be converted to a selenosulfide, lla, under the conditions of the assay. This compound would then serve as a source of benzeneselenolate ion, 12, the active electron transfer reagent formed in analogy with the equilibrium reaction of Eq. 3. When Ebselen, pretreated with N-acetylcysteine, or the known selenosulfide lib, or diselenide 2a, were added as catalysts, the similar reaction characteristics were observed as for the untreated Ebselen (Fig. 1). On the other hand, selenosulfide l i e turned out to be a poorer catalyst than Ebselen. This may be due to a less favourable equilibrium for selenolate formation. The previously reported catalysis of the thiol reduction of ferric cytochrome c by selenocystine, selenite and glutathione peroxidase [15,16] is explainable in view of the capacity of all three materials to generate a selenol, or in the case of selenite, a selenide ion. In analogy with the equilibrium reactions shown in Eqs. 2-3, diphenyl ditellurides 5 are expected to be converted to benzenetellurolate ions, 13, under the conditions of the assay. These species would then rapidly reduce ferric cytochrome c. The inhibition of the reaction after the rapid formation of a stoichiometric amount of ferrous cytochrome c suggests that the ditelluride catalysts are somehow inactivated. Although the UV spectrum of ferric cytochrome c thus obtained did not differ significantly from that of an authentic sample, we consider irreversible complexation to iron as a plausible explanation to the non-catalytic behaviour of diphenyl ditellurides. The tellurosulfides 14 and 15 with the potential to generate tellurolate ions turned out to be essentially inactive as catalysts. In conclusion, we have demonstrated that the clinically evaluated compound Ebselen has a low capacity to catalyse the potentially toxic thiol mediated reduction of ferric cytochrome c. In contrast, other glutathione peroxidase mimetics and related compounds with the potential to release benzeneselenolate ion (diphenyl diselenide, selenosulfides, a-(phenyselenenyl)-acetophenone, N-(phenylseleno)phthalimide) are very efficient catalysts for this redox reaction. We feel that this effect may contribute to the observed toxicity of certain organoselenium compounds and that it must be considered in future design and application of new glutathione peroxidase mimetics.
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L. Engman et al. ~Chem.-Biol. Interact. 93 (1994) 129-137
Acknowledgements Financial support by the Swedish Natural Science Research Council and the Swedish National Board for Industrial and Technical Development are gratefully acknowledged.
References 1 H. Sies (Ed.), Oxidative stress, Academic Press, London, 1985. 2 C. Vigo-Pelfrey (Ed.), Membrane lipid oxidation, vol. 1, CRC Press, Boca Raton, 1990. 3 B. Halliwell and J.M.C. Gutteridge, Free radicals in biology and medicine, Clarendon Press, Oxford, 1989. 4 A. Miiller, E. Cadenas, P. Graf and H. Sies, A novel biologically active seleno-organic compound I. Glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ 51 (Ebselen), Biochem. Pharmacol., 33 (1984) 3235-3239. 5 H. Sies, Ebselen, a selenoorganic compound as glutathione peroxidase mimic, Free Rad. Biol. Med., 14 (1993) 313-323. 6 S.R. Wilson, P.A. Zucker, R-R.C, Huang and A. Spector, Development of synthetic compounds with glutathione peroxidase activity, J. Am. Chem. Soc., 111 (1989) 5936-5939. 7 K.L. House, R.B. Dunlap, J.D. Odom, Z.-P. Wu, and D. Hilvert, Structural characterization of selenosubtilisin by 77Se NMR spectroscopy, J. Am. Chem. Soc., 114 (1992) 8573-8579. 8 I.A. Cotgreave, P. Mold6us, R. Brattsand, A. Hallberg, C.M. Andersson and L. Engman, t~(Phenylselenenyl)-acetophenone derivatives with glutathione peroxidase-like activity, Biochem. Pharmacol., 43 (1992) 793-802. 9 L. Engman, D. Stern, I.A. Cotgreave and C.M. Andersson, Thiol peroxidase activity of diaryl ditellurides as determined by a IH NMR method, J. Am. Chem. Soc., 114 (1992) 9737-9743. 