180
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
centered radicals to coordinate with electron-rich centers. Specific electronic structures such as the 2o'/Icr* three-electron bonds provide a rationale for many physical and chemical characteristics of the radical species. Another important finding is the frequent involvement of sulfur-centered radicals in thermodynamic equilibria. This appears to be of particular significance for the role of thiyl radicals since it could explain, for example, some of the insufficiencies in the repair and protection mechanism of thiols. In fact, the equilibria open the possibility that thiyl radicals indirectly could even contribute to biological damage. Finally, it must be recognized that thiyl radicals can undergo many more reactions than just recombination to their respective disulfides. From product analysis alone this is often difficult to realize since most of the thiyl radical reactions ultimately lead also to either disulfides or thiols. It must clearly be recognized that thiyl radicals are able to undergo a large variety of reactions, and depending on the environmental conditions, that is, local concentrations of possible reaction partners, may thus become imbedded in complex reaction schemes.
[16] Q u i n o i d C o m p o u n d s : H i g h - P e r f o r m a n c e L i q u i d Chromatography with Electrochemical Detection
By ENRIQUE CAI~ENASand LAgS EgNSTER Introduction The carbonyl groups in quinones are found in the same or separate rings and are conjugated with the double bond. The particular arrangement of double bonds inside and outside the six-membered ring of quinones is largely responsible for their chemical properties. The chemistry of quinones is, in many aspects, similar to that of ot,/3-unsaturated ketones, and most of their redox properties are based on the electrophilic reactivity determined by the carbonyl groups and the reaction of the polarized double bonds with nucleophiles. The redox features of quinones are essential to the understanding of their overall biological activity, which encompasses functional, toxicological, mutagenic, and antitumor actions. Quinones can participate in redox transitions involving (1) pure electron transfer, such as in the enzymatic reduction of quinones or in the several nonenzymatic sources of semiMETHODS IN ENZYMOLOGY, VOL. 186
Copyright @ 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
[16]
QUINOID COMPOUNDS
181
quinones, and (2) nucleophilic addition reactions, ~implying either oxidation or reduction of the quinone ring. The former is exemplified by the oxidation of the - - C 2 ~ C 3 - - bond to quinone epoxides by 02 nucleophiles and the latter by the reductive addition via sulfur nucleophiles to yield thioether-hydroquinone adducts. Since hydroquinones and quinones can be oxidized and reduced electrochemically,2,3 it appears reasonable to attempt to detect and quantify them by high-performance liquid chromatography (HPLC) with electrochemical methods (HPLC-EC). The sensitivity afforded by electrochemical detection (picomole range) along with the separation methods of HPLC 4 seems ideal for detection and quantification of quinoid compounds in biological systems) HPLC with oxidative electrochemical methods has been successfully applied to the detection of aromatic alcohols and amines, indoles, phenothiazines, purines, as well as other substances, such as ascorbic acid and thiols. 6,7HO., a species often responsible for cellular damage, can also be identified by HPLC-EC by measuring the adduct resulting from the reaction of HO. with 5,5-dimethylpyrroline N-oxide (DMPO) 8 or the oxidation products of salicylate9,~°; in the latter instances, however, some reservations should be taken into account, for the high oxidation state of certain hemoproteins, e.g., ferrylmyoglobin, can yield a hydroxylation pattern of salicylate similar to that observed with hydroxyl radical. 9,11 K. T. Finley, in "The Chemistry of Quinonoid Compounds" (S. Patai, ed.), p. 877. Wiley, London, 1974. 2 j. Q. Chambers, in "The Chemistry of Quinonoid Compounds" (S. Patai, ed.), p. 737. Wiley, London, 1974. 3 G. Dryhurst, K. M. Kadish, F. Scheller, and R. Renneberg, in "Biological Electrochemistry" (G. Dryhurst et al., eds.), vol. 1, p. 1. Academic Press, New York, 1982. 4 p. T. Kissinger, K. Bratin, W. P. King, and J. R. Rice, ACS Symp. Ser. 136, 57 (1981). 5 D. S. Fluck, S. M. Rappaport, D. A. Eastmond, and M. T. Smith, Arch. Biochem. Biophys. 235, 351 (1984). 6 T. Kurahashi, H. Nishino, S. Parvez, H. Parvez, K. Kojima, and T. Nagatsu, in "Progress in HPLC, Volume 2: Electrochemical Detection in Medicine and Chemistry" (H. Parvez, M. Bastart-Malsot, S. Parvez, T. Nagatsu, and G. Carpentier, eds.), p. 3. VNU Science Press, Utrecht, 1987. 7 H. Parvez, M. Bastart-Malsot, S. Parvez, T. Nagatsu, and G. Carpentier (eds.), "Progress in HPLC, Volume 2: Electrochemical Detection in Medicine and Chemistry." VNU Science Press, Utrecht, 1987. s R. A. Floyd, C. A. Lewis, and P. K. Wong, this series, Vol. 105, p. 231. 9 R. A. Floyd, R. Henderson, J. J. Watson, and P. K. Wong, J. Free Radicals Biol. Med. 2, 13 (1986). 10D. Mira, U. Brunk, A. Boveris, and E. Cadenas, Free Radical Biol. Med. 5, 155 (1988). H D. Galaris, D. Mira, A. Sevanian, E. Cadenas, and P. Hochstein, Arch. Biochem. Biophys. 262, 221 (1988).
182
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
The principle and versatile applications of HPLC-EC have been authoritatively reviewed. 4,6,12 The scope of this chapter encompasses only the amperometric detection of quinones of the p-benzo- and 1,4-naphthoquinone series and the effect of substituents on the electrochemical properties of these compounds. Principle of HPLC with Electrochemical Detection The principle of HPLC-EC is based on the redox properties of the compounds of interest; hence, the chemical reaction, namely, electron transfer, as well as its dependence on environmental factors (e.g., composition and physicochemical properties of the mobile phase) are essential to the detection p r o c e s s . 4'6'12 HPLC-EC is an amperometric determination that measures the potential difference between the electrode (embedded in one wall of the electrochemical cell) and the bulk of the solution. This determines the reactivity of the electrochemically active compound at the electrode surface. The detector used in this work, a three-pole potentiostat system, was a TL-5A three-electrode glassy carbon flow cell (Bioanalytical Systems Inc., West Lafayette, IN). On the basis of the potential of a reference electrode (Ag/AgCI), constant potential electrolysis is given between the working electrode (glassy carbon) and the auxiliary electrode (stainless steel tube). There are two modes of electrochemical detection: electron transfer from the solute to the electrode surface is known as oxidative electrochemical detection, whereas electron transfer from the electrode surface to the solute is known as reductive electrochemical detection. The former requires a positive applied potential, and the latter requires a negative applied potential (Fig. I). The interfacial current measured at a fixed electrode potential is proportional to the concentration of electrochemically active material and is converted to voltage by means of an amplifier current-to-voltage converter (Model LC-4, Bioanalytical Systems). The gain of the converter (nA V -l) over the recorder sensitivity (V cm -1) sets the units of the recorder scale (nA cm-~). Figure 2 shows a chromatogram with reductive applied potential of a mixture of various 1,4-naphthoquinones and naphthoquinone epoxides. Other quinones of biological interest, such as vitamin E quinone, can also be measured by this technique. 13Table I ~-~7 12 p. T. Kissinger, K. Bratin, G. C. Davis, and L. A. Pachla, J. Chromatogr. Sci. 17, 137 (1979). 13 S. Ikenoya, K. Abe, T. Tsuda, Y. Yamano, O. Hiroshima, M. Ohmae, and K. Kawabe, Chem. Pharm. Bull. 27~ 1237 (1979). ~4A. Brunmark, E. Cadenas, J. Segura-Aguilar, C. Lind, and L. Ernster, Free Radical Biol. Med. 5, 133 (1988).
[16]
183
QUINOID COMPOUNDS Negative applied potential Oxidative Electrochemistry OH
0
OH
0
[ Electrode ]
Positive applied potential Reductive Electrochemistry
FIO. 1. Schematic representation of electrochemical oxidation and reduction during HPLC with amperometric detection of 1,4-naphthoquinone.
lists the retention times observed for several p-benzo- and 1,4-naphthoquinone derivatives with different mobile phases. The mobile phases were chosen to have a high buffer capacity and a high ionic strength by addition of sodium sulfate. Within the same mobile phase (e.g., phase A, Table I), methyl substitution increases the retention time of the quinone, whereas the quinones substituted with the more polar hydroxyl and methoxyl groups have a shorter retention time. The same applies to naphthoquinone epoxides, whose methyl-substituted derivatives are retained longer in the column. Since amperometric detection involves heterogenous electron transfer between an electrochemically active material (solute or analyte) and the electrode surface, the fundamental redox process occurring in the cell will obviously be affected by the composition and pH of the mobile phase as well as the applied potential at the electrode. The selection of the mobile phase is an important factor, allowing the electrode reaction to occur; although the mobile phase should be electrochemically inert at the electrode surface, the amperometric character of the determination requires the presence of electrolytes to convey charge through the electrochemical cell and a solvent with a sufficiently high dielectric constant to permit dissociation of the electrolyte. Therefore, amperometric detection is most satisfactory in mobile phases of high ionic strength (>50 mM) which contain a minimum amount of nonaqueous solvent. 4 The pH of the mobile phase is another important consideration, for the heterogenous electron transfer between the electrochemically active molecule and the electrode surface is affected by pH, as are those chemical reactions linked to the 15 A. Brunmark and E. Cadenas, Free Radical Biol. Med. 3, 169 (1987). t6 A. Brunmark and E. Cadenas, Free Radical Biol. Med. 6, 149 (1989). |7 G. Buffinton, K. Ollinger, A. Brunmark, and E. Cadenas, Biochem. J. 257, 561 (1989).
