Electrochemical Oxidation of Ochratoxin A - American Chemical Society

Ochratoxin A (OTA, 1A) is a mycotoxin implicated in human kidney carcinogenesis, in which oxidative activation is believed to play a key role. To gain...
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Chem. Res. Toxicol. 2001, 14, 1266-1272

Electrochemical Oxidation of Ochratoxin A: Correlation with 4-Chlorophenol M. Wade Calcutt, Ivan G. Gillman, Ronald E. Noftle,* and Richard A. Manderville* Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109-7486 Received April 24, 2001

Ochratoxin A (OTA, 1A) is a mycotoxin implicated in human kidney carcinogenesis, in which oxidative activation is believed to play a key role. To gain an understanding of the oxidative behavior of the toxin, we have carried out an electrochemical study and have observed a direct correlation between the electrochemistry of OTA and 4-chlorophenol (4-ClPhOH). Cyclic voltammetry (CV) of OTA in acetonitrile (MeCN) showed that the toxin exhibits an irreversible oxidative half-peak potential (Ep/2) of 1.81 V vs saturated calomel electrode (SCE); the corresponding value for 4-ClPhOH is 1.59 V. For both compounds, subsequent scans revealed the appearance of the respective hydroquinone/benzoquinone couple, which formed from the oxidation of the parent para-chlorophenol moiety. The hydroquinone of OTA (OTHQ, 2) exhibited Ep/2 ) 1.21 V in MeCN. Deprotonation of OTA to form the phenolate (OTA-) lowered the potential to Ep/2 ) 1.0 V in MeCN. However, from the oxidation of OTA-, no evidence for the OTHQ(2)/OTQ(3) redox couple was found. In aqueous phosphate buffer (pH 6-8), the electrochemical behavior of OTA mimicked that observed for OTA- in MeCN; Ep/2 was ∼0.8 V vs SCE and subsequent scans did not generate the OTHQ/OTQ redox couple. From capillary electrophoresis (CE), a diffusion coefficient (D) of 0.48 × 10-5 cm2 s-1 was determined for OTA in phosphate buffer, pH 7.0. Combining this value with electrochemical data suggested that OTA undergoes a 1H+/1e oxidation in aqueous media. The biological implications of these findings with respect to the oxidative metabolism of OTA, and other chlorinated phenols, are discussed.

Introduction Ochratoxin A (OTA,1 1A, Figure 1) is a mycotoxin produced by several species of Penicillium and Aspergillus fungi and is found primarily in cereal and grain products (1, 2). OTA is nephrotoxic, hepatotoxic, and a potent carcinogen in rodents (1-4). Human health risks stemming from OTA exposure have historically been localized in the Balkans, where OTA has been associated with Balkan Endemic Nephropathy (BEN), a widespread * To whom correspondence should be addressed. (R.E.N.) Phone: (336) 758-5520. Fax: (336) 758-4656. E-mail: [email protected]. (R.A.M.) Phone: (336) 758-5513. Fax: (336) 758-4656. E-mail: manderra@ wfu.edu. 1 Abbreviations: OTA, ochratoxin A (N-{[(3R)-5-chloro-8-hydroxy3-methyl-1-oxo-7-isochromanyl]carbonyl}-3-phenyl-L-alanine); BEN, Balkan Endemic Nephropathy; PGHS, prostaglandin-H-synthetase; SOD, superoxide dismutase; FeTPPS, iron(III) meso-tetrakis(4-sulfonato-phenyl)porphyrin; OTHQ, ochratoxin hydroquinone (N-{[(3R)5,8-dihydroxy-3-methyl-1-oxo-7-isochromanyl]carbonyl}-3-phenyl-Lalanine); OTQ, ochratoxin quinone (N-{[(3R)-3-methyl 1,5,8-trioxo-7isochromanyl]carbonyl}-3-phenyl-L-alanine); TCP, 2,4,6-trichlorophenol; PCP, pentachlorophenol; MeCN, acetonitrile; 4-ClPhOH, 4-chlorophenol; BHQ, benzohydroquinone; TBAP, tetrabutylammonium perchlorate; CV, cyclic voltammetry; SCE, saturated calomel electrode; RDV, rotating disk voltammetry; CE, capillary electrophoresis; Ep/2, half peak potential; ν, electrochemical potential scan rate; iAp , anodic peak current; it, mass-transported current as a function of time; Ep, peak potential; BQ, benzoquinone; OTA-, deprotonated OTA (phenolate form); 4-ClPhO-, deprotonated 4-chlorophenol (phenolate form); iAL , limiting (steady state) anodic current; ω, rotating disk electrode rotation rate (rad/s); n, number of electrons; D, diffusion coefficient; σ2, peak variance; 4-(R)-OH-OTA, 4-(R)-hydroxy-OTA; 4-(S)-OH-OTA, 4-(S)-hydroxy-OTA; CYP450, cytochrome P-450; HRP, horseradish peroxidase.

