3583
J . Phys. Chem. 1990, 94, 3583-3588
One-Electron Reduction Potentials of 5- Indoxyl Radicals. A Pulse Radiolysis and Laser Photolysis Study Slobodan V. Jovanovic,*.' Steen Steenken,2and Michael G . Sink3 Laboratory 030, The Boris Kidric Institute of Nuclear Sciences, P.O. Box 522, 11001 Beograd, Yugoslavia, Max- Planck-lnstitut fur Strahlenchemie. 0 - 4 3 3 0 Miilheim a.d. Ruhr, Federal Republic of Germany, and Center f o r Radiation Research, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 (Received: May 23, 1989; In Final Form: Nouember 28, 1989) Reversible one-electron-transfer reactions between 5-hydroxyindole derivatives as donors and Fe(CN):- and IrCI," as acceptors were investigated by pulse radiolysis and laser flash photolysis. The reduction potentials vs NHE of the 5-indoxyl radicals are E < 0.867 V at pH 3, E = 0.55 & 0.02 V at pH 9.1, and E = 0.3 f 0.02 at pH 13.5, based on E(IrC152-/IrC163-) = 0.867 V at pH 3 and E(Fe(CN)&/Fe(CN):-) = 0.358 V at pH 9.1 and 13.5 as reference redox couples. The reduction potentials of the indoxyl radicals generated from the 5-hydroxyindole derivatives are independent of the side chain. From the pH dependence of the reduction potentials and taking into account the pK, values of the species involved, the reduction potential of the 5-indoxyl radicals at pH 7, E,, is derived as 0.64 V vs NHE. Reduction potentials were also measured for (TMPD), promethazine, and 4-methoxyphenol. Except the radicals derived from N,N,N',N'-tetramethyl-p-phenylenediamine for TMPD", for which the measured reduction potential = 0.27 f 0.02 V is equal to the previously reported value of 0.27 V, the reduction potentials of the promethazine radical cation, E, = 0.98 f 0.03 V, and the 4-methoxyphenoxyl radical, E,,,, = 0.54 & 0.02 V, were found to be higher than the previously reported and widely used values of 0.86 and 0.4 V, respectively.
Introduction 5-Hydroxytryptophan and its derivatives have been suggested as potential endogenous antioxidant^?^ on the basis of their ability to repair via one-electron transfer oxidizing free-radical intermediates, such as indolyl of tryptophan4 and guanyl of DNA., 5-Hydroxytryptophan is metabolized in the brain to the wellknown neurotransmitter 5-hydroxytryptamine (serotonin), which plays a role in regulating circadian rhythms. Serotonin is further metabolized to 5-hydroxyindole-3-acetic acid which is excreted into the urine. 5-Hydroxytryptophan and its metabolites appear a t biological pH 7 as zwitterionic (5-hydroxytryptophan), monopositive (5-hydroxytryptamine), and mononegative (5hydroxyindole-3-acetic acid) forms. This diversity of charge in the side chain may be important in helping to overcome repulsive electrostatic effects in reactions with biopolymers or aggregates, which are often multiply charged as either negative or positive. The protective role of the 5-hydroxytryptophan derivatives may be particularly important for nerves and brain cells which may be easily oxidized due to their high content of unsaturated lipids and metals6 The antioxidant activity of 5-hydroxyindoles and other endogenous antioxidants depends strongly on their reducing properties. Consequently, in order to assess the efficacy of 5hydroxyindoles as endogenous antioxidants, it is necessary to know the reduction potentials of the corresponding 5-indoxyl radicals. In this study we used the techniques of laser photolysis and pulse radiolysis to determine the reduction potentials of 5-indoxyl radicals. Inorganic redox couples, hexachloroiridate(lV/III), with E = 0.867 V,' and ferricyanide/ferrocyanide,with E = 0.358 V,8 were chosen as primary redox standards, since their reduction potentials are well-established and refer to fully reversible electron transfer as known from classical electrochemical measurements. In order to obtain even more reliable values for the one-electron reduction potentials of 5-indoxyl radicals, the potentials of the reference redox couples (secondary standards) promethazine radical cation/promethazine, TMPD'+/TMPD, and 4-methoxyphenoxy1 radical/4-methoxyphenol were reinvestigated.
-
( I ) The Boris Kidric Institute of Nuclear Sciences. (2) Max-Planck-lnstitut fur Strahlenchemie. (3) Center for Radiation Research. (4) Jovanovic, S. V.; Simic, M. G. Life Chem. Rep. 1985, 3, 124. (5) Jovanovic, S.V.: Simic, M. G.Biochim. Biophys. Acta 1989, 1008, 39. (6) Autor, A. P., Ed. Pathology of Oxygen; Academic Press: New York, 1982. (7) Margerum, D. W.; Chellappa, K. L.;Bossu, F. P.; Burce, G. L. J . Am. Chem. SOC.1975. 97. 6894. (8) Hanania, G . I: H.; Irvine, D. H.; Eaton, W . A,; George, P.J . Phys. Chem. 1967, 71, 2022.