184
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
initial electron-transfer process. Stationary phases or columns for amperometric detection include all type of reversed phase materials, for they are compatible with polar mobile phases containing dissolved electrolytes. 4 60(1
IV
nA V
II
3(Yd III VI
OI
[
I
I
0
10
20
30
Retention time (min) FIG. 2. Analysis of quinones by HPLC with electrochemical detection. Chromatographic conditions: Stationary phase: 15 cm × 3.9 mm Novapak 4/~M Cls reversed phase (WatersMillipore, AB, V. Fr61unda, Sweden). Mobile phase: 35% 2-propanol-65% H20 buffered to pH 6.5 with 50 mM sodium phosphate. Mobile phase flow rate: 0.5 ml min-I. Detector: LC-4 with a TL-5A three-electrode glassy carbon flow cell and a Ag/AgCI reference electrode (Bioanalytical Systems). Applied potential: -0.9 V versus Ag/AgC1. Amount injected: 2 nM of each compound. Peaks: I, 2,3-epoxy-l,4-naphthoquinone; II, 1,4-naphthoquinone; III, 2-methyl-2,3-epoxy-l,4-naphthoquinone; IV, 2-methyl-l,4-naphthoquinone; V, 2,3-dimethyl-2,3-epoxy-l,4-naphthoquinone; VI, 2,3-dimethyl-l,4-naphthoquinone.
[16]
QUINOID COMPOUNDS
185
TABLE I HPLC WITH ELECTROCHEMICALDETECTION ANALYSISOF 1,4-NAPHTHOQUINONE DERIVATIVES O
R2 R3
Retention time (min); mobile phase a
O
R1b
R2
R3
A
B
H CH3 CH3 OCH3 OH CH3 H CH3 H SG NAC H OH
H H CH3 OCHa H OH H H SG SG H SG SG SG SG NAC O
H H H H H H OH OH H H H OH H H OH H
6.8 12.4 27.4 4.7 2.1
4.6
H H H
4.9 7.8 13.8
CH3 CH3 CH3
C
D
2.3 6.3 36.0 10.1
16.0 5.4 16.5 16.0
1.8 38.0 46.0 26.0
4.1
0 R2 R3 H CH3 CH3
0 H H CH3
17.8 13.3
a (A) 35% 2-propanol-65% H20, buffered to pH 6.5 with 50 mM sodium phosphate. 5,14,t5 (B) Five milliliters phosphoric acid diluted to 1 liter with H20 and adjusted to pH 7 with Tris base; 10% of this buffer, 25% methanol, and 65% H20 containing 50 mM sodium sulfate was used as mobile phase. ~6 (C) 35% Methanol-65% H20-0.2 mM Tris buffer, 50 m M sodium sulfate, pH 7.0? 7 (D) As mobile phase B, but methanol content increased to 37%. 16 b CH3, Methyl; OCH3, methoxyl; OH, hydroxyl; SG, glutathionyl; NAC, N-acetylcysteinyl.
186
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
The primary problem in reductive electrochemical detection is the removal of dissolved 02 in the mobile phase, because of the interference caused by 02 on reduction to peroxide and, eventually, to water. This problem can be circumvented partly by purging the mobile phase and sample with helium or argon and by using an all-steel liquid chromatograph construction. However, even under these conditions, it is difficult to keep a driftless baseline in a reducing reaction, not only because of the interference of dissolved 02, but also because of the presence of traces of metal ions leached by the eluent from the stainless steel parts of the chromatograph. Alternative ways to avoid this problem are the redox mode TM and postcolumn mobile phase changes. 12 The redox mode has been successfully applied to the determination of vitamin KI compounds and consists of two sequential generator/detector electrodes arranged in a way such that one is directly upstream of the other. The principle involves the initial reduction of vitamin KI to vitamin K~ hydroquinone (Q + 2 e+ 2 H ÷ --> QH2) by applying a sufficiently negative potential at the generator electrode; the subsequent electrochemical oxidation of vitamin K~ hydroquinone (QH2 ---> Q + 2 e- + 2 H ÷) takes place at the detector electrode, which is set at a positive potential. ~8 By this method, pure standards of vitamin K~ can be detected down to 100 pg with the further advantage that it does not require the removal of 02 from the system, because while the negative potential at the generator electrode is sufficient to reduce vitamin K1 and 02, the positive potential at the detector electrode can oxidize K~ hydroquinone but not peroxide or water) 3 On the other hand, postcolumn mobile phase changes ~2 obtained by using postcolumn mixing to alter the mobile phase pH (1 M HC104 mixed with the mobile phase prior to detection) allow an optimal determination of vitamin K3 at lower detector potential and without interference from dissolved 02. Multielectrode electrochemical detection 19 offers greater versatility than a three-pole detector, because it helps to overcome the shortcomings inherent in conventional electrochemical detection by allowing the simultaneous determination of oxidized and reduced species. This can be obtained with a detector with three working electrodes, two of which can be set to desired potentials. By this means, reduced and oxidized coenzyme Q was detected simultaneously by setting the potentials of the first and second electrodes at +0.7 and - 0 . 3 V, respectively. 9,2° 18y. Haroon, C. A. W. Schubert, and P. V. Hauschka, J. Chromatogr. Sci. 22, 89 (1984). ~90. Hiroshima, S. Ikenoya, T. Naitoh, K. Kusube, M. Ohmae, K. Kawabe, S. Ishikawa, H. Hoshida, and T. Kurahashi, Chem. Pharm. Bull. 31, 3571 (1983). 2o M. Takada, S. Ikenoya, T. Yuzuriha, and K. Katayama, this series, Vol. 105, p. 147. (1984).
[16]
QUlNOID COMPOUNDS
187
The use of amperometric detection combined with HPLC is not restricted to compounds with redox properties. Electrochemically inactive compounds can be derivatized to electrochemically active compounds to be analyzed by HPLC-EC. In this regard, nitrophenyl, aniline, quinoneimine, and phenolate species are ideal candidates for sensitive amperometric methods. Effect of Substituents on Half-Wave Reduction Potentials of Quinones Determined by HPLC with Electrochemical Detection There is a substantial body of data on half-wave potentials (E~/2) of quinones originating from electrochemical measurements corresponding to the addition of first and second electrons in nonaqueous solvents. 2,3 These measurements are usually devoid of the complications introduced by protonation steps. Values obtained with a standard calomel electrode can be converted to standard hydrogen electrode by adding 241 mV and those obtained with a Ag/AgC1 electrode by adding 221 mV. These values cannot be compared directly with those observed in aqueous solvents, as obtained with fast techniques as pulse radiolysis, because of differences in solvation energies and occurrence of junction potentials. 2~ The electrochemical reduction of quinones in aqueous or protic media exhibits a complex behavior, which has the appearance of reversible couples3; a cyclic voltammetric study of the aqueous electrochemistry of some quinones 22 indicates a strong pH dependence of the redox process. The half-wave reduction potential (E1/2) values of the 1,4-naphthoquinone derivatives in aqueous solutions examined here have been determined by means of hydrodynamic voltamograms against a Ag/AgCI reference electrode. The peak heights are plotted as a function of applied potential for each compound, resulting in the usual sigmoidal voltammetric wave. Although these El~2 values cannot be equated directly with those derived from pulse radiolysis approaches in aqueous media or cyclic voltammetry in protic or aprotic media, they provide a general indication of the redox properties of quinones within the same system, which permits one to predict the likelihood of redox interactions between different molecules. The effects exerted by different substituents on the E~/2 values of pbenzo- and 1,4-naphthoquinones are tabulated in Table II. Figure 3 illustrates the hydrodynamic voltamograms (used to calculate E~/2 values) of 21 A. J. Swallow, in "Function of Quinones in Energy Conserving Systems" (B. L. Trumpower, ed.), p. 59. Academic Press, London, 1982. 22 S. I. Bailey and I. M. Ritchie, Electrochim. Acta 30, 3 (1985).
188
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
TABLE II HALF-WAvEPOTENTIALS(El/2) OF p-BENZO- AND 1,4-NAPHTHOQUINONEDERIVATIVESa Quinone p-Benzoquinone series Unsubstituted p-Benzoquinone Hydroxyl-substituted 2-Hydroxy-p-benzoquinone Methyl-substituted 2-Methyl-p-benzoquinone 2,6-Dimethyl-p-benzoquinone p-Benzoquinone epoxides 2,3-Epoxy- 1,4-p-benzoquinone 2,3-Epoxy-2-methyl-p-benzoquinone 2,3-Epoxy-2,6-dimethyl-p-benzoquinone 1,4-Naphthoquinone series Unsubstituted
1,4-Naphthoquinone Methyl-substituted 2-Methyl- 1,4-naphthoquinone 2,3-Dimethyl-l,4-naphthoquinone Methoxyl-substituted 2,3-Dimethoxyl-l,4-naphthoquinone Hydroxyl-substituted (quinone ring) 2-Hydroxy-1,4-naphthoquinone 2-Methyl-3-hydroxy- 1,4-naphthoquinone Hydroxyl-substituted (benzene ring) 5-Hydroxy-1,4-naphthoquinone 2-Methyl-5-hydroxy- 1,4-naphthoquinone Glutathionyl-substituted 3-Glutathionyl- 1,4-naphthoquinone 2,3-Diglutathionyl- 1,4-naphthoquinone 2-N-Acetylcysteinyl- 1,4-naphthoquinone 2-Methyl-3-glutathionyl- 1,4-naphthoquinone 2-Methyl-3-N-acetylcysteinyl- 1,4-naphthoquinone Glutathionyl- and hydroxyl-substituted 3-Glutathionyl-5-hydroxy- 1,4-naphthoquinone 2-Methyl-3-glutathionyl-5-hydroxy- 1,4-naphthoquinone 2-Hydroxy-3-glutathionyl- 1,4-naphthoquinone 1,4-Naphthoquinoneepoxides 2,3-Epoxy-1,4-naphthoquinone 2-Methyl-2,3-epoxy-l,4-naphthoquinone 2,3-Dimethyl-2,3-epoxy- 1,4-naphthoquinone Data from Refs. 14-17.