Figure 1. Structure and nomenclature of Ochratoxin A (OTA, 1A) and its analogues.

inflammatory and degenerative kidney disease in which patients suffer from urinary tract tumors (5-7). The

10.1021/tx015516q CCC: $20.00 © 2001 American Chemical Society Published on Web 08/16/2001

Electrochemical Oxidation of OTA

Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1267 Scheme 1. Chemical Oxidation of OTA

mutagenicity of OTA was confirmed recently using kidney microsomal fractions as metabolic activators in the Salmonella typhimurium reverse mutation test (8). While the mechanism of OTA carcinogenicity has not been elucidated, the toxin is known to facilitate singlestrand DNA cleavage (9, 10) and DNA adduction in vivo (11-13). Here an oxidative process is believed to play a key role, as OTA-induced DNA adducts have been detected after incubation with kidney microsomes, which contain prostaglandin-H-synthetase (PGHS) and lipoxygenase enzymes (14-16) that participate in the oxidation of certain xenobiotics (17). Superoxide dismutase (SOD) and catalase have also been shown to reduce the genotoxicity of OTA (14). Moreover, antioxidants greatly decrease OTA-mediated DNA adduct formation (18). Using 32P-postlabeling, Obrecht-Pflumio et al. demonstrated that incubation of OTA with kidney microsomes in vitro resulted in the formation of guanine-specific DNA adducts (19). However, from more recent studies involving [3H]OTA, it is not clear whether OTA is directly involved in adduct formation (20). In our studies on the in vitro activation of OTA, we have used chemical (21) and photochemical (22) means to study OTA-induced DNA damage. Using iron(III) meso-tetrakis(4-sulfonato-phenyl)porphyrin (FeTPPS) and hydrogen peroxide to mimic enzymatic activation of the toxin (21), the hydroquinone species (OTHQ, 2, Figure 1) was generated in the presence of sodium ascorbate. This suggested that the oxidation of OTA by FeTPPS/ H2O2 produced the quinone derivative (OTQ, 3, Figure 1), which was subsequently reduced to OTHQ by ascorbate (Scheme 1). The photoactivation of OTA in the presence of ascorbate also resulted in the formation of OTHQ (21). On this basis, we proposed that the genotoxicity of OTA is inherently dependent upon its ability to form a quinone species, which is very common for other chlorinated phenolic toxins, such as 2,4,6-trichlorophenol (TCP) (23) and pentachlorophenol (PCP) (24). This hypothesis was fully supported earlier by in vitro structureactivity relationships, which stressed the importance of the phenolic chlorine atom (25, 26). To further address the oxidative nature of OTA, we have utilized electrochemical methods, which can provide quantitative information on the redox processes. We now present studies on the electrochemistry of OTA in acetonitrile (MeCN) and aqueous buffer,2 where a direct correlation with the oxidative behavior of 4-chlorophenol (4-ClPhOH) has been established. Cyclic voltammetric, chronoamperometric, and UV-vis spectroelectrochemical studies are presented, and the implications of our find2 Presented in part at the 52nd Southeast/56th Southwest Joint Meeting of the American Chemical Society, New Orleans LA, Dec. 6-8, 2000.

ings to the oxidative properties of OTA in a biological system are discussed.