0022-3654/90/2094-3583$02.50/0
TABLE I: pK, Values of 5-Hydroxyindoles R
7
H
PK.
hvdroxvindole 5-hydroxyindole 5-hydroxytryptamine
R H CH2CH2NH2
5-hydroxyindole-3acetic acid 5-hydroxytryptophan
5-hydroxy groupa
R
11.1 1 1.2b 11.1
9.Sb
CH,COOH
11.4
4.15c
CH2CHNH2COOH
11.4
2.38; 9.39c
a Determined spectrophotometrically. Estimated to be accurate to f0.05. bFrom ref 17. cFrom ref 15. Values refer to indole-3-acetic acid and tryptophan, respectively. It is assumed that the hydroxy substitution at C5 has little influence on the ionization in the side chain.
Materials and Methods The compounds were of the highest purity commercially available and were used as received. 5-Hydroxyindole, 5hydroxytryptamine hydrochloride, 5-hydroxyindole-3-acetic acid, promethazine hydrochloride, and 4-methoxyphenol were obtained from Sigma? 5-hydroxytryptophan was obtained from Sigma and from Fluka; N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) was a product of Fluka; sodium hexachloroiridate( I l l ) was obtained from Ventron; bis( 1,4,7-triazacyclononane)nickel(lI) perchlorate was a generous gift of Karl Wieghardt from the Ruhr-Universitat Bochum; all other chemicals used in this study were products of Merck. Water was purified by a Millipore Milli-Q system. All solutions were prepared freshly before experiment and saturated with high-purity gases ([O,] 3 PPm). The pulse radiolysis experiments were performed on the 2-MeV electron accelerator Febetron 707 a t the Boris Kidric Institute, which was described in ref IO, and on the 3-MeV Van de Graaff accelerator at the Max-Planck-Institut, which was described in ref 1 I , For the determination of absorbed dose the thiocyanate (9) The mention of commercial products does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the products identified are the best available for the purpose. (IO) Markovic, V.; Nikolic, D.; Micic, 0. I . Int. J . Radiat. Phys. Chem. 1974, 6, 227.
0 1990 American Chemical Societv
3584 The Journal of Physical Chemistry, Vol. 94, No. 9, 1990
dosimetry was used, taking G[(SCN),'-] = 6.0 in a I O mM N20-saturated aqueous solution of KSCN and e480 = 7600 M-' cm-I . I 2 Fully computerized time-resolved laser photolysis equipment at the Max-Planck-ln~titut~~ was used in photoionization experbis(l,4,7-triazacyiments ( A = 248 nm) on Fe(CN),4-, ), and the 5-hydroxyclononane)nickel( 11) ( N i ( t a ~ n ) , ~ +TMPD, indoles. The pK, values of the 5-hydroxyindole derivatives are listed in Table I. A study of electron-transfer reactions by the pulse radiolysis technique utilizes selective transient oxidants for exclusive generation of desired free radicals. In this study we used dithiocyanate, (SCN),'-, azide, ON3, formyl, CH2=CHO', and trichloromethylperoxyl, Cl3C0O', radicals as primary oxidants. Dithiocyanate, azide, and formyl radicals were generated by the 'OH radical reactions with SCN- (0.1 M KSCN), N,- (0.1 M NaN3), and ethylene glycol (0.9 M H O C H 2 C H 2 0 H ) ,respectively. N 2 0 (25 mM) was used to convert hydrated electrons into *OH radicals. The reactions of the 'OH radical used in this study are the following: 'OH
+ SCN-
k = 1.1
-
SCN'
+ OH-
X l o i o M-' s-I
-
'OH
TABLE 11: Dependence of the Rate of Electron Transfer from TMPD to Fe(CN)6* on the Concentration of Monovalent Cations CcationSL11 M EFe(lll,blv kc/M-' s-' 0.022 0.464 0.529
"Concentration of added K+ or N a + plus concentration of K+ from K,[Fe(CN),]. bTaken from Figure 2 of ref 8. CEstimated to be accurate to f IO%.
facilitates their one-electron oxidation. Both laser excitation at 248 nm and the reactions of strong transient oxidants, X', such as the dithiocyanate radical anion and the formyl radical, were found to yield 5-indoxyl radical, via the following reactions
X'
+ R-lndH-0R-IndH-0-
k =1 HOCHCH20H
-
+ OH-
)(
-
R-IndH-0'
16
+ H20
-+
R-Iiid-0' OH-
CH,=CHO'
-
+ 0,
-
C1,COO'
Results Alkaline Media. Deprotonation of hydroxyindoles in alkaline media, according to the reaction R
+ e-
(3)
-?+ R-IndH-0'
R
fl-rnd-0.-
H
= 12.219
R
+ e- + H+
-
H
pK,(R-IndH-OH)
H R-I~~H-o-
= 1 1.1-11.4418
( 1 1 ) Fujita, S.;Steenken, S . J . Am. Chem. Soc. 1981, 103, 2540. (12) Baxendale, J . H.; Bevan. P. L. T.; Scott, D. A. Trans. Faraday Soc. 1968, 64. 2389. ( 1 3 ) Anklam, E.; Steenken, S . J . Photochem. Phofobiol. 1988, 43A, 2 3 3 . ( 14) (a) Perrin, D. D. Dissociation Constants of Organic Bases in Aqueous Solution; Butterworths: London, 1965. (b) Fasman, G. D., Ed. Handbook of Biochemistry and Molecular Biology, 3rd Ed.: CRC Press: Cleveland, 1976: Vol. I , p 305. (15) Land, E. J.; Priitz, W. A. Int. J . Radiat. Biol. 1979, 36, 75. (16) Steenken, S. J . Phys. Chem. 1979, 83, 595. (17) Packer. J . E.: Willson, R. L.: Bahnemann, D.; Asmus. K.-D. J . Chem. Soc.. Perkin Trans. 2 1980. 296.