EI~ (-mY) versus Ag/ AgC1
30 440 50 120 510 510 580 180 225 300 300 460 500 140 200 225 265 255 265-300 310 195 220 680 720 770-790 820
[16]
QUINOIDCOMPOUNDS
189
I 0"051
0i
I 0.2
I 0.4
I i lt.O 0.6 08 Oxidation Potential(-volts) FIG. 3. Hydrodynamic voltamograms showing the effect of substituents on half-wave potentials of 1,4-naphthoquinones. Chromatographic conditions: Stationary phase: 15 cm x 3.9 mm Novapak 4/~M Ct8 reversed phase (Waters-Millipore). Mobile phase: 35% 2-propanol-65% H20 buffered to pH 6.5 with 50 mM sodium phosphate. Mobile phase flow rate: 0.5 ml min-t. Detector: LC-4 with a TL-5A three-electrode glassy carbon flow cell and a Ag/ AgC1 reference electrode (Bioanalytical Systems). Other assay conditions in Refs. 14-17. Hydrodynamic voltamograms: II, 1,4-naphthoquinone (Ela = -180 mV); O, glutathionyl1,4-naphthoquinone (El/2 ~- -220 mV); El, 2-hydroxy-1,4-naphthoquinone (Ev2= -460 mV); O, 2,3-epoxy-l,4-naphthoquinone (Ev2 = -720 mV).
unsubstituted 1,4-naphthoquinone and derivatives bearing glutathionyl and hydroxyl substituents as well as 1,4-naphthoquinone epoxide. Most quinones can be reduced at working electrode potentials of -300 to -400 mV) The El~2 values for the unsubstituted parent compounds of the pbenzo- and 1,4-naphthoquinone series are - 3 0 and -180 mV, respectively. In general, substitution produces discrete changes in the E1/2 values (Table II) with exception of hydroxyl substituents in the quinone ring and quinone epoxides, compounds with Ei/2 values 300-400 mV more negative than the parent unsubstituted quinones. The introduction of methyl groups in p-benzoquinone or the quinone ring of 1,4-naphthoquinone decreases the Ei/2 values in an additive manner. 23 Mono- and dimethyl substitution of 1,4-naphthoquinone decreases the Ev2 by about 45 and 120 mV, respectively (Table II). 14'17 23p. Zuman, "Substituent Effects in Organic Polarography." Plenum, New York, 1967.
190
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
Hydroxyl Substitutents Hydroxyl-substituted p-benzoquinones can originate from different sources including monooxygenase-mediatedmetabolism of benzene,24 enzymatic reduction ofp-benzoquionone e p o x i d e s , 14,25,26 and sulfur nucleophilic addition to quinone epoxides. 16,27The hydroxyl substituent considerably lowers the Ev2 values of p-benzoquinones in aqueous solutions, 14,22,28 an effect explained on the basis of mesomeric or inductive effects introduced by the hydroxyl group (similar considerations apply to methoxyl substituents). 28 Reduced hydroxy-p-benzoquinones are autoxidized at rates substantially higher than the parent compounds.14,25 The effect of hydroxyl substituents on the E1/2 values of 1,4-naphthoquinones varies depending on whether the hydroxyl group is situated in the quinone ring (C-2) or in the adjacent benzene ring (C-5). In the former instance, hydroxyl substitution exerts an effect similar to that observed with p-benzoquinones, that is, a marked decrease in the EVE value; in addition, the hydroxyl substituent in the quinone ring raises the pKa value with regard to the parent quinone. 29On hydroxyl substitution at C-2, both 1,4-naphthoquinone and 2-methyl-l,4-naphthoquinone decrease their EVE by 280 and 275 mV, respectively.14.17 A similar decrease of 1,4-naphthoquinone was observed in the half-wave reduction potential (calculated against a standard calomel electrode) (AE1/2 = -280 mV)3° and in the oneelectron reduction potential [E(Q/Q-:)7] (calculated by pulse radiolysis) (AE1/2 = -275 mV) (data from Ian Wilson quoted in Ref. 31). Introduction of methoxyl groups causes a similar effect as the hydroxyl substituents vicinal to the carbonyl groups: the E1/2 value of 2,3dimethoxyl-1,4-naphthoquinone is 120 mV more negative than that of the parent compound. A 119 mV more negative E~/2 value (against standard calomel electrode) was found with the monomethoxy derivative of 1,4naphthoquinone in aqueous solutions) ° Thus, methoxyl substitution seems not to decrease the reduction potential in an additive manner; similar considerations may explain the not very negative one-electron 24 W. F. Greenlee, J. D. Sun, and J. S. Bus, Toxicol. Appl. Pharmacol. 59, 187 (1981). z5 A. Brunmark, E. Cadenas, C. Lind, J. Segura-Aguilar, and L. Ernster, Free Radical Biol. Med. 3, 181 (1987). 26 E. Cadenas, D. Mira, A. Brunmark, J. Segura-Aguilar, C. Lind, and L. Ernster, Free Radical Biol. Med. 5, 71 (1988). 27 A. Brunmark and E. Cadenas, Chem.-Biol. Interact. 68, 273 0 9 8 8 ) . W. Flaig, H. Beutelspacher, H. Riemer, and E. K~ilke, LiebigsAnn. Chem. 719, 96 (1968). 29 T. Mukherjee, Radiat. Phys. Chem. 29, 455 (1987). 3o E. M. Hodnett, C. Wongewiechintana, W. J. Dunn, and P. Marrs, J. Med. Chem. 26, 570 (1983). 31 M. d'Arcy Doherty, A. Rodgers, and G. M. Cohen, J. Appl. Toxicol. 7, 123 (1987).
[16]
QUINOID COMPOUNDS
191
reduction potential of 2,3-dimethoxyl-l,4-naphthoquinone [E(Q/Q-,) = 183 mV].32 When the hydroxyl substituent is in the adjacent benzene ring, it causes an increase in the E1/2 value, an effect understood as a polar effect caused by the hydroxyl group in the adjacent benzene ring. At variance with 2-hydroxy-l,4-naphthoquinone, a hydroxyl substituent in the benzene ring lowers the pKa with respect to the parent compound. 29 Another property of hydroxyquinones is their tendency to undergo strong intramolecular hydrogen bonding, which leads to stabilization of the semiquinone transient species involving displacement toward the left of the disproport i o n a t i o n r e a c t i o n 3 3 , 3 4 : 2 Q ~ ~-- Q + Q 2 - . On hydroxyl substitution at the aromatic ring, the E~/2values versus Ag/AgC1 of 1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone increase by 40 and 25 mV, respectively (Table II). 17This is in agreement with previous reports on the first and second electron reduction potential (calculated by cyclic voltammetry) of 5-hydroxy-l,4-naphthoquinone in apolar media33 and in aqueous media. 29 -
Glutathionyl Substituents Reduced glutathione (GSH) can be measured in tissue extracts by HPLC-EC with a method based on the separation of GSH from other components by cation-exchange chromatography coupled to the electrochemical oxidation of the thiol to the corresponding disulfide. 35 The oxidation of GSH is accomplished with a graphite paste electrochemical detector with an applied potential of 1.0 V versus Ag/AgC1. A simultaneous detection of thiols and disulfides can be obtained with a dual Hg/Au electrode thin-layer cell to perform both the reduction and detection functions. The electrodes are arranged in series with reduction of oxidized glutathione (GSSG) to GSH at the generator electrode and oxidation at the detector electrode. 36 Glutathionyl substitution is the result of the 1,4-reductive addition of the thiol across the double bond of the quinone. Since glutathionylquinone conjugates retain the redox properties inherent to the quinone moiety, they can be separated and detected by HPLC-EC. The glutathionyl substituent affects both the retention time and the Ell2 value of the quinone. ~5-17 Because of the relatively weak electron-withdrawing 35 T. W. Gant and G. M. Cohen, personal communication. 33 A. Ashnagar, J. M. Bruce, P. L. Dutton, and R. C. Prince, Biochim. Biophys. Acta 801, 351 (1984). 34 N. F. J. Dodd and T. Mukherjee, Biochem. Pharmacol. 33, 379 (1984). 35 I. Mefford and R. N. Adams, Life Sci. 23, 1167 (1978). 36 L. A. Allison, J. Keddington, and R. E. Shoup, J. Liq. Chrornatogr. 6, 1785 (1983).
192
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
properties of thioether substituents, only minute changes in the quinone reduction potential are expected on glutathionyl substitution. The EI/2 values of thioether adducts ofp-benzoquinone are slightly more negative than the parent compounds. 37 Despite the minor changes in the Ev2 on glutathionyl substitution, significant alterations in the oxidation equilibrium, involving both cross-oxidation and autoxidation reactions, are observed. 27 Electrochemical oxidation of glutathionyl-p-benzohydroquinone shows E1/2 values (versus a Ag/AgC1 electrode) of +220 mV, 20 mV more positive than those of the unsubstituted compound.15 Unsubstituted 1,4-naphthoquinone and derivatives bearing a methyl and/or hydroxyl (in the benzene ring) substituent undergo 1,4-reductive addition with thiols as GSH [reaction (1)]. Glutathionyl substitution of 1,4-naphthoquinone decreases additively the Ev2 value from -180 to -225 mV for the monoconjugate and to -265 mV for the diconjugate (Table II). The glutathionyl substitution of 2-methyl-l,4-naphthoquinone decreased the Ell2 value by 40 mV. The nucleophilic addition of N-acetylL-cysteine to 1,4-naphthoquinone and 2-methyl-l,4-naphthoquinone results in a decrease of the E1/2 value more pronounced (75 and 85 mV, respectively) than that observed with GSH.17 The decrease in E1/2 value caused by the thiol addition may be interpreted as a mesomeric effect similar to that produced by hydroxyl and methoxyl substituents. Minor changes in the one-electron reduction potential of 2-methyl-l,4-naphthoquinone [E(Q/Q:)7 = -203 mV] are observed on GSH addition [E(GS--Q/GS--Q :)7 = -192 mV]. 38 In spite of the small variations in reduction potential, the autoxidation of glutathionylnaphthohydroquinone derivatives, following the reduction of the oxidized counterpart by DT-diaphorase, is 12- to 16-fold higher than that of the parent naphthohydroquinones.17 O
OH RI
+ GS-+
H+
,.