Experimental Procedures Caution: The work described involves the synthesis and handling of hazardous agents and was therefore conducted in accordance with NIH guidelines for the Laboratory use of Chemical Carcinogens. Materials. Ochratoxin A (OTA, 1A) was purchased from Dr. Ronald R. Marquardt (Department of Animal Sciences, University of Manitoba) and used without further purification. The hydroquinone analogue of ochratoxin A (OTHQ, 2) was chemically synthesized as previously described (21). 4-Chlorophenol (4-ClPhOH), acetonitrile (MeCN) (ACROS), benzohydroquinone (BHQ) (Eastman), and tetrabutylammonium perchlorate (TBAP) (Alfa-Aesar) were obtained commercially. MeCN (99.9%) was distilled over CaH2, stored for short times over 3 Å molecular sieves, and transferred under dry N2. BHQ (99%) was recrystallized from acetone. 4-ClPhOH ()99.8%) and TBAP ()99%) were used without further purification. OTA and 4-ClPhOH were converted into their corresponding anions in MeCN by adding one equivalent of NaOMe in MeOH, evaporating to dryness, and redissolving in MeCN. NaOMe/MeOH solutions were prepared by reaction of Na with HPLC grade MeOH; the concentration was determined by potentiometric titration with HCl. Phosphate buffer solutions were prepared by dissolving KH2PO4 in deionized water. The resulting solution was then degassed, and the pH was adjusted by addition of aqueous KOH. Methods. Cyclic voltammetry (CV) was carried out in a three-electrode mini-cell (2-3 mL) containing a glassy carbon working electrode (diameter 1.5 mm), a platinum flag counter electrode, and a pseudoreference electrode (Ag wire or Pt wire). Potentials were checked against a saturated calomel electrode (SCE), and are given relative to this reference. Samples were typically 5 × 10-3 mol dm-3 in MeCN with TBAP (0.4-0.5 mol dm-3) as the supporting electrolyte. Solutions were purged with dry N2 prior to use and kept under N2 during the experiment. The glassy carbon working electrode was polished with 1.0, 0.3, and 0.05 µm R-alumina (Buehler Corp.), rinsed with deionized water, dried, and sonicated for several minutes in fresh MeCN prior to each experiment. This cleaning treatment was repeated using 0.05 µm alumina for polishing between runs. Data were collected using a Pine AFCBP1 (Pine Instrument Company, Grove City, PA) computer-controlled bi-potentiostat/waveform generator and PineChem 2.7 graphical user interface software. UV-vis spectroelectrochemistry was carried out with a smallvolume reflectance cell constructed “in-house” and similar to one previously described (27). The complete system included a deuterium light source (Analytical Instrument Systems, Inc.), a UV-vis detector (Ocean Optics, Inc.), and a bifurcated fused silica fiber optic dip probe/quartz window (Ocean Optics). Data were collected using the OOI Base software package (Ocean Optics) and processed using a standard spreadsheet program. Rotating disk voltammetry (RDV) was carried out using a Pine Instrument Company MSRX speed control/analytical rotator along with a Pine AFMT29TGCGC glassy carbon rotating ringdisk electrode (disk diameter 0.221 in.). The electrochemical RDV cell (25 mL) consisted of a Pt coil counter electrode and

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Figure 2. Cyclic voltammetry in MeCN. Voltammograms recorded using a glassy carbon (diameter 1.5 mm) working electrode and a Ag wire pseudoreference electrode. (A) 5.1 × 10-3 mol/dm3 OTA and 5.2 × 10-1 mol/dm3 TBAP, ν ) 0.100 V/s. (B) 5.9 × 10-3 mol/dm3 4-chorophenol (4-ClPhOH) and 2.17 × 10-1 mol/dm3 TBAP, ν ) 0.500 V/s. an SCE reference. Capillary electrophoresis (CE) experiments were performed using a Bio-Rad (Hercules, CA) Biofocus 3000 CE system with UV-vis detection at λ ) 210 nm (28). A fusedsilica capillary was used (60 cm × 50 µm i.d. uncoated, Polymicro Technologies, effective length 55.4 cm, thermostatically controlled, 20 °C). Samples were hydrodynamically injected at 5 psi s. Between each sample injection, the capillary was rinsed sequentially with 0.1 M NaOH, water, and electrophoretic buffer (20 mM NaH2PO4 in Milli-Q water adjusted to pH 7 by addition of 0.1 M NaOH). Before each experiment, the electrophoretic buffer was filtered using a 0.20-µm nylon syringe filter. Samples were prepared by diluting a stock solution of OTA/MeOH (33 mM) with electrophoretic buffer to a concentration of 13 µM.