R-lndH-0-
(5)
The ferricyanide/ferrocyanide redox couple with E = 0.358 V8 was used as a reference in the measurements of the reduction potentials of the 5-indoxyl radicals at pH 9.1 and 13.5. The reduction potential of the ferricyanide/ferrocyanide couple increases with the concentration of monovalent cations8 from 0.358 V at c'12 = 0 to 0.455 V at c1I2 = 0.7. The effect of the concentration of added monovalent cation, as either Na+ or K+, on the forward rate constant of the following redox equilibrium was studied in the pH range from 9.1 to 13.5 Fe(CN),3-
+ TMPD
TMPD"
+ Fe(CN)64-
(6)
Fe(CN)63- was generated either by pulse radiolysis from 10 mM Fe(CN)64- using ON3 as an oxidant
+ N 3 - 4 OH- + "3 + Fe(CN)64- N3- + Fe(CN),3'OH
'N,
-
k = 3.4 X IO9 M-I R-IndH-OH
(4)
Consequently, the half-cell reaction of any 5-hydroxyindole redox couple is
+ (CH3)2CHOH (CH3l2COH+ H 2 0 eaq- + (CH3I2CO+ H 2 0 (CH&COH + OH( C H 3 ) 2 C 0 H+ CC1, 'CCI3 + CI'CCI,
(2)
+ H+
The trichloromethylperoxyl radical was generated in an aerated aqueous solution of 1 M 2-propano1, 50 mM acetone, and 50 mM CCI, by the following reaction sequence" 'OH
+ R-IndH-0'
R
H 2 0 + -OCHCH20H O=CHCH2
O=CHCH2
X-
pK,(R-IndH-0')
HOCHCH20H
1010 M-1 s-1
-
The indoxyl radical loses a proton in alkaline media,I9 according to
k = 1 x 1010 M-1 s-1 ' 5 *OH + H O C H 2 C H 2 0 H
5.4 x 106 6.1 X IO6 9.9 x 106 9.7 x 106
0.397 0.437 0.455 0.457
0.181
l2
+ SCN- + (SCN);. + N3- ON3 + OH-
SCN'
Jovanovic et al.
S-I
(7) (8)
l9
or upon photoionization following laser excitation in Ar-saturated aqueous solutions of 2-4 mM K,[Fe(CN),] Fe(CN),,-
Fe(CN)63- + eaq-
(9)
4(e-) = 0.6720 I n the laser photolysis experiments, 0.1 M 2-chloroethanol was (18) See Table I in Materials and Methods Section. 0 9 ) Simic, M. G.: Steenken. S.; Jovanovic, S . V . Manuscript in prepara-
tion. (20) Airey, P. L. Quoted in: Calvert, J . G.;Pitts. J. N. Phorochemistry; Wiley: New York, 1966; p 271. (21) Steenken, S.; Neta, P. J . Phys. Chem. 1982, 86, 3661. ( 2 2 ) Wardman, P. J . Phys. Chem. ReJ Data, in press.
The Journal of Physical Chemistry, Vol. 94, No. 9, 1990 3585
Reduction Potentials of 5-Indoxyl Radicals TABLE 111: Electron-Transfer Reactions in Alkaline Solutions a t 20 "C
S-
X' Fe(CN)63-
X' generated by
5-Trp-O5-Trp-0HIAA* 5-Ind-0serotonin Fe(CN)b4Fe(CN)645-Trp-0TMPD TMPD TMPD TMPD TMPD TMPD
5-Trp-0' 4-CH30PhO' Fe(CN)& 5-Trp-0' 5-Ind-0'
hv
ON3 hv hu hv (SCN),'CHZCHO CH2CHO CHZCHO hu "3
CH~CHO CH2CHO CHZCHO
pH 13.5 13.5 13.5 13.5 13.7 9.1 13.5 13.5 13.5 9.1 13.5 13.7 12.2 12.2
k,"
k? 2.1 X IO6 1.6 X IO6 2.7 X IO6 4.0 X IO6 5.9 x 106 2.8 X IO6 1.06 X IO6 9.6 X IO8 1.5 x 109 5.4 x 106 9.8 X IO6 1.1 x 108f 6.3 X IO8 8.0 X IO8
Kkinb 75 59
2.8 x 104 2.7 x 104
AEC/V
K,bb 62
0.1 1
I x 105 2.5 x 104 2.8 x 104 5 x 1054
59 I I8 38
100 32 1920d
0.12 0.09 0.19d
1200 4.1 1 I8 140
0.09 0.18 0.035 0.12 0.12
e
1.4 X IOs
39
7 x 106 7 x 106
100 1I 4
" R a t e constants in M-I s-l. Estimated to be accurate to &IO% for the reactions in favored directions and i 2 0 % for the reverse reactions. Equilibrium constants derived from the kinetics, K,,, and equilibrium absorbances of the radicals, Kab. Redox potential difference calculated from the mean equilibrium constant, (Kak + Kkin)/2,using the Nernst equation AE = 0.059 log K. Estimated to be accurate to f0.02 V. dFrom ref 21. Irreversible. /Similar to the data in ref 21. ~5-Hydroxyindole-3-aceticacid.
