[5] Perthiols as antioxidants: Radical-scavenging andprooxidative mechanisms

[5]

PERTHIOLS AS ANTIOXIDANTS

55

ina. 4s It is also always useful to control the purity of a pooled fraction either by rechromatography under different HPLC conditions or by employing a second analytical technique such as, for example, CE (for an overview see Ref. 49). The respective analytes can be conveniently monitored by UV detection. 42-46 In general, reduced low molecular weight thiols and peptides (chromophore is the peptide bond) can be detected at h -< 214 rim, if other chromophores within the peptide, such as tryptophan or tyrosine, do not permit detection at higher wavelengths. Disulfide bonds show relatively broad absorption bands between 230 and 260 nm, with the actual Amax being dependent on the substituents in the a-position to the sulfur atoms as well as on the conformation of the disulfide bridge (dihedral angle of the C - S - S - C unit). 5° Oxidized dithiothreitol shows an exceptionally redshifted maximum at 285 nm. Other on-line methods that have been used to quantify sulfhydryls and disulfides include electrochemical detection 51-54 and FAB (fast atom bombardment) mass spectrometry. 5s

48 C. T. Mant, T. W. L. Burke, N. E. Zhou, J. M. R. Parker, and R. S. Hodges, J. Chromatogr. 485, 365 (1990). 49 S. R. Rabel and J. F. Stobaugh, Pharm. Res. 10, 171 (1993). 5o N. A. Rosenthal and G. Oster, J. Am. Chem. Soc. 83, 4445 (1961). 51 M. Ozcimder, A. J, H. Louter, H. Lingeman, W. H. Voogt, R. W. Frei, and M. Bloemendal, J. Chromatogr. 570, 19 (1991). 52 p. LllO, F. Zhang, and R. P. Baldwin, Anal, Chem. 63, 1702 (1991). 53 T. J. O'Shea and S. M. Lunte, Anal. Chem. 65, 247 (1993). 54 L. Dou and I. S. Krull, Anal. Chem. 62, 2599 (1990). 55 y. Sun, P. C. Andrews, and D. L. Smith, J. Protein Chem. 9, 151 (1990).

[51 P e r t h i o l s a s A n t i o x i d a n t s : R a d i c a l - S c a v e n g i n g Prooxidative Mechanisms

and

B y STEVEN A . EVERETT a n d PETER W A R D M A N

Introduction Thiols (RSH) are recognized for their radical-scavenging role in protection against cellular oxidative stress 1,2 and in the repair of radical-induced 1 T. M. Buttke, Immunol. Today 15, 1 (1994). 2 C. C. Winterbourn, Free Radical Biol. Med. 14, 85 (1993).

METHODS IN ENZYMOLOGY, VOL. 251

Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

56

THIYL RADICALS

[51

D N A damage? -6 The administration of exogenous thiol drugs has been shown to offer defense against many free radical-associated diseases 7'8 and to protect normal tissues in cancer therapy using radiation 9,1° or drugs.II, 12 The structurally related disulfur analogs or perthiols (RSSH) have significantly different free radical-scavenging and acid/base properties compared to thiols because of relative differences in S - H bond energies. 13,~4 The disulfide bond in perthiols weakens the R S S - H bond by ~90 kJ M -~ relative to R S - H . 15'16 Perthiols are stronger acids than thiols and at physiological pH (at which many thiols are predominantly in the protonated form) a significant proportion of RSSH exists as the deprotonated perthiolate anion (RSS-), depending on pKa: RSSH ~

RSS- + H +

(1)

Equation (1) has important implications for the ability of perthiols to scavenge free radicals by either hydrogen atom donation in Eq. (2) or by electron donation in Eq. (3) (where A is an electron acceptor): RSSH + R ' - C H 2 . ~ RSS. + R'CH3 RSS- + A . ~ RSS. + A-

(2) (3)