10 C.M. Andersson, A. Hallberg, R. Brattsand, I.A. Cotgreave, L. Engman and J. Persson, Glutathione peroxidase-like activity of diaryl tellurides, Bioorg. Med. Chem. Lett. 3 (1993) 2553-2558. 11 L. Engman, D. Stern, M. Pelcman and C.M. Andersson, Thiol peroxidase activity of diorganyl tellurides J. Org. Chem, 59 (1994) 1973-1979. 12 D.T. Sawyer, C. Kang, A. Llobet and C. Redman, Fenton reagents (1:1 FelILJHOOH) react via [LxFelIOOH(BH+)] (1) as hydroxylases (RH -- ROH), not as generators of free hydroxyl radicals, J. Am. Chem. Soc., 115 (1993) 5817-5818. 13 R.A. Sheldon and J.K. Kochi, Metal-catalyzed oxidations of organic compounds, Academic Press, New York 1981; chapters 2 and 3. 14 J.M.C. Gutteridge, Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by peroxides, FEBS Len., 201 (1986) 291-295. 15 O.A. Levander, V.C. Morris and D.J. Higgs, Selenium as a catalyst for the reduction ofcytochrome c by glutathione, Biochemistry, 12 (1973) 4591-4595. 16 M-L. Hu and A.L. Tappel, Selenium as sulfhydryl redox catalyst and survey of potential seleniumdependent enzymes, J. lnorg. Biochem., 30 (1987) 239-248. 17a G.W. Pettigrew and G.R. Moore, in: A. Rich (Ed.), Cytochromes c. Biological aspects. SpringerVerlag, Berlin, 1987. 17b G.R. Moore, and G.W. Pettigrew, Cytochromes c. Evolutionary, structural and physicochemical aspects. Springer-Verlag, Berlin, 1990. 18 L. Engman and A. Hallberg, Expedient synthesis of Ebselen and related compounds, J. Org. Chem., 54 (1989) 2964-2966. 19 H.M. Leicester, Diphenyl selenide, Org. Synth. Coll., 2 (1943) 238-240. 20 J. Matti, D6riv6s organiques du s616nium, Bull. Soc. Chim. France, (1940) 617-621. 21 H. Rheinboldt, in: E. Mfiller, (Ed.), Methoden der Organischen Chemie, Houben-Weyl, vol. 9, Georg Thieme Verlag, Stuttgart, 1955, p. 991. -
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22 H. Rheinboldt, in: E. Miiller, (Ed.), Methoden der Organischen Chemie, Houben-Weyl, vol. 9, Georg Thieme Verlag, Stuttgart, 1955, p. 993. 23 I.D. Sadekov, A.Ya. Bushkov and V.I. Minkin, Synthesis and structure of aromatic and heterocyclic tellurium compounds V. Synthesis of diaryl tellurides from diaryl ditellurides, Zh. Obsh. Khim., 43 (1973) 815-817. 24 L. Engman and J. Persson, Improved preparation ofdiaryl ditellurides, J. Organomet. Chem., 388 (1990) 71-74. 25 L. Engrnan, C. Andersson, R. Morgenstern, I.A. Cotgreave, C-M. Andersson and A. Hallberg, Evidence for a common selenolate intermediate in the glutathione peroxidase-like catalysis of oL(phenylselenenyl) ketones and diphenyl diselenide, Tetrahedron, 50 (1994) 2929-2938. 26 L. Engrnan, Synthesis of 2,3,7,8-tetramethoxydibenzotellurophene and its thio and seleno analogues, J. Heterocyclic Chem., 21 (1984) 413-416. 27 H. Fischer and N. Dereu, Mechanism of the catalytic reduction of hydroperoxides by Ebselen: a selenium-77 NMR study, Bull. Soc. Chim. Belg., 96 (1987) 757-768. 28 K. Sj6din, E. Nilsson, A. Hallberg and A. Tunek, Metabolism of N-acetyl-L-cysteine. Some structural requirements for the deacetylation and consequences for the oral bioavailability, Biochem. Pharmacol., 38 (1989) 3981-3985. 29 J.P. Danehy and K.N. Parameswaran, Acidic dissociation constants of thiols, J. Chem. Eng. Data, 13 (1968) 386-389. 30 W.H.H. Giinther, Methods in selenium chemistry. Ili. The reduction of diselenides with dithiothreitol, J. Org. Chem., 32 (1967) 3931-3933. 31 R.C. Dickson and A.L. Tappel, Reduction of selenocystine by cysteine or glutathione, Arch. Biochem. Biophys., 130 (1969) 547-550. 32 C. Schfneich, V. Narayanaswami, K-D. Asmus and H. Sies, Reactivity of ebselen and related selenoorganic compounds with 1,2-dichloroethane radical cations and halogenated peroxyl radicals, Arch. Biochem. Biophys., 282 (1990) 18-25. 33 N. Kamigata, M. Takata, H. Matsuyama and M. Kobayashi, Novel ring opening reaction of 2-aryl1,2-benzisoselenazol-3(2H)-one with thiols, Heterocycles, 24 (1986) 3027-3030.