used to scavenge the photogenerated electron. The results are summarized in Table 11. At pH 9.1 the indoxyl radical was generated by the thiocyanate radical induced oxidation of 5-hydroxytryptophan (5-Trp-OH) (SCN)2'-
+ 5-TrpOH
-
5 - T r p O ' +H+
k = (5.5 f 0.5) X IO8 M-l
+ 2SCN-
A)
(10)
s-I
The indoxyl-type radical was found to oxidize ferrocyanide in the system 5 and 8 mM 5-Trp-OH and 0.2-2 mM K4[Fe(CN),], according to the following reversible reaction 5-Trp-0'
k + Fe(CN)64- + H + & 5 - T r p O H + Fe(CN)63k,
(11)
350
460
5b
450
the concentration of H+.For instance, at pH 13.5 the equilibrium is such that Fe(CN)63- oxidizes the (ionized) R-IndH-0-; Le., the equilibrium position is inverted relative to that of eq 1 1. This was shown by using ferricyanide generated from ferrocyanide either by *N3,reaction 8, or by laser photolysis, reaction 9. The absorbances of the indoxyl radical anion depended on the ratio of concentrations of ferrocyanide and hydroxyindole in a way characteristic for the equilibrium reaction Fe(CN)63- + R-Ind-0-
2.50 )'
2.00 -
if. 1 6*lO6M1 s.'
(S(0pe)
kr=2.7x104M4s.' (Intercem)
'
~
1.50 -
k
0.00
+ 4-(CH30)C6H,O- + H+
--*
+
~-(CHY,O)C~H~O' CH3CHO (13)
k = 9.8
X
lo8 M-I
s-I
l6
The reaction of the formyl radical with ferrocyanide in concentrations from 0 to 14 mM was found to be 2 orders of magnitude slower CH2=CHO'
600
& R-Iiid-0' + Fe(CN)64- + H+ k,
The approach to equilibrium in reaction 12 and the results of the kinetic analysis of the experimental data are illustrated in Figure I. In addition to hydroxyindoles, the reduction potentials of the radicals derived from 4-methoxyphenol and TMPD were measured by using the ferricyanide/ferrocyanideredox couple as a reference. The 4-methoxyphenoxyl radical was generated in the reaction of the formyl radical with the 4-methoxyphenoxide anion CH2=CHO'
550
WAV E t E VGTH, nm
As can be seen, the position of the equilibrium is dependent on
+ Fe(CN)64- + H+ k = (8.7 f 0.8)
X
-+
Fe(CN)63- + C H 3 C H 0 (14)
IO6 M-l
s-I
020
0.40
060
0.80
1.00
[5-Tre-C]l[Fe(CN$-]
Figure 1. (A) Spectra of the radicals obtained upon the azide radical induced oxidation of Fe(CN)6' (0)(see reaction 8), upon establishment of equilibrium in electron transfer from 5-hydroxytryptophan to Fe(CN)C- (0)(see reaction I2), and upon the azide radical induced oxidation of 5-hydroxytryptophan (A). Dose per pulse = 1 Gy, I = 2 cm, pH(0.34 M KOH) = 13.5, 0.1 M NaN,, 20 "C. (B) Kinetic analysis of the data obtained for the electron-transfer equilibrium between Fe(CN):-/+ and 5-TTp-0' using pulse radiolysis and the azide radical as a "primary oxidant". At doses of
-
10 Gy per pulse, the approach t o equilibrium in the reaction of the 4-methoxyphenoxyl radical with ferrocyanide
4-(CH3O)C6H40'
+ Fe(CN)64-
k
kr
4-(CH3O)C6H@-
+ Fe(CN),3-
(1 5)
was partially obscured by the fast decay of the 4-methoxyphenoxyl radical. However, the use of a low dose rate, -0.8 Gy, at which the concentration of radicals is 0.5 MM,and high concentrations of solutes (41 mM 4-methoxyphenol and 0.3-1 1 mM Fe(CN),&)
3586 The Journal of Physical Chemistry, Vol. 94, No. 9, 1990 TABLE IV: One-Electron Redox Potentials versus NHE at 20 OC organic comDound reference comDound" DH E/V R-lndH-OH Fe(CN):(0.427) 9.1 0.55 0.29b R-IndH-0Fe(CN),'- (0.45) 13.5 4-CH3PhO- (0.54) 13.5 0.35c T M P D (0.27) 13.7 0.3Ic 0.39c T M P D (0.27) 12.2 0.27d TMPD Fe(CN),'- (0.45) 9. I 0.27d Fe(CN),'- (0.455) 13.5 0.54d 4-CH'OPhOFe(CN),4- (0.45) 13.5 0.73c 7 .O 0.96' 3 .O 0 1.14' 0.98c promethazine 4 - C H 3 0 P h O H (0.96)e 3.0 "Values given in parentheses refer to the redox potentials in volts of the reference compounds. bCorrected for ionic strength, I = 0.34 M, according to the formulazz E,,, = Em - AE,,,, AE,,,, = 59.1(-1 + 3)flf) 54.3 mV, where -1 and -3 are charges of reactants (see reaction 12 in text) and f(/)is calculated fromf(/) = 1.02 (/1/2(1 I1l2)-l 0 . 2 4 . ?These values are derived from secondary redox standards (organic redox couplex). They are less accurate than those obtained by using inorganic compounds as references (see Discussion). dCorrected for the ionic strength experimentally. The effect of ionic strength on the electron-transfer equilibria was studied in the range from I = 0.3 M to I = 0.6 M. The rate of reverse reactions was found to be unaffected by the variation of the ionic strength within f20%. FCalculated from the value at pH 13.5 via the formulaz2 EpH= Eo 0.059 log (lO-pKa IO-P"), where pKa(4-CH,0PhOH) = 10.2.15