~
RI
(1)
SG O
R 1 = H, CH3
OH
Hydroxyl and Glutathionyl Substituents Hydroxyl- and glutathionyl-disubstituted quinones can result from nucleophilic addition of GSH to (1) aromatic-, hydroxyl-substituted naphthoquinones, (2) hydroxy-p-benzoquinones, and (3) p-benzo- or 1,437 E. R. Brown, K. T. Finley, and R. L. Reeves, J. Org. Chem. 36, 2849 (1971). 38 I. Wilson, P. Wardman, T.-S. Lin, and A. C. Sartorelli, J. Med. Chem. 29, 1381 (1986).
[16]
193
QUINOID COMPOUNDS
naphthoquinone epoxides. The primary molecular products resulting from these reductive additions differ in all three instances. In the first case, the product bears a hydroxyl substituent in the benzene ring and a glutathionyl substituent in the quinone ring. In the two latter cases, the hydroxyl and glutathionyl substituents are found in the quinone ring. The occurrence of the new sulfur substituent in these hydroxyquinones controls to a large extent the subsequent redox chemistry in terms of crossoxidation, autoxidation, and disproportionation reactions. The nucleophilic addition of GSH to the 5-hydroxyl derivatives of 1,4naphthoquinone and 2-methyl-l,4-naphthoquinone [reaction (2)] results in compounds with E~/2 values close to those of 1,4-naphthoquinone and 2methyl-l,4-naphthoquinone; the difference amounts to -15 and +5 mV, respectively. This may be understood as the counterbalance of the rise in E~/2 value caused by the hydroxyl substituent at C-5 and the decrease in E~/2 value caused by the glutathionyl substituent. 0
OH + GS- + H+
(2)
~ ~ S O
OH O
OH OH
The nucleophilic addition of GSH to either 2-hydroxy-p-benzoquinone or 2,3-epoxy-p-benzoquinone yields the same primary molecular product, 2-hydroxy-5-glutathionyl-p-benzoquinone[reaction (3)], a compound that autoxidizes at rates substantially higher than the parent compound lacking a hydroxyl substituent. 27The rate of nucleophilic addition is considerably slowed down on methyl substitution. 16p-Benzohydroquinones with both hydroxyl and glutathionyl substituents autoxidize at rates 350-fold higher than the unsubstituted p-benzohydroquinone.27 It should be noted that the individual effects of glutathionyl and hydroxyl substituents are not additive when present in the same quinoid molecule, but they seem to potentiate the overall autoxidation. O
OH
O --
O
GS O
OH
(3)
O
The electron-donating properties of the hydroxyl substituent in the quinone ring increases the electron density at C-3, thereby preventing the nucleophilic addition of GSH to 2-hydroxy-l,4-naphthoquinone. However, a hydroxyglutathionyl adduct of 1,4-naphthoquinone, namely,
194
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
2-hydroxy-3-glutathionyl-l,4-naphthoquinone, was obtained during the cleavage of the epoxide ring of 2,3-epoxy-l,4-naphthoquinone on GSH nucleophilic addition 25 [reaction (4)]. A fraction of the product of reaction (4) undergoes oxidative elimination to yield glutathionylnaphthoquinone. 16
~
O
OH O + GS- + H+
~ ~[~OH
(4) SG
O
OH
Quinone Epoxides Vitamin K1 2,3-epoxide originates from the coupled activity of a twoenzyme system. Vitamin Kl epoxide is not active in prothrombin synthesis, but it is readily converted back to vitamin K by vitamin K epoxide reductase. 39 Chemical models for the molecular mechanism of vitamin K epoxide reductase emphasize the importance of a primary thiol addition to open the epoxide ring; reaction with a second thiolate results in reductive cleavage of the intermediate, followed by elimination of HzO to yield the quinone. 4°,41 Although simple quinone epoxides, such as 2,3-epoxy-p-benzoquinone and 2,3-epoxy-1,4-naphthoquinone and their methyl derivatives, are not of direct biological significance, they provide suitable experimental models to understand the electrochemical properties of biologically important quinone epoxides. The formation of quinone epoxides is attained in the addition of Oz nucleophiles to p-benzoquinones and 1,4-naphthoquinones, ~ as implied in the HzO2-dependent oxidation of the 2,3-double bond, 14,15 which is analogous to the reaction of sodium hydroperoxides with naphthoquinones. 42 The reaction is suggested to occur through a nucleophilic addition of HOO- at the/3 carbon of the unsaturated system to give an enolate ion as an intermediate. The carbanionic t~ carbon atom on the enolate ion displaces HO-, breaking the O - - O bond in the hydroperoxyl group and rearranging to an epoxide [reaction (5)]. Quinone epoxides also result from the reaction of O2 ~ with several vitamin K analogs.43 39 j. W. Suttie, CRC Crit. Rev. Biochem. 8, 191 (1980). 40 R. B. Silverman, J. Am. Chem. Soc. 103, 5939 (1981). 41 p. C. Preusch and J. W. Suttie, J. Org. Chem. 48, 3301 (1983). 42 L. F. Fieser, W. P. Campbell, E. M. Fry, and M. D. Gates, J. Am. Chem. Soc. 61, 3216 (1939). 43 I. Saito, T. Otsuki, and T. Matsura, Tetrahedron Lett. 19, 1693 (1979).
[16]
QUINOIDCOMVOUNOS
o
lo o
0
195
O -HO- ~ O O-OH
0
O-OH
(5)
O
The epoxidation ofp-benzo- and 1,4-naphthoquinones is accompanied by changes in their physicochemical properties in terms of electrophilicity, polarity, and reduction potential. The more hydrophilic quinone epoxide is a compound with a weaker electrophilic character than the parent quinone. Because quinone epoxides retain the redox properties of the quinones, they can be analyzed and identified by HPLC-EC. 14-16The Ell2 values for quinone epoxide reduction are about 300-400 mV more negative than those for the parent quinone compounds lacking an epoxide ring (Table II; Fig. 3). The reduction of a quinone epoxide is an irreversible event, which precludes any equilibrium with other redox couples and, consequently, the determination of the one-electron reduction potential by pulse radiolysis approaches. DT-diaphorase reduces various quinone epoxides at different rates.14,25 The two-electron reduction of quinone epoxides catalyzed by DT-diaphorase results in epoxide ring opening, yielding a 2-hydroxyhydroquinone as primary product. These hydroxyl derivatives show a higher rate of autoxidation than do the parent hydroquinones lacking a hydroxyl substituent, 14,25 similar to the autoxidation following the 1,4reductive addition of GSH to quinone epoxides.16,27
Concluding Remarks The development of sensitive and selective electrochemical detectors4 for use in liquid chromatography has created a new technology applicable to many problems of clinical, pharmacological, and environmental importance. In addition to high sensitivity, the electrochemical detector exhibits good selectivity; it responds only to electrochemically active substances with functional groups or to those compounds derivatized to electrochemically active species. The utility of HPLC combined with electrochemical detection for the investigation of quinone metabolite formation has been previously shown5 and extended to those derivatives resulting from the addition of oxygen or sulfur nucleophiles. 14-~7 This technique can also be used to detect small quantities of physiological quinones, such as vitamin E quinone ~3 as well as coenzyme Q20 and its
196
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
related changes in mitochondrial membranes on oxidative stress. 44 The constant improvement of amperometric detectors will allow a more efficient evaluation of the cellular redox metabolism of quinones. The overall process of electron transfer is a complex event influenced by the interaction of the inborn chemical properties of quinoid compounds with environmental factors, which contemplate homogeneous and heterogeneous systems. This applies also to the electrochemical reduction or oxidation of quinones and conveys the requirements for the optimal conditions for amperometric detection, that is, for the heterogenous electron transfer between an electrochemically active compound and the electrode surface. Although reduction potentials are a critical factor in the metabolism of quinones by one- or two-electron transfer flavoproteins, no statistical correlation was found between the reduction potential of the quinone and its rate of reduction by NADPH-cytochrome-P-450 reductase45 or DTdiaphorase.17 Moreover, autoxidation of unsubstituted- or methyl-substituted naphthoquinones, following their two-electron reduction by DTdiaphorase, was significantly different from those naphthoquinone derivatives bearing glutathionyl, hydroxyl (in the benzene ring), or methoxyl substituents. In the latter instances, autoxidation was 6-12 times higher than the unsubstituted 1,4-naphthoquinone, despite small changes in the reduction potential of the substituted naphthoquinones. Acknowledgments This research was supported by Grant 4481 from the Swedish Medical Research Council, Grant 2703-B89-01XA from the Swedish Cancer Foundation, and a grant from the Swedish Natural Science Research Council.
M. T. Smith, C. G. Evans, H. Thor, and S, Orrenius, in "Oxidative Stress" (H. Sies, ed.), p. 91. Academic Press, London, 1985. 45 G. Powis and P. L. Appel, Biochem. Pharmacol. 29, 2567 (1980).