3 p. Wardman, in "The Early Effects of Radiation on D N A " (E. M. Fielden and P. O'Neill, eds.), Vol. H54, p. 249. Springer-Verlag, Berlin and Heidelberg, 1991. 4 M. V. M. Lafleur and J. Retel, Mutat. Res. 295, 1 (1993). 5 C. von Sonntag and H.-P. Schuchman, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D. Asmus, eds.), p. 409. Plenum, New York, 1990. 6 D. Schulte-Frohlinde, in "Radiation Research: A Twentieth-Century Perspective" (W. C. Dewey, M. Edington, R. J. Fry, E. J. Hall, and G. F. Whitmore, eds.), Vol. 2, p. 104. Academic Press, London, 1992. 7 C. Rice-Evans and A. Diplock, Free Radical Biol. Med. 15, 77 (1993). 8 B. Halliwell, J. M. C. Gutteridge, and C. E. Cross, Jr. Lab. Clin. Med. 119, 598 (1992). 9 j. Denekamp and A. Rojas, in "Anticarcinogenesis and Radiation Protection" (P. A. Cerutti, O. F. Nygaard, and M. G. Simic, eds.), p. 421. Plenum, New York and London, 1987. 10M. Tamba, Z. Naturforsch. C." Biosci. 44C, 857 (1989). 11 G. J. Peters, C, L. van der Wilt, F. Gyergyay, J. A. M. van Laar, M. Treskes, van der Vijgh, and H. M. Pinedo, Int. J. Radiat. Oncol. BioL Phys. 22, 785 (1992). 12M. Teskes, L. Niflams, A. M. Fichtinger-Schepman, and W. J. van der Vijgh, Anticancer Res. 12, 2261 (1992). 13 p. Wardman, L. P. Candeias, S. A. Everett, and M. Tracey, lnt. J. Radiac BioL 65, 35 (1994). 14 S. A. Everett, C. Sch6neich, J. H. Stewart, and K.-D. Asmus, J. Phys. Chem. 96, 306 (1992). 15 S. D. Benson, Chem. Rev. 78, 23 (1978). 16 D. Griller, J. A. Martinho Simoes, and D. D. M. Wayner, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D. Asmus, eds.), p. 37. Plenum, New York, 1990.

[5]

PERTHIOLS AS ANTIOXIDANTS

57

The antioxidant efficiency of perthiols will reflect not only the free radical-scavenging ability of perthiols but also any prooxidative effects associated with perthiyl radical (RSS .) formation. The methods used to investigate thiol free-radical reactivity17'18 can be equally applied to assess the radical-scavenging and prooxidative properties of perthiols.

Synthesis of Perthiols A versatile method has been developed to prepare perthiols on the basis of a facile alkoxide-induced displacement of the RSS- anion from a methoxycarbonyl disulfide (RSSO2CH3) in Eq. (4). 19 RSSO2CH3 was prepared by the condensation of a thiol with methoxycarbonyl disulfonyl chloride (C1SCO2CH3) in Eq. (5). 2°'21 R'O- + RSSCO2CH3---~ RSS + R'OCO2CH3 RSH + C1SCO2CH3--~ RGSCO2CH3 + HC1

(4) (5)

The 1H nuclear magnetic resonance (NMR) spectra of RSSCO2CIt3 recorded in 2H20 are characterized by a singlet peak with a chemical shift 8 = 3.95 ppm relative to 2,2-dimethyl-2-silapentane 5-sulfonate (DSS), which corresponds to the three uncoupled hydrogens of the methoxycarbonyl grouping ( C H 3 O z C - ) . 21 The progress of the reaction [Eq. (4)] can be monitored by observing the decline in intensity of the singlet peak. Unequivocal identification of the perthiols was achieved by combined gas chromatography/mass spectroscopy (GC/MS) with chemical ionization (CI). Prior to GC separation the perthiols were converted to their trimethylsilyl (TMS) derivatives using bis(trimethylsilyl)trifluoroacetamide (BSTFA) as the derivatizing agent. 22 The CI mass spectra of the totally derivatized perthiol contained the molecular ions (M + H) +, which are 32 m/z (where m/z is the charge/mass ratio) greater than the corresponding thiols, as expected from the presence of an extra sulfur atom. The versatility 17 C. Chatgilialoglu and K.-D. Asmus (eds.), "Sulfur-Centered Reactive Intermediates in Chemistry and Biology," Vol. 197. Plenum, New York, 1990. 18 C. yon Sonntag, "The Chemical Basis of Radiation Biology." Taylor & FranCis, London, 1987. 19 S. A. Everett, L. K. Folkes, K.-D. Asmus, and P. Wardman, Free Radical Res. 20, 387 (1994). 20 A. Mott and G. Barany, Synthesis 51, 657 (1984). 21 G. Barany, A. L. Schroll, A. W. Mott, and D. A. Halsrud, J. Org. Chem. 48, 4750 (1983). z2 K. R. Leimer, R. H. Rice, and C. W. Oehrke, Z Chromalogr. 141, 355 (1977).