+
+
+
made a reliable measurement possible. The measured rate and equilibrium constants of reactions 6 , 1 I , 12, and 15 obtained under various experimental conditions are presented in Table 111. The data on electron-transfer reactions between organic redox couples of hydroxyindoles, 4-methoxyphenol, and TMPD are also given in Table 111. The reduction potentials of organic redox couples in alkaline media which were derived from the data presented in Table 111 are summarized in Table IV. Acidic Media. Hexachloroiridate(IV/III)with the reduction potential E = 0.867 V7 was used as a reference redox couple for the measurements in acidic media at pH 3. Hexachloroiridate(IV), IrC162-*was generated from hexachloroiridate(III), IrCI:-, in an Ar-saturated aqueous solution of 2-3 mM IrC163-containing 0.1 M 2-chloroethanol by 248-nm laser photolysis
-
IrC163-
hu
Jovanovic et al. fusion-controlled rate constant Pz*+
-
+ 1rc1,~-
k = (4.2 f 0.4)
Pz
+ IrCI,2-
(19)
lo9 M-l s-]
X
which means that its reduction potential must be higher than 0.867 V . In addition, the promethazine radical cation generated in reaction 18 from 7 mM promethazine at pH 3.3 was found to oxidize Ni(tacn),,+ in concentrations from 2 to 12 mM slowly and irreversibly according to the reaction Pz'+
+ Ni(tacn),,+ k = (4 f 1)
X
Pz
+ Ni(ta~n),~+
(20)
IO5 M-Is-l
I t was also possible to photoionize Ni(tacn),2+. Based on photoionization yield of K1 in aqueous solution of 0.29, the quantum yield for photoionization of Ni(tacn)?+ was measured to be $(e-) = 0.16. Since the reduction potential of the nickel complex is E(Ni(tacn)23+/Ni(tacn)2+)= 0.95 V,24the reduction potential of the promethazine radical cation must be higher than 0.95 V. The ability of the promethazine radical to oxidize Ir(II1) and Ni(I1) (reactions 19 and 20) is in disagreement with the previous report^^^^,^ of E3 = 0.86 V for the promethazine radical. In order to resolve this discrepancy, the redox reaction between the promethazine radical cation and 4methoxyphenol was studied. The promethazine radical cation was generated from 11 mM promethazine at pH 2.9 (reaction 18). It was found to oxidize 4-methoxyphenol ( E 3= 0.96 f 0.02 V ) in concentrations from 1.5 to 23 mM reversibly according to the reaction
Pz'+
+ 4-(CH30)C6H,0H
+
Pz
+ 4-(CH30)C6H40'+ H+
(21) From the equilibrium constant of reaction 21 (see Table V) and the calculated reduction potential of the 4-methoxyphenoxyl radical, E3 = 0.96 f 0.02 V (see Table IV), the reduction potential of the promethazine radical is derived as E = 0.98 f 0.03 V. The promethazine radical cation generated by the Cl3C0O' induced oxidation of 10 mM promethazine was then reacted with the 5-hydroxyindoles. At pH 3 electron transfer from hydroxyindoles in concentrations from 0.1 to 2 mM to the promethazine radical cation was found to proceed under equilibrium conditions R
+
IrC162- eaq-
d(e-) = 0.14 R-IndH-OH
However, the oxidation of the hydroxyindoles in concentrations from 0.1 to 1 mM by lrC162-was found to be slow IrCI6*- + R-IndH-OH
-
IrC163-+ R-IndH-0'
k = (3.8 f 0.3)
X 10,
M-'
+ H+
+ Pz + H+
-
Pz"
R
PZ*+
( 1 7)
s-I
A reverse reaction was not seen but cannot be excluded. This allows a rough estimate of an upper limit of the reduction potential of the hydroxyindoles, that is, E < 0.87 V. The promethazine radical cation, Pz", with a reported reduction potential E 3 = 0.86 V,23ais also a moderately strong oxidant in acidic media. I t was generated by the trichloromethylperoxyl radical, CI,COO', induced oxidation of -4 mM promethazine, PZ
C1,COO'
(R' = CHZCH(CH~)N(CH~)Z)
+ C13COOH
(18)
k = 6.0 X IO8 M-l s-l
The promethazine radical cation was found to oxidize Ir111C163in concentrations from 0.04 to 0.3 mM irreversibly, with a dif-
R' Pz
The measured rate and equilibrium constants of reactions 21 and 22 are summarized in Table V. On the basis of the reduction potential of the promethazine radical, E = 0.98 f 0.03 V, and the measured redox potential difference relative to the hydroxyindoles (see Table V), the reduction potential of the 5-indoxyl radicals E, = 0.86 f 0.05 V vs NHE at pH 3. Discussion The Method. The reduction potential determinations are based on the following e q u i l i b r i ~ m ~ ~ ~ ~ ~