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
centered radicals to coordinate with electron-rich centers. Specific electronic structures such as the 2o'/Icr* three-electron bonds provide a rationale for many physical and chemical characteristics of the radical species. Another important finding is the frequent involvement of sulfur-centered radicals in thermodynamic equilibria. This appears to be of particular significance for the role of thiyl radicals since it could explain, for example, some of the insufficiencies in the repair and protection mechanism of thiols. In fact, the equilibria open the possibility that thiyl radicals indirectly could even contribute to biological damage. Finally, it must be recognized that thiyl radicals can undergo many more reactions than just recombination to their respective disulfides. From product analysis alone this is often difficult to realize since most of the thiyl radical reactions ultimately lead also to either disulfides or thiols. It must clearly be recognized that thiyl radicals are able to undergo a large variety of reactions, and depending on the environmental conditions, that is, local concentrations of possible reaction partners, may thus become imbedded in complex reaction schemes.
[16] Q u i n o i d C o m p o u n d s : H i g h - P e r f o r m a n c e L i q u i d Chromatography with Electrochemical Detection
By ENRIQUE CAI~ENASand LAgS EgNSTER Introduction The carbonyl groups in quinones are found in the same or separate rings and are conjugated with the double bond. The particular arrangement of double bonds inside and outside the six-membered ring of quinones is largely responsible for their chemical properties. The chemistry of quinones is, in many aspects, similar to that of ot,/3-unsaturated ketones, and most of their redox properties are based on the electrophilic reactivity determined by the carbonyl groups and the reaction of the polarized double bonds with nucleophiles. The redox features of quinones are essential to the understanding of their overall biological activity, which encompasses functional, toxicological, mutagenic, and antitumor actions. Quinones can participate in redox transitions involving (1) pure electron transfer, such as in the enzymatic reduction of quinones or in the several nonenzymatic sources of semiMETHODS IN ENZYMOLOGY, VOL. 186
Copyright @ 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
[16]
QUINOID COMPOUNDS
181
quinones, and (2) nucleophilic addition reactions, ~implying either oxidation or reduction of the quinone ring. The former is exemplified by the oxidation of the - - C 2 ~ C 3 - - bond to quinone epoxides by 02 nucleophiles and the latter by the reductive addition via sulfur nucleophiles to yield thioether-hydroquinone adducts. Since hydroquinones and quinones can be oxidized and reduced electrochemically,2,3 it appears reasonable to attempt to detect and quantify them by high-performance liquid chromatography (HPLC) with electrochemical methods (HPLC-EC). The sensitivity afforded by electrochemical detection (picomole range) along with the separation methods of HPLC 4 seems ideal for detection and quantification of quinoid compounds in biological systems) HPLC with oxidative electrochemical methods has been successfully applied to the detection of aromatic alcohols and amines, indoles, phenothiazines, purines, as well as other substances, such as ascorbic acid and thiols. 6,7HO., a species often responsible for cellular damage, can also be identified by HPLC-EC by measuring the adduct resulting from the reaction of HO. with 5,5-dimethylpyrroline N-oxide (DMPO) 8 or the oxidation products of salicylate9,~°; in the latter instances, however, some reservations should be taken into account, for the high oxidation state of certain hemoproteins, e.g., ferrylmyoglobin, can yield a hydroxylation pattern of salicylate similar to that observed with hydroxyl radical. 9,11 K. T. Finley, in "The Chemistry of Quinonoid Compounds" (S. Patai, ed.), p. 877. Wiley, London, 1974. 2 j. Q. Chambers, in "The Chemistry of Quinonoid Compounds" (S. Patai, ed.), p. 737. Wiley, London, 1974. 3 G. Dryhurst, K. M. Kadish, F. Scheller, and R. Renneberg, in "Biological Electrochemistry" (G. Dryhurst et al., eds.), vol. 1, p. 1. Academic Press, New York, 1982. 4 p. T. Kissinger, K. Bratin, W. P. King, and J. R. Rice, ACS Symp. Ser. 136, 57 (1981). 5 D. S. Fluck, S. M. Rappaport, D. A. Eastmond, and M. T. Smith, Arch. Biochem. Biophys. 235, 351 (1984). 6 T. Kurahashi, H. Nishino, S. Parvez, H. Parvez, K. Kojima, and T. Nagatsu, in "Progress in HPLC, Volume 2: Electrochemical Detection in Medicine and Chemistry" (H. Parvez, M. Bastart-Malsot, S. Parvez, T. Nagatsu, and G. Carpentier, eds.), p. 3. VNU Science Press, Utrecht, 1987. 7 H. Parvez, M. Bastart-Malsot, S. Parvez, T. Nagatsu, and G. Carpentier (eds.), "Progress in HPLC, Volume 2: Electrochemical Detection in Medicine and Chemistry." VNU Science Press, Utrecht, 1987. s R. A. Floyd, C. A. Lewis, and P. K. Wong, this series, Vol. 105, p. 231. 9 R. A. Floyd, R. Henderson, J. J. Watson, and P. K. Wong, J. Free Radicals Biol. Med. 2, 13 (1986). 10D. Mira, U. Brunk, A. Boveris, and E. Cadenas, Free Radical Biol. Med. 5, 155 (1988). H D. Galaris, D. Mira, A. Sevanian, E. Cadenas, and P. Hochstein, Arch. Biochem. Biophys. 262, 221 (1988).
182
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
The principle and versatile applications of HPLC-EC have been authoritatively reviewed. 4,6,12 The scope of this chapter encompasses only the amperometric detection of quinones of the p-benzo- and 1,4-naphthoquinone series and the effect of substituents on the electrochemical properties of these compounds. Principle of HPLC with Electrochemical Detection The principle of HPLC-EC is based on the redox properties of the compounds of interest; hence, the chemical reaction, namely, electron transfer, as well as its dependence on environmental factors (e.g., composition and physicochemical properties of the mobile phase) are essential to the detection p r o c e s s . 4'6'12 HPLC-EC is an amperometric determination that measures the potential difference between the electrode (embedded in one wall of the electrochemical cell) and the bulk of the solution. This determines the reactivity of the electrochemically active compound at the electrode surface. The detector used in this work, a three-pole potentiostat system, was a TL-5A three-electrode glassy carbon flow cell (Bioanalytical Systems Inc., West Lafayette, IN). On the basis of the potential of a reference electrode (Ag/AgCI), constant potential electrolysis is given between the working electrode (glassy carbon) and the auxiliary electrode (stainless steel tube). There are two modes of electrochemical detection: electron transfer from the solute to the electrode surface is known as oxidative electrochemical detection, whereas electron transfer from the electrode surface to the solute is known as reductive electrochemical detection. The former requires a positive applied potential, and the latter requires a negative applied potential (Fig. I). The interfacial current measured at a fixed electrode potential is proportional to the concentration of electrochemically active material and is converted to voltage by means of an amplifier current-to-voltage converter (Model LC-4, Bioanalytical Systems). The gain of the converter (nA V -l) over the recorder sensitivity (V cm -1) sets the units of the recorder scale (nA cm-~). Figure 2 shows a chromatogram with reductive applied potential of a mixture of various 1,4-naphthoquinones and naphthoquinone epoxides. Other quinones of biological interest, such as vitamin E quinone, can also be measured by this technique. 13Table I ~-~7 12 p. T. Kissinger, K. Bratin, G. C. Davis, and L. A. Pachla, J. Chromatogr. Sci. 17, 137 (1979). 13 S. Ikenoya, K. Abe, T. Tsuda, Y. Yamano, O. Hiroshima, M. Ohmae, and K. Kawabe, Chem. Pharm. Bull. 27~ 1237 (1979). ~4A. Brunmark, E. Cadenas, J. Segura-Aguilar, C. Lind, and L. Ernster, Free Radical Biol. Med. 5, 133 (1988).
[16]
183
QUINOID COMPOUNDS Negative applied potential Oxidative Electrochemistry OH
0
OH
0
[ Electrode ]
Positive applied potential Reductive Electrochemistry
FIO. 1. Schematic representation of electrochemical oxidation and reduction during HPLC with amperometric detection of 1,4-naphthoquinone.
lists the retention times observed for several p-benzo- and 1,4-naphthoquinone derivatives with different mobile phases. The mobile phases were chosen to have a high buffer capacity and a high ionic strength by addition of sodium sulfate. Within the same mobile phase (e.g., phase A, Table I), methyl substitution increases the retention time of the quinone, whereas the quinones substituted with the more polar hydroxyl and methoxyl groups have a shorter retention time. The same applies to naphthoquinone epoxides, whose methyl-substituted derivatives are retained longer in the column. Since amperometric detection involves heterogenous electron transfer between an electrochemically active material (solute or analyte) and the electrode surface, the fundamental redox process occurring in the cell will obviously be affected by the composition and pH of the mobile phase as well as the applied potential at the electrode. The selection of the mobile phase is an important factor, allowing the electrode reaction to occur; although the mobile phase should be electrochemically inert at the electrode surface, the amperometric character of the determination requires the presence of electrolytes to convey charge through the electrochemical cell and a solvent with a sufficiently high dielectric constant to permit dissociation of the electrolyte. Therefore, amperometric detection is most satisfactory in mobile phases of high ionic strength (>50 mM) which contain a minimum amount of nonaqueous solvent. 4 The pH of the mobile phase is another important consideration, for the heterogenous electron transfer between the electrochemically active molecule and the electrode surface is affected by pH, as are those chemical reactions linked to the 15 A. Brunmark and E. Cadenas, Free Radical Biol. Med. 3, 169 (1987). t6 A. Brunmark and E. Cadenas, Free Radical Biol. Med. 6, 149 (1989). |7 G. Buffinton, K. Ollinger, A. Brunmark, and E. Cadenas, Biochem. J. 257, 561 (1989).