Chemico-Biological Interactions 93 (1994) 129-137
Catalytic effects of glutathione peroxidase mimetics on the thiol reduction of cytochrome c Lars Engman ~a , Anders Tunek b, Mathias Hallberg b, Anders Hallberg c aUppsala University, Institute of Chemistry, Department of Organic Chemistry, Box 531, S-751 21 Uppsala, Sweden bDepartment of Pharmacology, Astra Draco AB, P.O. Box 34, S-221 O0 Lund, Sweden CDepartment of Organic Pharmaceutical Chemistry, Uppsala University, P.O. Box 574, S-751 23 Uppsala, Sweden
Received 8 December 1993; revision received 28 January 1994; accepted I February 1994
Abstract
The reduction of ferric cytochrome c by various thiols was studied. It was found that Lcysteine, t-cysteine methyl ester and D-penicillamine were very efficient reductants for cytochrome c, whereas N-acetylated aminoacids (N-acetyl-L-cysteine and N-acetyl-Dcysteine) reacted considerably slower. A series of glutathione peroxidase mimetics and related compounds were studied as catalysts for the N-acetyl-t-cysteine reduction of ferric cytochrome c. Diphenyl diselenide, t-butylthio phenyl selenide, S-(phenylseleno)-glutathione, N-(phenylseleno)-phthalimide and a-(phenylselenenyl)-acetophenone were all efficient reduction catalysts. Diphenyl disulfide, Ebselen and several derivatives thereof were less potent catalysts whereas diaryl selenides and diphenyltelluride did not affect the rate of reduction when present in catalytic amounts. The catalysis of diphenyl diselenide, selenosulfides, a(phenylselenenyl)acetophenone, N-(phenylseleno)-phthalimide and Ebselen and derived compounds was suggested to involve the formation of areneselenolate ions as redox-active species capable of transferring one electron to the ferric cytochrome c. The resulting selenium centered arylseleno radicals would then dimerize to regenerate the catalyst in the diselenide form. In the presence of diaryl ditellurides and N-acetyl-L-cysteine, ferric cytochrome c was also rapidly reduced. This reaction was stoichiometric with respect to the ditelluride reagent. Keywords: Cytochrome c; Thiols; Diphenyl diselenide; Selenosulfides; Ebselen; a-(Phenyl-
selenenyl)-acetophenone; Diaryl ditellurides; Selenolate ions; Electron transfer; Catalytic mechanism * Corresponding author. 0009-2797/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009-2797(94)03277-F
130
L. Engman et al. ~Chem.-Biol. Interact. 93 (1994) 129-137
1. Introduction
Aerobic cells are susceptible to oxidative damage once their antioxidant capabilities are overwhelmed. Due to their high reactivity towards important biomolecules, oxygen species formed (superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen) are particularly harmful to the cell [1]. Another kind of oxidative stress damage is due to peroxidative degradation of unsaturated lipids in cell membranes [2]. Biological systems have developed several lines of protection against oxidants. These defence systems include antioxidants/radical scavengers like c~tocopherol and ascorbic acid, enzyme systems like glutathione peroxidases, superoxide dismutases and catalase, metal chelating proteins like ferritin, transferrin, lactoferrin