X' (23) (a) Pelizetti, E.; Mentasti, E. fnorg. Chem. 1979, 18, 583. (b) Bahnemann. D.; Asmus, K.-D.; Willson, R. L. J . Chem. Sor., Perkin Trans. 2 1983, 1669. (c) Bahnemann, D.; Asmus. K.-D.; Willson, R. L. J . Chem. SOC., Perkin Trans 2 1983, 1661
R-IndH-0'-
k
+ s-& x- + S' k,
(24) Wieghardt. K.; Schmidt, W.; Herrmann, W.; Kiippers, H.-J. Inorg. Chem. 1983, 22. 2953.
The Journal of Physical Chemistry, Vol. 94, No. 9, 1990 3587
Reduction Potentials of 5-Indoxyl Radicals
TABLE V: Electron-Transfer Reactions in Acidic Solutions Investigated by Pulse Radiolysis at p H 3, 20 'C"
X'
S-
Pz" (0.98)c
4.2 x 109 4 x 1058 1.1 x 108 1.8 X IO8
1rCI:Ni(tacn)$+ 5-Trp-OH 5-lnd-OH HIAA
serotonin 4-CHjOPhOH
2.0 x 108 6.3 x 107 2.2 x 106
k,b
KhC
KSkC
1.0 x 106 1.6 X IO6
1 IO 112
61
0.12
114
0.1 1
2.6 x 105 1.3 X IOs
242 3
165
0.14 0.02*
AE~IV
f f
2
"CI,COO' is used as a "primary oxidant"; see eq 18. bRate constants in M-' s-'. Estimated to be accurate to &IO% for the reactions in favored directions and &20%for the reverse reactions. CEquilibriumconstants derived from the kinetics, Kki,, and equilibrium absorbances of the radicals, K,b. dRedox potential difference calculated from the mean equilibrium constant, (Kab + Kkin)/2,using the Nernst equation, AE = 0.059 log K. Estimated to be accurate to f0.02 V. CRedoxpotential at pH 3; taken from Table IV. /Irreversible. ZError margins are larger, &25%,because of the low rate of this reaction. *Estimated to be accurate to f0.005V .
with K = k f / k , and K = {[X-][S']/[X'][S-]}. The unknown reduction potential E(X'/X-) is related to the reduction potential of the standard, E ( S ' / S - ) , by E(X'/X-) = E ( S ' / S - )
+ AE
where AE = 0.059 log K [VI (the Nernst equation). The electron exchange is often in competition with the second-order decay of IO9 M-I s-I. Under our organic free radicals with typically 2k experimental conditions the free-radical concentrations were 10" M. Therefore, in order to compete effectively with the decay of the radical, the pseudounimolecular rate of the electron exchange should be > I O 4 s-l. Hence, at solutes concentrations of M the second-order rate of electron transfer should be >lo7 M-' s-I. Such a high rate of electron transfer in an unfavorable direction is unlikely, and complications due to radical/radical decay must therefore be anticipated. The difficulties arising from the second-order decay of radicals can be minimized by lowering the concentration of radicals. However, this method is limited by the finite sensitivity of their detection. Inorganic compounds, notably transition-metal complexes, are advantageous as redox standards over organic redox couples because both the oxidized and the reduced forms are long-lived. Hence, the reduction potentials of metal complexes are known with a high degree of accuracy from the classical electrochemical measurements and pertain to fully reversible electron transfer. The electron-transfer reactions involving metal complexes, however, are usually slow due to high reorganization energies.26 An additional complication is that the generation of redox-active forms in pulse radiolysis of aqueous solutions of metal complexes may be difficult due to side reactions of oxidizing radicals which may interact with the ligands rather than oxidize the metal center, as in the case of the 'OH radical.27 In these cases it is advantageous to generate the oxidized forms of inorganic complexes by photoionization, using laser photolysis. Hence, the results obtained by laser photolysis were compared to those from pulse radiolysis, where the azide radical was used as a selective oxidant. Both methods were found to give similar results (see Tables 111 and IV). The advantages of the pulse radiolysis method are reflected in more accurate determination of equilibrium absorbances of reactants. On the other hand, the laser photolysis generates the oxidized forms directly, which simplifies the experimental system. This is particularly important in those cases where the ionic strength of the medium should be kept low. The Accuracy of Measurement. The reduction potentials of the radicals derived from hydroxyindoles, TMPD, 4-methoxy-
-
-
( 2 5 ) Meisel, D.; Czapski, G. J . Phys. Chem. 1975, 79, 1503. Ilan, Y . A,; Csapski, G.; Meisel, D. Biochim. Biophys. Acta 1976,430, 209. Wardman, P.; Clarke, E. D.J . Chem. Soc., Faraday Trans. I 1976, 72, 1377. (26) Cannon, R. D.Electron Transfer Reactions; Butterworths: London,
1980.
p.\.