58

THIYL RADICALS

[5]

-ooo\

H /H S I Sx.H (1)

S I S-.H

--S

I

S-..H

(2)

(3) H

Ha NQ.,.~COOH3c~ {--. HaC" "IS S~,~CH3 -OOC~ ~NH3+ (4)

HaCT

N.~COO" s/S "S OOC

N""QC H3 I H

(s)

FIG. 1. Structures of chemicals referred to in text: the perthiol analogs of (l), WR 1065; (2), cysteine; (3), 2-carboxyethanethiol; (4), penicillamine disulfide; (S), symmetrical trisulfide of N-acetylcysteine.

of this method facilitates the synthesis of perthiols with specific targeting abilities. The perthiol in Fig. 1, (1), contains a polyamino grouping that will aid the targeting of D N A through counterion condensation phenomena 23 in a manner similar to that demonstrated for the radioprotective thiol drug, W R 1065 (from which it was synthesized). A more lipophilic side grouping such as that in 2-carboxyethaneperthiol [Fig. 1, (2)] is likely to ensure incorporation into cellular biomembranes. Generation a n d Characterization of Perthiyl Radicals The electronic nature of RSS. radicals can be interpreted as a resonance hybrid of the normal sulfur-sulfur o-bonded state (2o-) and a five-electron bonded state (2o-/27r/17r*). 24 Delocalization of the perthiyl radical electron spin between the two sulfur atoms endows this species with optical properties clearly distinguishable from those of corresponding thiyl radicals

23 R. C. Fahey, B. Voynovic, and B. D. Michael, Inr J. Radiat. Biol. 59, 885 (1991). 24 j. L. Kice, in "Sulfur in Organic and Inorganic Chemistry" (A. Senning, ed.), Vol. 1, p. 153. Dekker, New York, 1971.

[5]

PERTHIOLS AS ANTIOXIDANTS

59

(RS .).25 Perthiyl radical absorption spectra are characterized by absorption maxima at ~374 nm and extinction coefficients e374 ~ 1700 M -1 cm -1, which are typically invariant with the nature of the side group R . 14'19'26-29 Thiyl radicals, on the other hand, have significantly lower extinction coefficients (e = 400-1200 M -1 cm -~) with absorption maxima at - 3 3 0 rim? °'31 The characterization of perthiyl radicals has been studied using electron spin resonance spectroscopy,32,33 flash photolysis, 14'27'34 and pulse radiolysis. 14'19'26'28'35 The generation of perthiyl radicals by photolysis of disulfides (RSSR) can be useful in some cases but relies heavily on photolytic cleavage of weak C-S bonds in disulfides, exemplified by the flash photolysis of penicillamine disulfide 27,34 [Fig. 1, (4)] as in Eq. (6a). Unfortunately, the photolysis of many biologically relevant disulfides result in homolytic cleavage of the S-S bond with the subsequent formation of thiyl radicals as in E q . (6b)a7:

RSSR + h v---~ RSS. + R. RSSR + hv--~ 2RS. t-BuSSC1 + hv-+ t-BuSS. + C1.

(6a) (6b) (7)

Laser photolysis of tert-butyldisulfonyl chloride ( t - B u S S C 1 ) 29 in Eq. (7) has also been demonstrated to yield perthiyl radicals but is inappropriate for studies in aqueous solution because of hydrolysis. Of the methods available to study the kinetics of perthiyl radical reactions, the radiation chemical technique of pulse radiolysis has proved the most versatile. Perthiyl radicals can be generated from a variety of independent sources, which include the one-electron reduction of organic trisul-

2s M. Guerra, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D. Asmus, eds.), p. 7. Plenum, New York, 1990. z6 Z. Wu, T. G. Back, R. Ahmad, R. Yamdagni, and D. A. Armstrong, J. Phys. Chem. 86, 4417 (1982). 27 G. H. Morine and R. R. Kuntz, Photoehem. Photobiol. 33, 1 (1981). 28 A. J. Elliot, R. J. McEachern, and D. A. Armstrong, J. Phys. Chem. 85, 68 (1981). ~9T. J. Burkey, J. A. Hawari, F. P. Lossing, J. Lusztyk, R. Sutcliffe, and D. Griller, J. Org. Chem. 50, 4966 (1985). 3o M. Z. Hoffman and E. Hayon, J. Am. Chem. Soc. 94, 7950 (1972). 31 K.-D. Asmus, this series, Vol. 186, p. 168. 32 B. C. Gilbert, in "Sulfur-Centered Reactive Intemediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D. Asmus, eds.), p. 135. Plenum, New York, 1990. 33 D. Becket, S. Swarts, M. Champagne, and D. Sevilla, Int. J. Radiat. Biol. 53, 767 (1988). 34 D. W. Grant and J. H. Stewart, Photochem. Photobiol. 40, 285 (1984). 35 D. A. Armstrong, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D. Asmus, eds.), p. 121. Plenum, New York, 1990.

60

THIYL RADICALS

[51

fides 14'19'26'35 as in Eq. (8) and the oxidation of disulfides 28,36 [Eq. (9)] or trisulfides 14 [Eq. (10)]: eaq-/[C02:

] + RSSSR--. RSS. + RS- + [CO2]

• O H + RSSR --~ RSS- + products • O H + RSSSR ~ RSS. + R S O H

(8) (9) (10)

The principles of pulse radiolysis methodology, including the design of radiolysis experiments seeking to generate specific radicals, have already been extensively reviewed. 35,37 4o The primary radical species from water ionization [Eq. (11)], that is, the hydroxyl radical (. OH), hydrogen atom (H-), and hydrated electron (eaq-), will react with other solutes present in high concentration to generate secondary radicals capable of either oxidizing or reducing the compound under study that is present in lower concentrations. H 2 0 --> eaq-, H ' , " O H . . .