and others [3]. The selenium containing glutathione peroxidases are believed to play a central role in the inactivation of peroxides, thus preventing the formation of alkoxyl, peroxyl and hydroxyl radicals. A few years ago, the organosetenium compound Ebselen (la) was described and shown to possess glutathione peroxidase-like activity [4,5]. Recently, a variety of organoselenium and organotellurium compounds were also shown to act as glutathione peroxidase mimetics. These compounds include diaryl diselenides [6], selenosubtilisin [7], c~-(phenylselenenyl)-acetophenone derivatives [8], diaryl ditellurides [9] and diaryl tellurides [10,11]. The highly reactive and biologically damaging free hydroxyl radical, or a reactive intermediate related to it [12], can be formed from hydrogen peroxide when suitable transition metals are present. The most abundant metal ion likely to catalyse this reaction is iron [13] (Fenton reaction; Eq. 1). There is also evidence that iron proteins act as Fenton catalysts, probably via induced release of ferrous iron from the proteins [14]. Thus, to minimize the toxic effects of hydroxyl radical formation in biological systems, it would seem desirable to keep the Fe(I0/Fe(lll) ratio low. It has been demonstrated [15,16] that certain selenium containing compounds (selenocysteine, selenite and glutathione peroxidase) catalyse the thiol mediated one-electron reduction of ferric cytochrome c [17a, 17b]. Since the ferric cytochrome c is probably a representative model for haeme proteins and its reduction can be monitored with great sensitivity and specificity by measuring the absorbance change at 550 nm, we decided to study the potentially toxic catalysis of its reduction by some recently developed glutathione peroxidase mimetics. 2. Materials and methods
2.1. Compounds All thiols used were commercially available except for Noacetyl-D-cysteine which was obtained from Astra Draco AB (Lund, Sweden). Cytochrome c from horse heart (type III) was obtained from Sigma. Catalysts tested were obtained from commercial sources or prepared according to literature methods Scheme 1 (1-15): Ebselen (la) [ 18], 2-phenyl- 1,2-benzisothiazol-3(2H)-one (1 b) [ 18], 2,2' -diselenobis(benzanilide) (2a) [18], diphenyl selenide (3a) [19], bis(4-aminophenyl) selenide (3b) [20], bis(4(dimethylamino)phenyl) selenide (3c) [21], bis(4-acetamidophenyl) selenide (3d) [20],
L. Engman et al./Chem.-Biol. Interact. 93 (1994) 129-137
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132
L. Engman et aL ~Chem.-Biol. Interact. 93 (1994) 129-137
bis(4-nitrophenyl) selenide (3e) [22], diphenyl telluride (4) [23], diphenyl ditelluride (5a) [24], bis(4-methoxyphenyl) diteiluride (5b) [24], bis(4-aminophenyl) ditelluride (5e) [10], bis(4-(dimethylamino)phenyl) ditelluride (5d) [24], t-butylthio phenyl selenide (8a) [9], S-(phenylselenenyl)-glutathione (8b) [25], ~-(phenylselenenyl)acetophenone (9) [8], 2,2'-diselenobiphenyl (10) [26], S-(N-phenyl(2-carboxamido)phenylselenenyl)-glutathione (llb) [27], l-octylthio-N-phenyl(2-carboxamido)phenyl selenide (lle) [9], t-butylthio phenyl telluride (14) [9].