Results Cyclic Voltammetry of OTA and 4-ClPhOH in MeCN. Cyclic voltammetry (CV) of OTA in acetonitrile (MeCN) revealed only one oxidation peak on the forward scan of the first cycle with a half-peak potential (Ep/2) of 1.81 V at a scan rate (ν) of 0.100 V s-1 (Figure 2A). The value of Ep/2 for the primary oxidation wave was dependent on scan rate, with an increase in Ep/2 of approximately 60 mV for each 10-fold increase in ν. The amplitude of the anodic peak current (iAp ) for the primary oxidation of OTA increased linearly with ν1/2 and chronoamperometric experiments confirmed that the oxidation was masstransport limited. A Cottrell plot for the oxidation of OTA in MeCN showed a linear relationship between the current (it) and t-1/2. On the reverse scan of the first cycle, a reduction peak at 0.58 V (Ep) was noted. As is evident in Figure 2A, the anodic peak current of the first cycle was significantly greater than that of the cathodic peak current, and the peak separation was considerable. With successive cycles, a secondary oxidation peak at 1.38 V (Ep) was observed, which grew in intensity as the primary oxidation peak current diminished. Truncated scans indicated that this secondary oxidation peak was produced from the oxidation of OTA. To investigate the hypothesis that the chlorophenolic moiety was the electrochemically active constituent of OTA, a comparison with the electrochemical oxidation of 4-chlorophenol (4-ClPhOH) was carried out (Figure 2B). For 4-ClPhOH, the oxidation occurred at 1.59 V

Figure 3. Spectroelectrochemistry in MeCN. Difference spectra were plotted after the onset of diffusion-limited oxidation. (A) 1.0 × 10-3 mol/dm3 OTA and 3.0 × 10-1 mol/dm3 TBAP, 2.0 V applied for 5 min, one spectrum recorded every 62 s. (B) 1.0 × 10-3 mol/dm3 OTHQ and 3.0 × 10-1 mol/dm3 TBAP, 1.5 V applied for 10 s, one spectrum recorded every second.

(Ep/2), with a reduction peak at 0.50 V (Ep). As with OTA, Ep/2 for the primary oxidation peak of 4-ClPhOH increased by approximately 60 mV for each 10-fold increase in ν, and the magnitude of the peak current was linearly dependent on ν1/2. Subsequent scans showed the appearance of a secondary oxidation peak at 1.28V (Ep). This result led us to speculate that the oxidation of 4-ClPhOH produced benzoquinone (BQ) and that the oxidation peak at 1.28 V was due to the oxidation of benzohydroquinone (BHQ). Consistent with this hypothesis, spiking the MeCN solution of 4-ClPhOH with BHQ produced a CV in which the oxidation and reduction peak potentials for the BHQ/BQ couple were coincident with the secondary redox peaks in the CV of 4-ClPhOH. To determine whether OTA also produced a quinone species, a small amount of authentic OTHQ (2, Figure 1) was added to the OTA/MeCN solution. The oxidation and subsequent reduction peaks of OTHQ fell in the same potential range as that of the species produced in the anodic oxidation of OTA. OTHQ exhibited an oxidation wave with Ep/2 ) 1.21 V, and a single reduction at ca. 0.30 V (data not shown). These potentials were coincident with the secondary redox peaks observed in the CV of OTA (Figure 2A). Spectroelectrochemistry. UV-vis spectroelectrochemistry was used to further identify the products of anodic oxidation of OTA (λmax ) 330 nm) and OTHQ (λmax ) 350 nm). The difference spectra observed upon oxidation of OTA in MeCN emphasize the changes in absorption resulting from the oxidative process (Figure 3A). At the onset of oxidation, the OTA absorbance at λ ) 330 nm decreased and two peaks appeared at λ ) 375 260 nm. Similar results were observed for the oxidation of OTHQ (Figure 3B), indicating that both are converted to similar products upon electrochemical oxidation in MeCN. These findings were analogous to our earlier UVvis spectrophotometric studies on the chemical oxidation of OTHQ by NOBF4 in MeCN (21).