(11)

The reactions in Eqs. (9)-(11) proceed with rate constants approaching the diffusion-controlled limit, 14,26,28m that is, -109-101° M -1 sec -~, and usually generate RSS. radicals almost instantaneously after the electron pulse. Unlike the reactions of thiyl radicals, those involving perthiyl radicals can usually be monitored directly because of the approximately sixfold higher extinction coefficients of the latter.

Hydrogen Transfer b y Perthiols Oxidative attack on biomolecules by hydrogen-abstracting free radicals (e.g., hydroxyl radicals) result in the formation of carbon-centered radicals that are the precursers to strand breaks in DNA. ¢1 Pulse radiolysis of N20saturated alcohol-water mixtures is a convenient method of generating carbon-centered alcohol radicals, which constitute simplified models for similar radical-damaged sites in DNA. 42 The primary radical species from the radiolysis of water are rapidly and quantitatively converted to alcohol radicals via Eqs. (11)-(13) in
[5]

PERTHIOLSAS ANTIOXIDANTS N20 + eaq- + H20 -->" OH + O H - + N2 • O H / H . + (CH3)2CHOH ~ (CH3)2C. O H +.CH2(CH3)CHOH + H20/H2

61

(12) (13)

At pH - 4 (at which most perthiols are undissociated) RSSH reacts with carbon-centered radicals by donating a hydrogen atom 19 as indicated by the characterization of perthiyl radicals (RSS-; Amax ~ 374 am, s374 1680 --- 20 M -1 cm -1) following Eq. (14): RSSH + (CH3)2C" O H ~ RSS- + (CH3)2CHOH

(14)

The assignment of RSS. radicals can be confirmed by generating the same radical from independent sources [e.g., Eq. (8)-(11)]. The rate constant k14 = (2.3 +- 0.1) × 109 M -t sec 1 for the perthiol analog of WR 1065 [Fig. 1, (1)] was determined either directly from the variation of the observed rate constant (kobs) for the first-order buildup of the perthiyl radical absorption (374 am) with RSSH concentration, or alternatively by applying competition kinetics with a suitable alcohol radical scavenger. ~9 The reduction of methyl viologen (MY > ) by alcohol radicals in Eq. (15) generates the strongly absorbing radical cation (MV +-; ~max -~- 600 nm, e60o = 1.37 × 104 M-1 cm-1)43. (CH3)2C" OH + MV 2+ ---->MV +' + (CH3)2C=O + H +

(15)

The competition between the carbon-centered radicals either reducing MV 2+ in Eq. (15) or being repaired by the perthiol in Eq. (14) can therefore be characterized by analysis of the MV +. chromophore at 600 am. t9,44 By applying standard competition kinetics in Eq. (16) it was possible to estimate the value for k14 from the slope of the linear plots of the initial yield of the MV +. radical cation ( A o / A ) (where A0 and A represent the absorbance yield in the absence and presence of RSSH, respectively), measured for different [RSSH]/(MV 2+] ratios. (Ao/A) = 1

+ k14[RSSH]/kls[(MV 2+]

(16)

Figure 2a shows a typical optical trace of the rapid generation of RSS. radicals following hydrogen transfer from the perthiol analog of cysteine [Fig. 1, (2)] to carbon-centered alcohol radicals [Eq. (14)]. At the low 2-propanol concentrations used in this experiment the perthiyl radicals decay by second-order kinetics, -d[RSS.]/dt = 2k17[RSS.]2 by radicalG. Mulazzani, M. D'Angelantonio, M. Venturi, and M. A. J. Rodgers, J. Phys. Chem. 90, 5347 (1986). 44p. Wardman, in Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D. Asmus, eds.), p. 415. Plenum, New York, 1990. 43 Q.