2.2. t-Butylthio-N-phenyl ( 2-carboxamido ) phenyl telluride (15a ) To a stirred solution of t-butyl mercaptan (0.020 g, 0.22 mmol) in CH2C12 (5 ml) was added N-phenyl(2-carboxamido)phenyltellurenyl iodide [18] (0.080 g, 0.18 mmol) and dry pyridine (30/~1, 0.18 mmol). After 16 h at ambient temperature, the reaction mixture was evaporated under reduced pressure in the presence of some silica gel and submitted to flash chromatography (SiO2/CH2C12:hexanes = 1:1) to afford the title compound, 0.066 g (90%), m.p. 134-135°C. Analysis calculated for C17H19NOSTe: C, 49.44; H, 4.64. Found: C, 49.42; H, 4.72. JH NMR 6(CDC13): 1.43 (s, 9H), 7.20 (t, 1H), 7.33-7.53 (several peaks, 4H), 7.61-7.65 (several peaks, 2H), 7.74 (d, 1H), 8.07 (s, 1H), 8.58 (d, 1H). 2.3. S-[N-phenyl(2-carboxamido)phenyltellurenyl] glutathione (15b) To a stirred solution of N-phenyl(2-carboxamido)phenyltellurenyl iodide (0.10 g, 0.22 mmol) in DMF (1.0 ml) was added glutathione (0.068 g, 0.22 mmol) and dry pyridine (18 #1, 0.22 mmol). Addition of water (1 ml) caused an exothermic reaction. Further addition of water (5 ml) to the resulting clear yellowish solution caused separation of an oil. This crystallized upon trituration with ethanol to give 0.106 g (72%) of yellowish crystals of the title compound, m.p. 205-208°C. The material could not be further purified by recrystallization. Analysis calculated for C23H26N4OySTe: C, 43.84; H, 4.16. Found: C, 42.77; H, 3.96. ~H NMR 6(DMSO-d6): 1.80-2.05 (several peaks, 2H), 2.29-2.48 (several peaks, 2H), 3.18 (d, 2H), 3.36 (t, IH), 3.67 (d, 2H), 4.40 (m, 1H), 7.20 (t, 1H), 7.38-7.74 (several peaks, 6H), 8.33-8.51 (several peaks, 3H), 8.67 (t, IH), 10.82 (s, 1H). 2.4. Cytochrome c reduction assay Instrumentation. A Varian DMS 100 double beam spectrophotometer was used. The absorbance at 550 nm was continuously recorded. Solutions. The assay buffer was 0.1 M KH2PO4, pH 7.5, containing 0.05% EDTA. Cytochrome c was dissolved in the assay buffer (18 mg/ml; 1.5 mM) and this stock solution kept one ice. The catalysts were dissolved in CH3CN (3 mM) and kept at ambient temperature. Stock solutions of thiols in the assay buffer were normally 240 mM. These were freshly prepared (on the day) and kept on ice. Assay. The cuvette holders and the assay buffer were maintained at 37°C. The reference cuvette contained only the assay buffer. To the sample cyvette were added assay buffer (2.75 ml) and cytochrome c solution (100 tA). After 3 rain, thiol solution (100 tA) appropriately diluted was added, followed after another 7-10 min by catalyst solution (50 ~1). Acetonitrile in the concentrations used did not influence the reduction rate which was expressed as absorbance increase/min.
L. Engrnan et aL / Chern.-BioL Interact. 93 (1994) 129-137
133
3. Results and discussion
A series of thiols were tested with respect to their ability to reduce ferric cytochrome c in the absence of added catalysts but in the presence of EDTA as a chelating agent. Without this chelator, the reduction of cytochrome c by any thiol was considerably faster and variable due to transition metal catalysis of the reaction. It was found that L-cysteine, L-cysteine methyl ester and D-penicillamine were very efficient reducing agents for cytochrome c (at 550 nm the absorbance increased > 0.2 absorbance units/min in 1.0 mM solutions of the thiols containing 50 #mol/l of ferric cytochrome c) whereas the corresponding N-acetylated aminoacids reacted considerably slower (the absorbance increased ---0.002 absorbance units/min for Nacetyl-L-cysteine and N-acetyl-D-cysteine). Since the D and L forms of Nacetylcysteine were equally reactive, the steric requirements in this electron transfer process do not seem essential. This is in contrast to the powerful chiral recognition observed with these compounds towards deacetylases [28]. The lower reactivity of N-acylated amino acids towards cytochrome c may be due to the higher pKa value of the thiol group, resulting in a lower concentration of the redox-active thiolate form of the compounds (pKa = 8.5 for the sulfhydryl group of cysteine and 9.5 for N-acetylcysteine [29]). By using N-acetyl-L-cysteine as a thiol reducing agent in the ferric cytochrome c system, we then investigated the catalytic activity of a series of glutathione peroxidase mimetics and related compounds with the potential of being good one-electron donors. It turned out that diphenyl selenide (3a) and a variety of 4-substituted derivatives 3b-3e thereof, as well as diphenyl telluride (4), had little, if any, catalytic activity when present at the 10 moi% level with respect to cytochrome c. However, diphenyl diselenide (2b) and diaryl ditellurides (5) were found to actively promote the reaction. Interestingly, the mode of action for the two classes of compounds is quite different, as shown in Fig. 1 for diphenyl diselenide and diphenyl ditelluride. Whereas the organoselenium compound acts in a truly catalytic fashion to reduce cytochrome c (initially very rapidly, then more slowly), all four organotellurium compounds seem to act in a stoichiometric fashion as judged by the amount of ferrous cytochrome c formed in the rapid initial reaction. Fig. 2 shows the