"0° 0.75
(27) Pohl, K.; Wieghardt, K.; Kaim, K.; Steenken, S. Inorg. Chem. 1988, 27, 440.
0.25
i u
0.00
3.0
6.0 9.0 PH
12.0
Figure 2. pH dependence of the reduction potential of the 5-indoxyl radicals. The points represent the measured values. See Discussion for
details. phenol, and promethazine were determined in this study versus the primary redox standards ferricyanide/ferrocyanide, hexachloroiridate(IV/III), and ( t a ~ n ) , N i * ~ ~The / ~ Istandard . deviations of the values of the reduction potential difference, which are f0.02 V, reflect the error limits of the equilibrium constants for the electron-transfer reactions and are approximately an order of magnitude higher than those of the electrochemically determined reduction potentials of the primary From the reactions between the organic redox couples, which may be regarded as secondary standards, it was found that the measured reduction potential differences agreed with those calculated from their reduction potentials measured versus the primary redox standards (see e.g. Table IV and Figure 2). It should be pointed out, however, that the error limits for the values based on secondary redox standards are larger, Le., f 0 . 0 4 V. One-Electron Reduction Potentials. The difference between the reduction potentials of the radicals derived from individual 5-hydroxyindolederivatives is found to be insignificant, both when Fe(CN):- (Table 111) and promethazine (Table V) are used as redox standards. It is therefore concluded that the reduction potential of 5-indoxyl radicals is independent of the side chain. This is not surprising since the redox-active moiety in the compounds and the radicals is the indole ring whose electronic structure is apparently only marginally affected by alkyl substitution in the C3 position. On the other hand, the rate constant for reaction of the promethazine radical cation at pH 3 with (positively charged) serotonin was found to be - 3 times lower than with (uncharged) 5-hydroxyindole (Table V). An analogous effect was observed in the reactions of Fc!(CN)~~-at pH 13.5 (Table H I ) . Unsubstituted 5-hydroxyindoleand serotonin (with uncharged side chain) react - 2 times faster with Fe(CN)63- than do 5hydroxytryptophan and 5-hydroxyindole-3-acetic acid which have a negatively charged carboxylic group in the side chain. These differences in reactivity are attributed to electrostatic effects. Obviously, the charge of the side chain of 5-hydroxyindole derivatives influences the kinetics of their reactions with charged
3588
The Journal of Physical Chemistry, Vol. 94, No. 9, 1990
substrates. However, in all cases the effect on kf and k , is similar (see eqs 12 and 22). As a result, thermodynamic quantities, such as reduction potentials and dissociation constants of the molecules and free radicals, are practically independent of the structure of the side chain. The reduction potentials of the 5-indoxyl radicals in this study are as follows: El,,, = 0.29 f 0.02 V at pH 13.5, E9,, = 0.55 f 0.02 V at pH 9. I , and E, = 0.86 f 0.05 V at pH 3. On the basis of these values and taking into account the dissociation constants of the species involved, the pH dependence of the reduction potential was calculated and is shown in Figure 2. The value at pH 7, E7 = 0.64 V, is lower than the reduction potentials of radicals derived from biological target molecules, for example those derived from tyrosine, E7 = 0.85 V,28 and DNA-guanine, E , = 1.04 V.5 This means that the 5-hydroxyindoles are able to repair the radicals of tyrosine and guanine by electron transfer which supports the hypothesis that 5-hydroxytryptophan, serotonin, and 5-hydroxyindole-3-acetic acid may function via electron donation as endogenous antioxidants in biological systems.29 The values of reduction potentials of 4-methoxyphenoxyl radicals, El3 = 0.43 V and E7 = 0.63 V, were recently obtained by cyclic ~ o l t a m m e t r y . ~Because ~ of a lack of experimental detail, the reason for the discrepancy between the above values and our values of E13.5 = 0.54 V and E, = 0.73 V cannot be addressed properly. A final comment may be made on the previously21determined E values for the radicals derived from 5-hydroxytryptophan and 4-methoxyphenol, which are lower than those measured in the present work. The value of 0.4 V for El3.5 for the 4-methoxyphenoxy1 radicals2' was based on .El3,,= 0.26 V for 5-hydroxytryptophan radicals. I f the new value of 0.30 V is taken for 5-hydroxytryptophan radicals as a standard, the derived E,,,, for 4-methoxyphenoxyl radicals is 0.49 V, not too different from the revised value of 0.54 V (Table 1V). The higher values of 0.54 V at pH 13.5 and 0.73 V at pH 7 obtained for 4-methoxyphenoxyl radicals in this study by pulse radiolysis are considered to be more reliable, because they are based on the comparison with the inorganic standard d e ~ c r i b e d . ~ ' - , ~ (28) Jovanovic, S . V.; Harriman, A.; Simic, M. G.J . Pbys. Cbem. 1986, 90, 1935. Butler, J.; Land, E. J.; Priitz, W. A.; Swallow, A . J. Biochim. Biopbys. Acfa 1982, 705, 150. Butler, J.; Land, E. J.; Priitz, W. A,; Swallow. A. J. J . Chem. Soc., Chem. Commun. 1986, 348. (29) However, in contrast to an earlier suggestion (Cadenas, E.; Simic, M. G.; Sies, H. Free Radical Res. Commun. 1989, 6, 1 l ) , the 5-hydroxyindoles are not able to repair by one-electron transfer the radical of vitamin E. (30) Harriman, A . J . Pbys. Cbem. 1987, 9 / , 6102. (31) See: ref 27 and Steenken, S., unpublished results. (32) Exactly the same value (E13,5= 0.54 V) has been determined for 4-methoxyphenol based on electron exchange with the CI02'/C102- couple: Eriksen, T. E.; Lind, J.; Merenyi, G. The One-Electron Reduction Potential of 4-Substituted Phenoxy1 Radicals in Water, 1989, private communication.