62

THIYL RADICALS

[5]

04 Time (200 #sec / di~

Time (100 ~sec / div

Fro. 2. Absorbance/time traces of (a) perthiyl radical formation and subsequent secondorder decay at 374 nm following hydrogen transfer from cysteine perthiol to a carbon-centered 2-propanol radical and (b) slow and delayed formation of the M V : radical cation at 600 nm obtained by controlled disturbance of the reversible hydrogen transfer equilibrium between cysteine perthiol and carbon-centered alcohol radicals.

radical recombination to the symmetrical tetrasulfide [Eq. (17)] with rate constant 2k17 ~- 1-4 × 109 M -1 sec <. 2RSS.--~ RS4R

(17)

However, at high alcohol concentrations (i.e., 2-propanol -> 50% by volume) the perthiyl radicals generated by Eq. (14) react exclusively with the alcohol by hydrogen abstraction to establish an overall equilibrium Eq. (14/- 14).44a Figure 2b is a typical optical trace showing the characteristic prompt and delayed formation of the MV .+ radical cation obtained by disturbing equilibrium Eq. (14/-14) for the perthiol analog of WR 1065 by competitive scavenging of the 2-propanol radicals by Eq. (15). The fast, initial buildup of the MV .+ radical cation is due to Eq. (15) and is lowered by increasing [RSSH] by Eq. (14). The subsequent slower buildup of the MV .+ radical cation is due to Eq. (-14), where the RSS. radicals regenerate (CH3)eC" OH through hydrogen abstraction from the alcohol. Kinetic analysis of the slow, first-order buildup of the MV -+ radical cation was based on Eq. (18) 42. [ ( C H 3 ) 2 C H O I - ~ ] / k o b s = 1 / k _ i 4 q-

1/k_14 x k15[RSSH]/kt4[MV + ] (18)

Linear plots of [(CH3)2CHOH]/koBs vs [RSSH]/[MV 2+] gave an intercept from which the rate constant k-14 = (3.8 + 0.3) × M -1 sec -1 was obtained for hydrogen abstraction by the RSS. radical derived from the perthiol analog of WR 1065.19 A comparison of the equilibrium constant K14 = (k14/k-14) -~ (6.3 ± 0.1) × 105 with K19 = (k19/k-19) --- (1.8 + 0.1) X 104 indicated that for the perthiol analog of WR 1065 the equilibrium lies further to the side of hydrogen transfer and therefore free radical repair. 44~Minus sign preceding an equation number indicates a reverse reaction.

[51

PERTHIOLS AS ANTIOXIDANTS

RSH + (CH3)2C" OH ~.~ RS. + (CHB)zCHOH

63

(19)

The difference in Gibbs free energies between the equilibrium reactions Eqs. (14/-14) and (19/-19) is a measure of resonance stabilization energy within the perthiyl radical, A G(RSS .-RS ")RSZ,relative to the RS. radical. Substituting the values obtained for K19 for WR 1065 and K14 for the perthiol analog into Eq. (20) gave a value of 8.8 kJ M -1 for the resonance stabilization energy of perthiyl radicals, 19which agrees well with previously published values obtained from the thermal decomposition of organic tetrasulfides. 24 2xG(RSS .-RS ")Rs~ = (AG(14/-14) - AG(19/-19)) = ( - R T l n K14 q- RTln K19)

(20)

This inherent stability of the perthiyl radical provide the thermodynamic driving force for increased efficiency in perthiol hydrogen transfer over their thiol counterparts.

Electron Transfer by Perthiols As well as facilitating fast hydrogen atom transfer a lower S - H bond energy also influences the acid/base properties of perthiols relative to thiols. For example, the introduction of the second sulfur atom in WR 1065 reduces the pKa (RS-H) = 7.6 + 0.1 to pKa (RSS-H) = 6.2 +- 0.1 so that at pH 7.4, while the thiol has 20% in the RS- form the perthiol analog has 95% in the RSS- form. 19,45However, RS- anions undergo rapid electron transfer reactions with, for example, peroxyl radicals 46 and some D N A base radicals. 47 Indirect evidence has been obtained for perthiyl radical formation from the reduction of Fe(III)-cytochrome c (cyt c) by the RSS- anion [Eq. (21)], 36 another illustration that perthiols can scavenge free radicals by electron transfer processes. RSS- + Fe(III)-cyt c --+ RSS. + Fe(II)-cyt c

(21)

The halogenated peroxyl radical (CC13OO .) can be conveniently generated by pulse radiolysis of air-saturated CC14/(CH3)2CHOH/H20 mixtures via Eqs. (11), (13), (22), and (23). 46 At pH 8 (where the perthiol analog of WR 1065 is 99% in the RSS- form) there is no RSSH present to form RSS. radicals via reactions in Eqs. (11), (13), and (14). 45 G. L. Newton, T. J. Dwyer, T. Kim, J. Ward, and R. C. Fahay, Radiat. Res. 131, 143 (1992). 46 M. G. Simic and E. P. L. Hunter, Free Radical BioL Med. 2, 227 (1986). 47 p. O'Neill, Radiat. Res. 96, 198 (1983).