increase of the absorption of ferrous cytochrome c at 550 nm with time as the concentration of diphenyl diselenide is varied. In the absence of thiols, diphenyl diselenide and other catalysts tested did not reduce ferric cytochrome c. As shown in Fig. 1, diphenyl disulfide (6) is a poorer catalyst than its corresponding selenium and tellurium analogues. Ebselen (la) catalysed the reduction of cytochrome c very poorly (Fig. 1). The sulfur analogue lb of Ebselen was completely void of any catalytic effect. It is well known that an equilibrium is rapidly established when diselenides and thiols are brought together [30,31] (Eqs. 2-3; diphenyl diselenide used as an example). It is likely that the benzeneselenolate ion 7 formed acts as a one-electron reductant towards cytochrome c as shown in Eq. 4. The resulting selenium centered radical could then dimerise to regenerate the catalyst in the diselenide form [15]. The spontaneous reduction of cytochrome c by N-acetyl-L-cysteine and other thiols probably also involves electron transfer from thiolate ions to iron. However, since
134
L. Engman et al. ~Chem.-Biol. Interact. 93 (1994) 129-137
Absorbance (550 nm) 1.11.00.9 0.8
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3
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Fig. 2. Absorbance as a function of time for the N-acetyl-L-cysteine (1 mM) reduction of cytochrome c (50/~M) in the presence ofdiphenyl diselenide (0.625 #M, 1.25 #M, 2.5 ~M, 5/zM and 10 #M final concentration). The catalyst was added at ,' = 1.5 rain to an aqueous solution (pH = 7.5) of the other reactants containing 0,05% EDTA.
L. Engman et al. ~Chem.-Biol. Interact. 93 (1994) 129-137
135
selenolate ions are stronger reductants than thiolate ions (Se is less electronegative than S) and selenols are more acidic than thiols (benzeneselenol is essentially dissociated at physiological pH, whereas, for example, N-acetylcysteine contains only a minute fraction of the thiolate form) the uncatalysed reduction of cytochrome c by thiols is relatively slow. In accord with the proposed mechanism, alkyl aryl selenosulfides 8a and 8b, selenenamide 8¢ and a-(phenylselenenyl)acetophenone (9), which all could be expected [25] to readily generate benzeneselenolate ion in the presence of thiol, showed the similar catalytic activity as diphenyl diselenide (not shown). Diselenide 10 turned out to be inactive as a catalyst. Due to the intramolecular nature of the backward reaction, the equilibrium with thiol is probably shifted strongly to the diselenide side with this compound. The high oxidation potential of Ebselen (1.59 V versus Ag/AgCI [32]) suggests that the compound does not exert its catalytic effect by direct one-electron transfer from selenium to iron. Since Ebselen is known to be rapidly ring-opened by any thiol present [33], it is likely to be converted to a selenosulfide, lla, under the conditions of the assay. This compound would then serve as a source of benzeneselenolate ion, 12, the active electron transfer reagent formed in analogy with the equilibrium reaction of Eq. 3. When Ebselen, pretreated with N-acetylcysteine, or the known selenosulfide lib, or diselenide 2a, were added as catalysts, the similar reaction characteristics were observed as for the untreated Ebselen (Fig. 1). On the other hand, selenosulfide l i e turned out to be a poorer catalyst than Ebselen. This may be due to a less favourable equilibrium for selenolate formation. The previously reported catalysis of the thiol reduction of ferric cytochrome c by selenocystine, selenite and glutathione peroxidase [15,16] is explainable in view of the capacity of all three materials to generate a selenol, or in the case of selenite, a selenide ion. In analogy with the equilibrium reactions shown in Eqs. 2-3, diphenyl ditellurides 5 are expected to be converted to benzenetellurolate ions, 13, under the conditions of the assay. These species would then rapidly reduce ferric cytochrome c. The inhibition of the reaction after the rapid formation of a stoichiometric amount of ferrous cytochrome c suggests that the ditelluride catalysts are somehow inactivated. Although the UV spectrum of ferric cytochrome c thus obtained did not differ significantly from that of an authentic sample, we consider irreversible complexation to iron as a plausible explanation to the non-catalytic behaviour of diphenyl ditellurides. The tellurosulfides 14 and 15 with the potential to generate tellurolate ions turned out to be essentially inactive as catalysts. In conclusion, we have demonstrated that the clinically evaluated compound Ebselen has a low capacity to catalyse the potentially toxic thiol mediated reduction of ferric cytochrome c. In contrast, other glutathione peroxidase mimetics and related compounds with the potential to release benzeneselenolate ion (diphenyl diselenide, selenosulfides, a-(phenyselenenyl)-acetophenone, N-(phenylseleno)phthalimide) are very efficient catalysts for this redox reaction. We feel that this effect may contribute to the observed toxicity of certain organoselenium compounds and that it must be considered in future design and application of new glutathione peroxidase mimetics.