184
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
initial electron-transfer process. Stationary phases or columns for amperometric detection include all type of reversed phase materials, for they are compatible with polar mobile phases containing dissolved electrolytes. 4 60(1
IV
nA V
II
3(Yd III VI
OI
[
I
I
0
10
20
30
Retention time (min) FIG. 2. Analysis of quinones by HPLC with electrochemical detection. Chromatographic conditions: Stationary phase: 15 cm × 3.9 mm Novapak 4/~M Cls reversed phase (WatersMillipore, AB, V. Fr61unda, Sweden). Mobile phase: 35% 2-propanol-65% H20 buffered to pH 6.5 with 50 mM sodium phosphate. Mobile phase flow rate: 0.5 ml min-I. Detector: LC-4 with a TL-5A three-electrode glassy carbon flow cell and a Ag/AgCI reference electrode (Bioanalytical Systems). Applied potential: -0.9 V versus Ag/AgC1. Amount injected: 2 nM of each compound. Peaks: I, 2,3-epoxy-l,4-naphthoquinone; II, 1,4-naphthoquinone; III, 2-methyl-2,3-epoxy-l,4-naphthoquinone; IV, 2-methyl-l,4-naphthoquinone; V, 2,3-dimethyl-2,3-epoxy-l,4-naphthoquinone; VI, 2,3-dimethyl-l,4-naphthoquinone.
[16]
QUINOID COMPOUNDS
185
TABLE I HPLC WITH ELECTROCHEMICALDETECTION ANALYSISOF 1,4-NAPHTHOQUINONE DERIVATIVES O
R2 R3
Retention time (min); mobile phase a
O
R1b
R2
R3
A
B
H CH3 CH3 OCH3 OH CH3 H CH3 H SG NAC H OH
H H CH3 OCHa H OH H H SG SG H SG SG SG SG NAC O
H H H H H H OH OH H H H OH H H OH H
6.8 12.4 27.4 4.7 2.1
4.6
H H H
4.9 7.8 13.8
CH3 CH3 CH3
C
D
2.3 6.3 36.0 10.1
16.0 5.4 16.5 16.0
1.8 38.0 46.0 26.0
4.1
0 R2 R3 H CH3 CH3
0 H H CH3
17.8 13.3
a (A) 35% 2-propanol-65% H20, buffered to pH 6.5 with 50 mM sodium phosphate. 5,14,t5 (B) Five milliliters phosphoric acid diluted to 1 liter with H20 and adjusted to pH 7 with Tris base; 10% of this buffer, 25% methanol, and 65% H20 containing 50 mM sodium sulfate was used as mobile phase. ~6 (C) 35% Methanol-65% H20-0.2 mM Tris buffer, 50 m M sodium sulfate, pH 7.0? 7 (D) As mobile phase B, but methanol content increased to 37%. 16 b CH3, Methyl; OCH3, methoxyl; OH, hydroxyl; SG, glutathionyl; NAC, N-acetylcysteinyl.
186
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
The primary problem in reductive electrochemical detection is the removal of dissolved 02 in the mobile phase, because of the interference caused by 02 on reduction to peroxide and, eventually, to water. This problem can be circumvented partly by purging the mobile phase and sample with helium or argon and by using an all-steel liquid chromatograph construction. However, even under these conditions, it is difficult to keep a driftless baseline in a reducing reaction, not only because of the interference of dissolved 02, but also because of the presence of traces of metal ions leached by the eluent from the stainless steel parts of the chromatograph. Alternative ways to avoid this problem are the redox mode TM and postcolumn mobile phase changes. 12 The redox mode has been successfully applied to the determination of vitamin KI compounds and consists of two sequential generator/detector electrodes arranged in a way such that one is directly upstream of the other. The principle involves the initial reduction of vitamin KI to vitamin K~ hydroquinone (Q + 2 e+ 2 H ÷ --> QH2) by applying a sufficiently negative potential at the generator electrode; the subsequent electrochemical oxidation of vitamin K~ hydroquinone (QH2 ---> Q + 2 e- + 2 H ÷) takes place at the detector electrode, which is set at a positive potential. ~8 By this method, pure standards of vitamin K~ can be detected down to 100 pg with the further advantage that it does not require the removal of 02 from the system, because while the negative potential at the generator electrode is sufficient to reduce vitamin K1 and 02, the positive potential at the detector electrode can oxidize K~ hydroquinone but not peroxide or water) 3 On the other hand, postcolumn mobile phase changes ~2 obtained by using postcolumn mixing to alter the mobile phase pH (1 M HC104 mixed with the mobile phase prior to detection) allow an optimal determination of vitamin K3 at lower detector potential and without interference from dissolved 02. Multielectrode electrochemical detection 19 offers greater versatility than a three-pole detector, because it helps to overcome the shortcomings inherent in conventional electrochemical detection by allowing the simultaneous determination of oxidized and reduced species. This can be obtained with a detector with three working electrodes, two of which can be set to desired potentials. By this means, reduced and oxidized coenzyme Q was detected simultaneously by setting the potentials of the first and second electrodes at +0.7 and - 0 . 3 V, respectively. 9,2° 18y. Haroon, C. A. W. Schubert, and P. V. Hauschka, J. Chromatogr. Sci. 22, 89 (1984). ~90. Hiroshima, S. Ikenoya, T. Naitoh, K. Kusube, M. Ohmae, K. Kawabe, S. Ishikawa, H. Hoshida, and T. Kurahashi, Chem. Pharm. Bull. 31, 3571 (1983). 2o M. Takada, S. Ikenoya, T. Yuzuriha, and K. Katayama, this series, Vol. 105, p. 147. (1984).
[16]
QUlNOID COMPOUNDS
187
The use of amperometric detection combined with HPLC is not restricted to compounds with redox properties. Electrochemically inactive compounds can be derivatized to electrochemically active compounds to be analyzed by HPLC-EC. In this regard, nitrophenyl, aniline, quinoneimine, and phenolate species are ideal candidates for sensitive amperometric methods. Effect of Substituents on Half-Wave Reduction Potentials of Quinones Determined by HPLC with Electrochemical Detection There is a substantial body of data on half-wave potentials (E~/2) of quinones originating from electrochemical measurements corresponding to the addition of first and second electrons in nonaqueous solvents. 2,3 These measurements are usually devoid of the complications introduced by protonation steps. Values obtained with a standard calomel electrode can be converted to standard hydrogen electrode by adding 241 mV and those obtained with a Ag/AgC1 electrode by adding 221 mV. These values cannot be compared directly with those observed in aqueous solvents, as obtained with fast techniques as pulse radiolysis, because of differences in solvation energies and occurrence of junction potentials. 2~ The electrochemical reduction of quinones in aqueous or protic media exhibits a complex behavior, which has the appearance of reversible couples3; a cyclic voltammetric study of the aqueous electrochemistry of some quinones 22 indicates a strong pH dependence of the redox process. The half-wave reduction potential (E1/2) values of the 1,4-naphthoquinone derivatives in aqueous solutions examined here have been determined by means of hydrodynamic voltamograms against a Ag/AgCI reference electrode. The peak heights are plotted as a function of applied potential for each compound, resulting in the usual sigmoidal voltammetric wave. Although these El~2 values cannot be equated directly with those derived from pulse radiolysis approaches in aqueous media or cyclic voltammetry in protic or aprotic media, they provide a general indication of the redox properties of quinones within the same system, which permits one to predict the likelihood of redox interactions between different molecules. The effects exerted by different substituents on the E~/2 values of pbenzo- and 1,4-naphthoquinones are tabulated in Table II. Figure 3 illustrates the hydrodynamic voltamograms (used to calculate E~/2 values) of 21 A. J. Swallow, in "Function of Quinones in Energy Conserving Systems" (B. L. Trumpower, ed.), p. 59. Academic Press, London, 1982. 22 S. I. Bailey and I. M. Ritchie, Electrochim. Acta 30, 3 (1985).
188
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
TABLE II HALF-WAvEPOTENTIALS(El/2) OF p-BENZO- AND 1,4-NAPHTHOQUINONEDERIVATIVESa Quinone p-Benzoquinone series Unsubstituted p-Benzoquinone Hydroxyl-substituted 2-Hydroxy-p-benzoquinone Methyl-substituted 2-Methyl-p-benzoquinone 2,6-Dimethyl-p-benzoquinone p-Benzoquinone epoxides 2,3-Epoxy- 1,4-p-benzoquinone 2,3-Epoxy-2-methyl-p-benzoquinone 2,3-Epoxy-2,6-dimethyl-p-benzoquinone 1,4-Naphthoquinone series Unsubstituted
1,4-Naphthoquinone Methyl-substituted 2-Methyl- 1,4-naphthoquinone 2,3-Dimethyl-l,4-naphthoquinone Methoxyl-substituted 2,3-Dimethoxyl-l,4-naphthoquinone Hydroxyl-substituted (quinone ring) 2-Hydroxy-1,4-naphthoquinone 2-Methyl-3-hydroxy- 1,4-naphthoquinone Hydroxyl-substituted (benzene ring) 5-Hydroxy-1,4-naphthoquinone 2-Methyl-5-hydroxy- 1,4-naphthoquinone Glutathionyl-substituted 3-Glutathionyl- 1,4-naphthoquinone 2,3-Diglutathionyl- 1,4-naphthoquinone 2-N-Acetylcysteinyl- 1,4-naphthoquinone 2-Methyl-3-glutathionyl- 1,4-naphthoquinone 2-Methyl-3-N-acetylcysteinyl- 1,4-naphthoquinone Glutathionyl- and hydroxyl-substituted 3-Glutathionyl-5-hydroxy- 1,4-naphthoquinone 2-Methyl-3-glutathionyl-5-hydroxy- 1,4-naphthoquinone 2-Hydroxy-3-glutathionyl- 1,4-naphthoquinone 1,4-Naphthoquinoneepoxides 2,3-Epoxy-1,4-naphthoquinone 2-Methyl-2,3-epoxy-l,4-naphthoquinone 2,3-Dimethyl-2,3-epoxy- 1,4-naphthoquinone Data from Refs. 14-17.