64

THIYL RADICALS

[5]

eaq-/[(CH3)zC'OH] + CCl 4 C C l 3. -}- C1- + [(CH3)2C---O + H +] CC13" + 02--~ CC13OO • CC13OO" + RSS- --+ CC13OO- + RSS"

(22) (23) (24)

The RSS- anion scavenged the peroxyl radical by a rapid electron transfer [Eq. (24)] as indicated by the first-order buildup of RSS. radical absorption at 374 nm. The rate constant k24 = (4.2 _+ 0.1) × 109 M -~ sec -1 was obtained from the slope of the linear plot of kobs vs [RSS-]. It is reasonable to assume that resonance stabilization within the RSS. radical will provide the thermodynamic driving force for electron transfer by the RSS- anion in the manner already demonstrated for the corresponding hydrogen transfer from RSSH.

Conjugation of Perthiyl Radicals with Perthiolate Anions and Thiolate Anions By analogy to the reduction of molecular oxygen to the superoxide radical anion 02 ~ by the disulfide radical anion RSSRL Eqs. (25) and (26) may also represent a possible pathway by which perthiols might induce cellular oxidative stress. RSS. + RSS- ,~ (RSSSSR): (RSSSSR) = + 02 --~ RSSSSR + 02:

(25) (26)

However, no evidence has been obtained for a stable tetrasulfide radical anion, (RSSSSR): which must therefore have a half-life of <1/xsec. 19 The central S-S bond in tetrasulfides is weak [D(RSS-SSR) ~ 147.6 kJ M-~]. 48 Thus, accommodating an extra electron in the antibonding orbital of this already weak bond would have a highly destabilizing effect, assuming that this species has a localized three-electron (.'.) bond that is (2o-/o-*) in electronic nature, that is, (RSS.'.SSR)-. The interaction of RSS. radicals with endogenous glutathione thiolate anions (GS-) is expected to generate a trisulfide radical anion (RSSSG)-. Despite the RS-SSR bond being significantly stronger than RSS-SSR [D(RS-SSR) ~ 217 kJ M-l] 4s no evidence has been obtained for the transient stabilization of trisulfide radical anions from the one-electron reduction of trisulfides [Eq. (8)], 14'19'26'35The possibility remains that molecular oxygen might drive Eq. (25) to the right by removing the RSSSSR: from the equilibrium by Eq. (25). 48 I. Kende, T. L. Pickering, and A. V. Tobolsky, J. A m . Chem. Soc. 877 5582 (1965).

[5]

PERTHIOLS AS ANTIOXIDANTS

65

Perthiyl Radical Reaction with Polyunsaturated Fatty Acids Hydrogen atom transfer and thiyl radical (RS .) formation are synonymous with thiol protection and repair. 5'1a'41'49'5°However, there is increasing evidence to indicate that RS. radicals are prooxidants capable of initiating potentially damaging processes within the biological environment. 17'31Polyunsaturated fatty acids (PUFAs) are major constituents of the lipid bilayer of cellular membranes and are susceptible targets easily damaged by cellular oxidative stress. 51 RS. radicals abstract a bisallylic hydrogen from PUFAs and are considered potential initiators of lipid peroxidation. 52-54 Although rate constants for RS. radical hydrogen abstraction from the activated C H bonds of alcohols are on the order of k-19 --~ 1-5 x 104 M -1 sec -1, the corresponding rate constants for abstraction by RS. of a weaker bisallylic hydrogen from PUFAs [Eq. (27)] 52,54,55are significantly higher (k27 ~ 106107 M -1 sec 1) and found to increase with both the number of P U F A bisallylic groups and the lipophilicity of the RS. radicals. 53 RS. + P U F A ( - H ) --~ RSH + P U F A . a s - + PUFA--~ [(RS)PUFA] • RSH + [(RS)PUFA]. --~ RS. + (RS)PUFA

(27) (28) (29)

RS. radicals were also found to add across the P U F A double bonds to form radical adducts [Eq. (27)], which in turn were scavenged by the thiol [Eq. (28)] thus regenerating RS. radicals to undergo Eq. (27). 54,55 The reaction of RSS. radicals with PUFAs can be directly monitored by time-resolved optical detection in pulse radiolysis experiments owing to the strong, characteristic absorption of the pentadienyl radical (PUFA. ; Amax = 290 nm, 13290 ~ 2.6 × 104 M -1 cm-1). 52 However, to differentiate between RSS. radical abstraction of a bisallylic hydrogen and possible radical adduct formation [(RSS)PUFA) • the RSS- radicals were generated from RSSSR [Eq. (8)], which would be unreactive toward [(RSS)PUFA]. 49 C. von Sonntag, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D, Asmus, eds.), p. 359. Plenum, New York, 1990. 50 p. Wardman, in "Activation of Drugs by Redox Processes" (G. E. Adams, A. Breccia, E. M. Fielden, and P. Wardman, eds.), Plenum, New York, 1990. Sl T. A. Dix and J. Aikens, Chem. Res. ToxicoL 6, 2 (1992). s2 C. Sch6neich, K.-D. Asmus, U. Dillinger, and F. V. Bruchhausen, Biochem. Biophys. Res. Commun. 161, 113 (1989). 53 C. Sch6neieh and K.-D. Asmus, Radiar Environ. Biophys. 29, 263 (1990). 54 C. Sch6neich, U. Dillinger, F. V. Bruchhausen, and K.-D. Asmus, Arch. Biochem. Biophys. 292, 456 (1992). 55 K.-D. Asmus, in "Active Oxygens, Lipid Peroxides, and Antioxidants" (K. Yagi, ed.), p. 57. CRC Press, Tokyo, 1993.