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L. Engman et al. ~Chem.-Biol. Interact. 93 (1994) 129-137
Acknowledgements Financial support by the Swedish Natural Science Research Council and the Swedish National Board for Industrial and Technical Development are gratefully acknowledged.
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22 H. Rheinboldt, in: E. Miiller, (Ed.), Methoden der Organischen Chemie, Houben-Weyl, vol. 9, Georg Thieme Verlag, Stuttgart, 1955, p. 993. 23 I.D. Sadekov, A.Ya. Bushkov and V.I. Minkin, Synthesis and structure of aromatic and heterocyclic tellurium compounds V. Synthesis of diaryl tellurides from diaryl ditellurides, Zh. Obsh. Khim., 43 (1973) 815-817. 24 L. Engman and J. Persson, Improved preparation ofdiaryl ditellurides, J. Organomet. Chem., 388 (1990) 71-74. 25 L. Engrnan, C. Andersson, R. Morgenstern, I.A. Cotgreave, C-M. Andersson and A. Hallberg, Evidence for a common selenolate intermediate in the glutathione peroxidase-like catalysis of oL(phenylselenenyl) ketones and diphenyl diselenide, Tetrahedron, 50 (1994) 2929-2938. 26 L. Engrnan, Synthesis of 2,3,7,8-tetramethoxydibenzotellurophene and its thio and seleno analogues, J. Heterocyclic Chem., 21 (1984) 413-416. 27 H. Fischer and N. Dereu, Mechanism of the catalytic reduction of hydroperoxides by Ebselen: a selenium-77 NMR study, Bull. Soc. Chim. Belg., 96 (1987) 757-768. 28 K. Sj6din, E. Nilsson, A. Hallberg and A. Tunek, Metabolism of N-acetyl-L-cysteine. Some structural requirements for the deacetylation and consequences for the oral bioavailability, Biochem. Pharmacol., 38 (1989) 3981-3985. 29 J.P. Danehy and K.N. Parameswaran, Acidic dissociation constants of thiols, J. Chem. Eng. Data, 13 (1968) 386-389. 30 W.H.H. Giinther, Methods in selenium chemistry. Ili. The reduction of diselenides with dithiothreitol, J. Org. Chem., 32 (1967) 3931-3933. 31 R.C. Dickson and A.L. Tappel, Reduction of selenocystine by cysteine or glutathione, Arch. Biochem. Biophys., 130 (1969) 547-550. 32 C. Schfneich, V. Narayanaswami, K-D. Asmus and H. Sies, Reactivity of ebselen and related selenoorganic compounds with 1,2-dichloroethane radical cations and halogenated peroxyl radicals, Arch. Biochem. Biophys., 282 (1990) 18-25. 33 N. Kamigata, M. Takata, H. Matsuyama and M. Kobayashi, Novel ring opening reaction of 2-aryl1,2-benzisoselenazol-3(2H)-one with thiols, Heterocycles, 24 (1986) 3027-3030.