EI~ (-mY) versus Ag/ AgC1
30 440 50 120 510 510 580 180 225 300 300 460 500 140 200 225 265 255 265-300 310 195 220 680 720 770-790 820
[16]
QUINOIDCOMPOUNDS
189
I 0"051
0i
I 0.2
I 0.4
I i lt.O 0.6 08 Oxidation Potential(-volts) FIG. 3. Hydrodynamic voltamograms showing the effect of substituents on half-wave potentials of 1,4-naphthoquinones. Chromatographic conditions: Stationary phase: 15 cm x 3.9 mm Novapak 4/~M Ct8 reversed phase (Waters-Millipore). Mobile phase: 35% 2-propanol-65% H20 buffered to pH 6.5 with 50 mM sodium phosphate. Mobile phase flow rate: 0.5 ml min-t. Detector: LC-4 with a TL-5A three-electrode glassy carbon flow cell and a Ag/ AgC1 reference electrode (Bioanalytical Systems). Other assay conditions in Refs. 14-17. Hydrodynamic voltamograms: II, 1,4-naphthoquinone (Ela = -180 mV); O, glutathionyl1,4-naphthoquinone (El/2 ~- -220 mV); El, 2-hydroxy-1,4-naphthoquinone (Ev2= -460 mV); O, 2,3-epoxy-l,4-naphthoquinone (Ev2 = -720 mV).
unsubstituted 1,4-naphthoquinone and derivatives bearing glutathionyl and hydroxyl substituents as well as 1,4-naphthoquinone epoxide. Most quinones can be reduced at working electrode potentials of -300 to -400 mV) The El~2 values for the unsubstituted parent compounds of the pbenzo- and 1,4-naphthoquinone series are - 3 0 and -180 mV, respectively. In general, substitution produces discrete changes in the E1/2 values (Table II) with exception of hydroxyl substituents in the quinone ring and quinone epoxides, compounds with Ei/2 values 300-400 mV more negative than the parent unsubstituted quinones. The introduction of methyl groups in p-benzoquinone or the quinone ring of 1,4-naphthoquinone decreases the Ei/2 values in an additive manner. 23 Mono- and dimethyl substitution of 1,4-naphthoquinone decreases the Ev2 by about 45 and 120 mV, respectively (Table II). 14'17 23p. Zuman, "Substituent Effects in Organic Polarography." Plenum, New York, 1967.
190
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
Hydroxyl Substitutents Hydroxyl-substituted p-benzoquinones can originate from different sources including monooxygenase-mediatedmetabolism of benzene,24 enzymatic reduction ofp-benzoquionone e p o x i d e s , 14,25,26 and sulfur nucleophilic addition to quinone epoxides. 16,27The hydroxyl substituent considerably lowers the Ev2 values of p-benzoquinones in aqueous solutions, 14,22,28 an effect explained on the basis of mesomeric or inductive effects introduced by the hydroxyl group (similar considerations apply to methoxyl substituents). 28 Reduced hydroxy-p-benzoquinones are autoxidized at rates substantially higher than the parent compounds.14,25 The effect of hydroxyl substituents on the E1/2 values of 1,4-naphthoquinones varies depending on whether the hydroxyl group is situated in the quinone ring (C-2) or in the adjacent benzene ring (C-5). In the former instance, hydroxyl substitution exerts an effect similar to that observed with p-benzoquinones, that is, a marked decrease in the EVE value; in addition, the hydroxyl substituent in the quinone ring raises the pKa value with regard to the parent quinone. 29On hydroxyl substitution at C-2, both 1,4-naphthoquinone and 2-methyl-l,4-naphthoquinone decrease their EVE by 280 and 275 mV, respectively.14.17 A similar decrease of 1,4-naphthoquinone was observed in the half-wave reduction potential (calculated against a standard calomel electrode) (AE1/2 = -280 mV)3° and in the oneelectron reduction potential [E(Q/Q-:)7] (calculated by pulse radiolysis) (AE1/2 = -275 mV) (data from Ian Wilson quoted in Ref. 31). Introduction of methoxyl groups causes a similar effect as the hydroxyl substituents vicinal to the carbonyl groups: the E1/2 value of 2,3dimethoxyl-1,4-naphthoquinone is 120 mV more negative than that of the parent compound. A 119 mV more negative E~/2 value (against standard calomel electrode) was found with the monomethoxy derivative of 1,4naphthoquinone in aqueous solutions) ° Thus, methoxyl substitution seems not to decrease the reduction potential in an additive manner; similar considerations may explain the not very negative one-electron 24 W. F. Greenlee, J. D. Sun, and J. S. Bus, Toxicol. Appl. Pharmacol. 59, 187 (1981). z5 A. Brunmark, E. Cadenas, C. Lind, J. Segura-Aguilar, and L. Ernster, Free Radical Biol. Med. 3, 181 (1987). 26 E. Cadenas, D. Mira, A. Brunmark, J. Segura-Aguilar, C. Lind, and L. Ernster, Free Radical Biol. Med. 5, 71 (1988). 27 A. Brunmark and E. Cadenas, Chem.-Biol. Interact. 68, 273 0 9 8 8 ) . W. Flaig, H. Beutelspacher, H. Riemer, and E. K~ilke, LiebigsAnn. Chem. 719, 96 (1968). 29 T. Mukherjee, Radiat. Phys. Chem. 29, 455 (1987). 3o E. M. Hodnett, C. Wongewiechintana, W. J. Dunn, and P. Marrs, J. Med. Chem. 26, 570 (1983). 31 M. d'Arcy Doherty, A. Rodgers, and G. M. Cohen, J. Appl. Toxicol. 7, 123 (1987).
[16]
QUINOID COMPOUNDS
191
reduction potential of 2,3-dimethoxyl-l,4-naphthoquinone [E(Q/Q-,) = 183 mV].32 When the hydroxyl substituent is in the adjacent benzene ring, it causes an increase in the E1/2 value, an effect understood as a polar effect caused by the hydroxyl group in the adjacent benzene ring. At variance with 2-hydroxy-l,4-naphthoquinone, a hydroxyl substituent in the benzene ring lowers the pKa with respect to the parent compound. 29 Another property of hydroxyquinones is their tendency to undergo strong intramolecular hydrogen bonding, which leads to stabilization of the semiquinone transient species involving displacement toward the left of the disproport i o n a t i o n r e a c t i o n 3 3 , 3 4 : 2 Q ~ ~-- Q + Q 2 - . On hydroxyl substitution at the aromatic ring, the E~/2values versus Ag/AgC1 of 1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone increase by 40 and 25 mV, respectively (Table II). 17This is in agreement with previous reports on the first and second electron reduction potential (calculated by cyclic voltammetry) of 5-hydroxy-l,4-naphthoquinone in apolar media33 and in aqueous media. 29 -
Glutathionyl Substituents Reduced glutathione (GSH) can be measured in tissue extracts by HPLC-EC with a method based on the separation of GSH from other components by cation-exchange chromatography coupled to the electrochemical oxidation of the thiol to the corresponding disulfide. 35 The oxidation of GSH is accomplished with a graphite paste electrochemical detector with an applied potential of 1.0 V versus Ag/AgC1. A simultaneous detection of thiols and disulfides can be obtained with a dual Hg/Au electrode thin-layer cell to perform both the reduction and detection functions. The electrodes are arranged in series with reduction of oxidized glutathione (GSSG) to GSH at the generator electrode and oxidation at the detector electrode. 36 Glutathionyl substitution is the result of the 1,4-reductive addition of the thiol across the double bond of the quinone. Since glutathionylquinone conjugates retain the redox properties inherent to the quinone moiety, they can be separated and detected by HPLC-EC. The glutathionyl substituent affects both the retention time and the Ell2 value of the quinone. ~5-17 Because of the relatively weak electron-withdrawing 35 T. W. Gant and G. M. Cohen, personal communication. 33 A. Ashnagar, J. M. Bruce, P. L. Dutton, and R. C. Prince, Biochim. Biophys. Acta 801, 351 (1984). 34 N. F. J. Dodd and T. Mukherjee, Biochem. Pharmacol. 33, 379 (1984). 35 I. Mefford and R. N. Adams, Life Sci. 23, 1167 (1978). 36 L. A. Allison, J. Keddington, and R. E. Shoup, J. Liq. Chrornatogr. 6, 1785 (1983).
192
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
properties of thioether substituents, only minute changes in the quinone reduction potential are expected on glutathionyl substitution. The EI/2 values of thioether adducts ofp-benzoquinone are slightly more negative than the parent compounds. 37 Despite the minor changes in the Ev2 on glutathionyl substitution, significant alterations in the oxidation equilibrium, involving both cross-oxidation and autoxidation reactions, are observed. 27 Electrochemical oxidation of glutathionyl-p-benzohydroquinone shows E1/2 values (versus a Ag/AgC1 electrode) of +220 mV, 20 mV more positive than those of the unsubstituted compound.15 Unsubstituted 1,4-naphthoquinone and derivatives bearing a methyl and/or hydroxyl (in the benzene ring) substituent undergo 1,4-reductive addition with thiols as GSH [reaction (1)]. Glutathionyl substitution of 1,4-naphthoquinone decreases additively the Ev2 value from -180 to -225 mV for the monoconjugate and to -265 mV for the diconjugate (Table II). The glutathionyl substitution of 2-methyl-l,4-naphthoquinone decreased the Ell2 value by 40 mV. The nucleophilic addition of N-acetylL-cysteine to 1,4-naphthoquinone and 2-methyl-l,4-naphthoquinone results in a decrease of the E1/2 value more pronounced (75 and 85 mV, respectively) than that observed with GSH.17 The decrease in E1/2 value caused by the thiol addition may be interpreted as a mesomeric effect similar to that produced by hydroxyl and methoxyl substituents. Minor changes in the one-electron reduction potential of 2-methyl-l,4-naphthoquinone [E(Q/Q:)7 = -203 mV] are observed on GSH addition [E(GS--Q/GS--Q :)7 = -192 mV]. 38 In spite of the small variations in reduction potential, the autoxidation of glutathionylnaphthohydroquinone derivatives, following the reduction of the oxidized counterpart by DT-diaphorase, is 12- to 16-fold higher than that of the parent naphthohydroquinones.17 O
OH RI
+ GS-+
H+
,.