Electrochemical Oxidation of OTA

Figure 4. Effect of deprotonation in MeCN. Voltammograms were recorded using a Ag wire pseudoreference electrode and a glassy carbon (diameter 1.5 mm) working electrode, ν ) 0.100 V/s. (A) 5.45 × 10-3 mol/dm3 OTA- and 8.0 × 10-1 mol/dm3 TBAP. (B) 3.3 × 10-3 mol/dm3 4-ClPhO- and 3.3 × 10-1 mol/ dm3 TBAP.

Cyclic Voltammetry of OTA- and 4-ClPhO- in MeCN. Having established that the para-chlorophenolic group of OTA is the electrochemically active component, the effect of phenolic deprotonation on the oxidative mechanism was examined. A spectrophotometric titration of OTA in MeCN with NaOMe/MeOH revealed that 1 equiv of base almost completely converted protonated OTA (λmax ) 330 nm) to the phenolate ion (OTA-, λmax ) 385 nm). It was anticipated that 2 equiv of base would be required to generate the phenolate of OTA (i.e., the carboxylic acid would deprotonate first). However, early literature on apparent pKa values for phenols and carboxylic acids in dipolar aprotic solvents show that many phenols are more acidic (29, 30). As shown in Figure 4A, the CV of OTA- revealed one oxidation wave on the first forward scan with Ep/2 ) 1.0 V. When corrected for differences in concentration, the peak current of the anion was approximately half that observed for the protonated species (Figure 2A). Additionally, there were no secondary redox waves in the CV of OTA-, in contrast to the CV of the protonated species. A CV experiment was also carried out on the phenolate ion of 4-ClPhOH (4-ClPhO-). The voltammogram of 4-ClPhO- exhibited one primary oxidation wave with Ep/2 ) 0.74 V, and no apparent secondary redox couples were observed (Figure 4B). Cyclic Voltammetry in Aqueous Buffer. The electrochemistry of OTA in aqueous buffer was quite different from that observed in MeCN for the protonated species but was remarkably similar to the behavior of the phenolate, OTA-. Figure 5A shows the CV obtained for OTA in phosphate buffer, pH 7.4. Only a single irreversible oxidation (Ep/2 ) 0.80 V) accompanied by a small, broad reduction peak (Ep ) 0.1 V) was noted. Ep/2 shifted in the anodic direction by ca. 20 mV/10-fold increase in ν, and a linear relationship between the anodic peak current and ν1/2 was observed. In contrast, OTHQ (2) exhibited two distinct oxidation peaks: Ep ) 0.34 and 0.65 V, and reduction peaks Ep ) 0.07 V and -0.20 V in buffer (Figure 5B). These results indicated that the electrochemical oxidation of OTA in phosphate buffer at pH 7.4 did not yield the OTQ/OTHQ redox couple, as

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Figure 5. Cyclic voltammetry in aqueous (5% v/v dioxane) phosphate buffer. Voltammograms were recorded using an SCE reference and a glassy carbon (diameter 1.0 mm) working electrode, ν ) 0.100 V/s, pH 7.4. (A) 3.6 × 10-3 mol/dm3 OTA and 4.0 × 10-1 mol/dm3 KH2PO4. (B) 3.0 × 10-3 mol/dm3 OTHQ and 1.0 × 10-1 mol/dm3 KH2PO4. Table 1. Anodic Half-Peak Potentials for OTA and Structurally Similar Compounds analogue

solvent

Ep/2a,b

OTA OTHQ 4-ClPhOH OTA4-ClPhOOTA OTHQ

MeCN MeCN MeCN MeCN MeCN aq. buffer aq. buffer

1.81 1.21 1.59 1.00c 0.74c 0.80d 0.34, 0.65d,e

a In volts vs SCE. b ν ) 0.100 V s-1. c Formed by reaction with NaMeO-/MeOH. d pH 7.4. e Ep reported.