66

THIYL RADICALS

[51

radicals. Figure 3 shows the absorption spectrum recorded 10/zsec after pulse radiolysis of an N2-saturated butanol-water mixture (50% by volume) containing 1 m M trisulfide [Fig. 1, (5)] and 2 m M linolenic acid (C18 : 2). The decay of RSS- radicals at 374 nm (Fig. 3, inset a) is paralleled by an increase in absorption at 290 nm (Fig. 3, inset b) corresponding to the formation of the pentadienyl radical by Eq. (30). RSS. + P U F A ( - H ) ~ RSSH + P U F A .

(30)

The RSS. radicals were quantitatively converted to pentadienyl radicals, indicating that the interaction occurred almost exclusively by abstraction of a biallylic hydrogen. The kinetics of the pentadienyl radical formation followed an exponential rate law with the half-life dependent on the P U F A concentration. The rate constant k31 = (1.2 + 0.1) × 106 M -1 sec -1 for the N-acetylcysteine perthiyl radical reacting with linolenic acid was obtained from the linear plot of kobs vs [PUFA]. A comparison of k31 with k27 = 1.4 x 107 M -1 sec -1 for the analogous reaction with the thiyl radical 53 indicates that perthiyl radicals are less reactive toward PUFAs and therefore less likely to initiate the chain of lipid peroxidation. That perthiyl radicals are moderately good oxidants but weaker than thiyl radicals is also indicated

3ooo

i 3 nm 1

,2000 1500 "----11000O0

5000

3(30

350

4()0

450

(nm) FIG. 3. Transient absorption spectrum obtained on pulse radiolysis (1 Gy) of an N2saturated 50% tert-butanol-water mixture containing 1 mM linolenic acid and i m M trisulfide [Fig. 1, (5)], showing the decay of the perthiyl radical and build-up of the pentadienyl radical. Inset a: Optical trace at 374 nm showing fast formation of the perthiyl radical and its subsequent first-order decay in the presence of linolenic acid. Inset b: Optical trace at 290 nm showing slow first-order buildup of the pentadienyl radical following abstraction of a bisallylic hydrogen from linolenic acid.

[5]

PERTHIOLS AS ANTIOXIDANTS

67

25

0,~ 0

. 50

.

. . 100 150 Dose (Gy)

200

250

Fio. 4. Yields [SO42-] obtained using 6°Co 3' radiolysis of (0) an N20/O2 (80:20%)saturated aqueous solution containing i M formate and I mM trisulfide [Fig. 1, (4)] and 1 mM phosphate buffer at pH 5.2 and (©) N20/O2 (80 : 20%)-saturated aqueous solution containing 1 M formate and 1 mM N-acetylcysteinedisulfideand I mM phosphate buffer at pH 7. Inset: Typical HPIC chromatogram indicating the formation of SO42- anions followingirradiation (200 Gy) of the trisulfide. The large peak corresponds to formate and phosphate anions, which are unretained. by their reaction with ascorbate (k30 ~ 1-6 × 106 M -1 sec -1) in Eq. 31,14'55 which occurs one to two orders of magnitude slower than RS. radicals (k ~ 107-108 M -1 sec-1)56: RSS. + (ascorbate) A H - ~ RSSH + A =

(31)

The observed differences in reactivity can be rationalized in terms of the increased resonance stabilization of the perthiyl radicals. R e a c t i o n of Perthiyl Radicals with Molecular O x y g e n Perthiyl radicals undergo addition reactions with molecular oxygen [Eq. (32)] to form perthiyl peroxyl radicals (RSSOO .) with rate constants on the order of k32 ~ 5-8 x 106 M -1 sec-1.14,55 RSS. + 02 ~ R S S O O .

(32)

The detection by high-performance ion chromatography (HPIC) of inorganic sulfate ions (SO42-) following Eq. (32) has provided information regarding the rate of perthiyl radicals in the presence of molecular oxygen. 14 Figure 4 (solid circles) shows a linear plot of [SO42-] vs dose obtained by 56 C. Dunster and R. L. Willson, in Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D. Asmus, eds.), p. 377. Plenum, New York, 1990.