Jovanovic et al. An additional outcome of this study is the revised value E , = 0.98 i 0.03 V for the promethazine redox couple. This value is 0.12 V higher than the previously reported numbers of 0.8623aand 0.865 V.23b The former23awas determined with reference to the Fe(lll)/Fe(Il) couple. However, the distortion of the electrontransfer equilibrium by the decay34of the promethazine radical cations was neglected. The latter value23bwas determined by equilibrating promethazine with 12*-. However, in this reaction -;1 does not react exclusively with promethazine by outer-sphere electron transfer. In fact, the efficiency is less than Similarly, dithiocyanate radicals, which are stronger oxidants than I**-, react with promethazine by electron transfer with only 64% efficiency.23c Hence, when other than outer-sphere electrontransfer mechanisms are involved, the determination of the equilibrium constant for electron transfer is likely to be susceptible to errors. Our value for Pz'+ of 0.98 V, which is based on electron transfer with 4-methoxyphenol and which is substantiated by comparison with the (ta~n)~Ni"'/''system, is therefore considered to be more reliable than the previous ones.36-38 Acknowledgment. We are grateful to Prof. Karl Wieghardt from the Ruhr Universitat Bochum, who provided Ni(tacn),S.V.J. thanks the Max Planck Society and the Deutscher Akademischer Austauschdienst for fellowships. (33) The reduction potential of the 4-methylphenoxyl radical in neutral medium was further checked versus l,l'-ferrocenedicarboxylic acid with E , = 0.66 V (Farragi, M.; Weinraub. D.; Broitman, F.; DeFelippis, M. R. Radiat. Pbys. Cbem. 1988, 32, 293). In an N20-saturated aqueous solution of 7-9 mM 4-methoxyphenol, 0.1 M NaN,, and 0.35-2 mM ferrocene derivative at pH 7.5 the followin electron-transfer reaction was observed: 4-t F$+(C,H&OOH)2 -t H+ 4-(CH30)C6H,0H + (CH@)C&0' Fe3+(CsH4C0OH),,k = (6.6 f 1) X 106 M-' s-I. Consequently, the reduction potential of the 4-methoxyphenoxyl radicals must be higher than 0.66 V, which is in support of E , = 0.73 V obtained in this study. (34) Forni, L. G.; Monig, J.; Mora-Arellano, V. 0.;Willson, R. L. J. Cbem. SOC.,Perkin Trans. 2 1983, 961. The relative instability of the promethazine radical cation was also observed in the cyclic voltammetry measurement,)6 in which both electrochemical charge transfer from promethazine and chemical decay of the resulting promethazine radical cation were found to be fast. (35) This value is based on the yield of electron transfer from promethazine to 12'- in the system 10 mM KI, 0.5 mM promethazine, N20-saturated aqueous solution, measured as the absorbance of the promethazine radical cation at 525 nm, and taking c(Pz'+) = 9500 M'I cm-1.23c (36) A similar value, E = 0.94 f 0.02 V, was obtained by cyclic voltammetry with glassy carbon electrodes: Zecevic, S.; Simic, M.; Jovanovic, S . Manuscript in preparation. (37) While this manuscript was being reviewed, another cyclic voltammetry measurement gave E = 0.99 0.02 V for the promethazine redox couple: Harriman, A., unpublished results. (38) The electron-transfer reaction between the promethazine radical cation and the CI02'/C10; couple in the pH range from 5 to 7, which was suggested by one of the reviewers as additional proof for the reduction potential of the promethazine radical cation, could not be investigated because of the rapid thermal oxidation of promethazine. (When solutions of C102and promethazine were mixed at millimolar concentrations of solutes, an intense red color developed instantly.) +