~
RI
(1)
SG O
R 1 = H, CH3
OH
Hydroxyl and Glutathionyl Substituents Hydroxyl- and glutathionyl-disubstituted quinones can result from nucleophilic addition of GSH to (1) aromatic-, hydroxyl-substituted naphthoquinones, (2) hydroxy-p-benzoquinones, and (3) p-benzo- or 1,437 E. R. Brown, K. T. Finley, and R. L. Reeves, J. Org. Chem. 36, 2849 (1971). 38 I. Wilson, P. Wardman, T.-S. Lin, and A. C. Sartorelli, J. Med. Chem. 29, 1381 (1986).
[16]
193
QUINOID COMPOUNDS
naphthoquinone epoxides. The primary molecular products resulting from these reductive additions differ in all three instances. In the first case, the product bears a hydroxyl substituent in the benzene ring and a glutathionyl substituent in the quinone ring. In the two latter cases, the hydroxyl and glutathionyl substituents are found in the quinone ring. The occurrence of the new sulfur substituent in these hydroxyquinones controls to a large extent the subsequent redox chemistry in terms of crossoxidation, autoxidation, and disproportionation reactions. The nucleophilic addition of GSH to the 5-hydroxyl derivatives of 1,4naphthoquinone and 2-methyl-l,4-naphthoquinone [reaction (2)] results in compounds with E~/2 values close to those of 1,4-naphthoquinone and 2methyl-l,4-naphthoquinone; the difference amounts to -15 and +5 mV, respectively. This may be understood as the counterbalance of the rise in E~/2 value caused by the hydroxyl substituent at C-5 and the decrease in E~/2 value caused by the glutathionyl substituent. 0
OH + GS- + H+
(2)
~ ~ S O
OH O
OH OH
The nucleophilic addition of GSH to either 2-hydroxy-p-benzoquinone or 2,3-epoxy-p-benzoquinone yields the same primary molecular product, 2-hydroxy-5-glutathionyl-p-benzoquinone[reaction (3)], a compound that autoxidizes at rates substantially higher than the parent compound lacking a hydroxyl substituent. 27The rate of nucleophilic addition is considerably slowed down on methyl substitution. 16p-Benzohydroquinones with both hydroxyl and glutathionyl substituents autoxidize at rates 350-fold higher than the unsubstituted p-benzohydroquinone.27 It should be noted that the individual effects of glutathionyl and hydroxyl substituents are not additive when present in the same quinoid molecule, but they seem to potentiate the overall autoxidation. O
OH
O --
O
GS O
OH
(3)
O
The electron-donating properties of the hydroxyl substituent in the quinone ring increases the electron density at C-3, thereby preventing the nucleophilic addition of GSH to 2-hydroxy-l,4-naphthoquinone. However, a hydroxyglutathionyl adduct of 1,4-naphthoquinone, namely,
194
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
2-hydroxy-3-glutathionyl-l,4-naphthoquinone, was obtained during the cleavage of the epoxide ring of 2,3-epoxy-l,4-naphthoquinone on GSH nucleophilic addition 25 [reaction (4)]. A fraction of the product of reaction (4) undergoes oxidative elimination to yield glutathionylnaphthoquinone. 16
~
O
OH O + GS- + H+
~ ~[~OH
(4) SG
O
OH
Quinone Epoxides Vitamin K1 2,3-epoxide originates from the coupled activity of a twoenzyme system. Vitamin Kl epoxide is not active in prothrombin synthesis, but it is readily converted back to vitamin K by vitamin K epoxide reductase. 39 Chemical models for the molecular mechanism of vitamin K epoxide reductase emphasize the importance of a primary thiol addition to open the epoxide ring; reaction with a second thiolate results in reductive cleavage of the intermediate, followed by elimination of HzO to yield the quinone. 4°,41 Although simple quinone epoxides, such as 2,3-epoxy-p-benzoquinone and 2,3-epoxy-1,4-naphthoquinone and their methyl derivatives, are not of direct biological significance, they provide suitable experimental models to understand the electrochemical properties of biologically important quinone epoxides. The formation of quinone epoxides is attained in the addition of Oz nucleophiles to p-benzoquinones and 1,4-naphthoquinones, ~ as implied in the HzO2-dependent oxidation of the 2,3-double bond, 14,15 which is analogous to the reaction of sodium hydroperoxides with naphthoquinones. 42 The reaction is suggested to occur through a nucleophilic addition of HOO- at the/3 carbon of the unsaturated system to give an enolate ion as an intermediate. The carbanionic t~ carbon atom on the enolate ion displaces HO-, breaking the O - - O bond in the hydroperoxyl group and rearranging to an epoxide [reaction (5)]. Quinone epoxides also result from the reaction of O2 ~ with several vitamin K analogs.43 39 j. W. Suttie, CRC Crit. Rev. Biochem. 8, 191 (1980). 40 R. B. Silverman, J. Am. Chem. Soc. 103, 5939 (1981). 41 p. C. Preusch and J. W. Suttie, J. Org. Chem. 48, 3301 (1983). 42 L. F. Fieser, W. P. Campbell, E. M. Fry, and M. D. Gates, J. Am. Chem. Soc. 61, 3216 (1939). 43 I. Saito, T. Otsuki, and T. Matsura, Tetrahedron Lett. 19, 1693 (1979).
[16]
QUINOIDCOMVOUNOS
o
lo o
0
195
O -HO- ~ O O-OH
0
O-OH
(5)
O
The epoxidation ofp-benzo- and 1,4-naphthoquinones is accompanied by changes in their physicochemical properties in terms of electrophilicity, polarity, and reduction potential. The more hydrophilic quinone epoxide is a compound with a weaker electrophilic character than the parent quinone. Because quinone epoxides retain the redox properties of the quinones, they can be analyzed and identified by HPLC-EC. 14-16The Ell2 values for quinone epoxide reduction are about 300-400 mV more negative than those for the parent quinone compounds lacking an epoxide ring (Table II; Fig. 3). The reduction of a quinone epoxide is an irreversible event, which precludes any equilibrium with other redox couples and, consequently, the determination of the one-electron reduction potential by pulse radiolysis approaches. DT-diaphorase reduces various quinone epoxides at different rates.14,25 The two-electron reduction of quinone epoxides catalyzed by DT-diaphorase results in epoxide ring opening, yielding a 2-hydroxyhydroquinone as primary product. These hydroxyl derivatives show a higher rate of autoxidation than do the parent hydroquinones lacking a hydroxyl substituent, 14,25 similar to the autoxidation following the 1,4reductive addition of GSH to quinone epoxides.16,27
Concluding Remarks The development of sensitive and selective electrochemical detectors4 for use in liquid chromatography has created a new technology applicable to many problems of clinical, pharmacological, and environmental importance. In addition to high sensitivity, the electrochemical detector exhibits good selectivity; it responds only to electrochemically active substances with functional groups or to those compounds derivatized to electrochemically active species. The utility of HPLC combined with electrochemical detection for the investigation of quinone metabolite formation has been previously shown5 and extended to those derivatives resulting from the addition of oxygen or sulfur nucleophiles. 14-~7 This technique can also be used to detect small quantities of physiological quinones, such as vitamin E quinone ~3 as well as coenzyme Q20 and its
196
PRODUCTION, DETECTION, AND CHARACTERIZATION
[16]
related changes in mitochondrial membranes on oxidative stress. 44 The constant improvement of amperometric detectors will allow a more efficient evaluation of the cellular redox metabolism of quinones. The overall process of electron transfer is a complex event influenced by the interaction of the inborn chemical properties of quinoid compounds with environmental factors, which contemplate homogeneous and heterogeneous systems. This applies also to the electrochemical reduction or oxidation of quinones and conveys the requirements for the optimal conditions for amperometric detection, that is, for the heterogenous electron transfer between an electrochemically active compound and the electrode surface. Although reduction potentials are a critical factor in the metabolism of quinones by one- or two-electron transfer flavoproteins, no statistical correlation was found between the reduction potential of the quinone and its rate of reduction by NADPH-cytochrome-P-450 reductase45 or DTdiaphorase.17 Moreover, autoxidation of unsubstituted- or methyl-substituted naphthoquinones, following their two-electron reduction by DTdiaphorase, was significantly different from those naphthoquinone derivatives bearing glutathionyl, hydroxyl (in the benzene ring), or methoxyl substituents. In the latter instances, autoxidation was 6-12 times higher than the unsubstituted 1,4-naphthoquinone, despite small changes in the reduction potential of the substituted naphthoquinones. Acknowledgments This research was supported by Grant 4481 from the Swedish Medical Research Council, Grant 2703-B89-01XA from the Swedish Cancer Foundation, and a grant from the Swedish Natural Science Research Council.
M. T. Smith, C. G. Evans, H. Thor, and S, Orrenius, in "Oxidative Stress" (H. Sies, ed.), p. 91. Academic Press, London, 1985. 45 G. Powis and P. L. Appel, Biochem. Pharmacol. 29, 2567 (1980).