68

THIYL RADICALS

[5]

steady state y radiolysis of an N20/02 (80 : 20%)-saturated aqueous solution containing 1 M formate and 1 m M trisulfide (in this case the symmetrical trisulfide of N-acetylcysteine [Fig. 1, (4)] at pH 7. The inset in Fig. 4 shows a typical ion chromatogram obtained after a total radiation dose of 200 Gy. Under these experimental conditions perthiyl radicals are generated at a steady rate by Eq. (8) with a yield of G(RSS.) = 0.6 b~M j-1. The c o r r e s p o n d i n g G ( $ 0 4 2 - ) = 0.3 /xM J-1 from the slope of Fig. 4a accounts for 50% of the initial yield of RSS. radicals generated by this system. The mechanism of SO42- anion formation is suggested to proceed in analogy to the well-characterized reactions of thiyl and peroxyl radicals. The RSSOO • radicals generated by Eq. (31) undergo a structural rearrangement to a sulfonyl-type radical according to Eq. (33): RSSOO. - , RSS(O)(O) •

(33)

This transfer of the radical spin density from oxygen to sulfur facilitates a second addition of molecular oxygen to form a sulfonyl peroxyl radical in

(34): RSSO2. + 02

RSS(O)(O)OO.

(34)

Hydrolysis of the sulfonyl peroxyl radical yields SO42- anions and a sulfenyl radical by Eq. (35): R880200"

+ H 2 O ---'> RSSO. + 8 0 4 2 . -t- 2 H +

(35)

Most of the oxygenated intermediates formed as a consequence of Eqs. (32)-(35) must be considered effective oxidants (as with the corresponding radicals believed to be formed in the corresponding RS "/02 systems)57with the potential to inflict biological damage. Interestingly, in marked contrast to RSS "/02 systems, the generation of SO42- anions in RS "/O2 systems is of less importance. The selective generation of RS. radicals by the oneelectron reduction of N-acetylcysteine disulfide (see Fig. 4, open circles) gave significantly lower yields of 8042 anions, that is, G($042-) < 0.l /xM J-L The measurement of SO42- anions by HPIC may prove a useful mechanistic probe for RSS- radicals formed by perthiol radical scavenging reactions. Conclusions

Pulse radiolysis has proved to be a useful tool with which to quantify the free radical-scavenging ability of perthiols. Chemical models have been designed to probe the interaction of perthiols with free radical species 57 M. D. Sevilla, D. Becker, and M. Yan, Int. J. Radiat. Biol. 57, 65 (1990).

[6]

THIYL FREE RADICALS AND VITAMIN PROTECTION

69

commonly associated with cellular oxidative stress and to elucidate subsequent prooxidative reactions. Knowledge of these fundamental mechanisms will form the basis for the rational design of novel synthetic perthiol drugs with strategic targeting properties. Perthiols exhibit many of the attributes necessary in an antioxidant. The free radical-scavenging reactions of perthiols are qualitatively similar to, but quantitatively different from, those of the corresponding thiol antioxidants. Perthiols are not only more efficient hydrogen donors than thiols, but as perthiolate anions they are highly efficient electron donors. Moreover, the antioxidant-derived radical, in this case the perthiyl radicals, are significantly less reactive than their thiyl radical counterparts and are therefore less likely to pose a threat if generated within the cellular environment. In view of the essential role thiols play in controlling cellular oxidative stress, the encouraging performance of perthiols as free radical scavengers suggests they will be of use as exogenous antioxidants.

Acknowledgments This work is supported by the Cancer Research Campaign.

[61 T h i y l ( S u l f h y d r y l / T h i o l ) F r e e R a d i c a l R e a c t i o n s , Vitamins,/3-Carotene, and Superoxide Dismutase in Oxidative Stress: Design and Interpretation of Enzymatic Studies By

SUBHAS C. KUNDU a n d ROBIN L. WILLSON

Introduction Thiyl (sulfhydryl/thiol) free radicals are receiving increasing consideration as intermediates in processes that may be involved in the development of biological damage resulting from oxidative or reductive stress. These include cytotoxicity1'2 mutagenesis) DNA damage, 4 lipid peroxidation, s-8 1 R. L. Willson and A. J. F. 8earle, Nature (London) 255, 498 (1975). 2 G. Saez, P. J. Thornalley, H. A. O. Hill, R. Hems, and J. V. Bannister, Biochirn. Biophys. Acta 719, 24 (1982). 3 H. Glatt, M. Protic-Sabljic, and F. Oesch, Science 220, 961 (1983). 4 W. A. Prutz and H. Monig, Int. J. Radiat. Biol. 52, 677 (1987). 5 D. Sehulte-Frohlinde, Free Radical Res. Commun. 6, 181 (1989).

METHODS IN ENZYMOLOGY, VOL. 251

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