observed for the protonated species in MeCN. The results of these studies are summarized in Table 1. Rotating Disk Voltammetry. A Levich plot (iAL vs 1/2, where iA ) limiting (steady state) current; ω ) ω L rotation rate) was linear, indicating that the oxidation of OTA in buffer was mass-transport controlled (data not shown). At the highest rotation rates, the voltammograms were peaked which is consistent with the formation of an insulating film at the correspondingly higher currents. Also, the half-wave potential was linearly dependent on log(ω-1) with a slope of -32 mV. Determination of The Diffusion Coefficient of OTA. To determine the number of electrons (n) involved in the oxidation of the OTA in aqueous phosphate buffer, it was necessary to determine the diffusion coefficient (D) of OTA; an electrophoretic method was used (31). Conditions were chosen so that peak dispersion was due to solute diffusion, and D was determined from the linear dependence of the peak variance (σ2) on the solute migration time at low applied voltage (data not shown). A D of 0.48 × 10-5 cm2 s-1 was calculated from the slope of the resulting plot (slope ) 0.96 × 10-5 cm2 s-1 ) 2D). Using the Cottrell equation and chronoamperometric data, the product nD1/2 was calculated (nD1/2 ) 2.38 × 10-3 cm2 s-1). By combining this value with D ) 0.48 × 10-5 cm2 s-1, n was obtained (n ) 1.09). Variation of Ep/2 with pH. The anodic half-peak potential for the oxidation of OTA in aqueous media was found to be linearly dependent on pH (pH 6-8, Figure 6). Electrochemically reversible electron-transfer mechanisms that involve H+ equilibria exhibit a linear de-

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Figure 6. Anodic half-peak potential (EAp/2) dependence for electrochemical oxidation of OTA in aqueous buffer as a function of pH. 3.0 × 10-3 mol/dm3 OTA and 4.0 × 10-1 mol/dm3 aqueous KH2PO4 (5% dioxane), glassy carbon (diameter 1 mm) working electrode, SCE reference, ν ) 0.1V/s.

pendence of Ep/2 on pH with a slope of (nH+/ne) x 59.2 mV (32). The slope observed for the irreversible oxidation of OTA was 51 mV.

Discussion Much work has been done in an effort to determine the mode of OTA-mediated carcinogenicity. Structureactivity studies on OTA (1A) and OTB (1B, Figure 1) have indicated that the chlorine atom (Figure 1) plays an important role in its toxicity (25, 26). Recent efforts have established that OTA is mutagenic (8), promotes oxidative stress (33), and facilitates guanine-specific DNA adduct formation upon oxidative activation (19). However, known OTA metabolites, 4-(R)-OH-OTA (1C) and 4-(S)-OH-OTA (1D, Figure 1) that are oxidatively produced by CYP450 isoforms (34) are not toxic (15, 16, 20, 25, 26). Thus, the mechanism of OTA-mediated genotoxicity and mutagenicity has remained elusive (20). Recently, we showed that FeTPPS, a water soluble Fe(III) porphyrin, was capable of oxidizing OTA in aqueous buffered media (21). Upon oxidation and subsequent reduction by ascorbate (Scheme 1), OTHQ (2, Figure 1) was detected by reversed-phase HPLC and electrospray mass spectrometry. This led us to suspect that the quinone OTQ (3, Figure 1) of OTA may play an important role in OTA-mediated genotoxicity, either through a direct interaction with DNA, or by oxidative stress as a result of redox cycling (21). The extension of this study to include the electrochemical oxidation of OTA has provided new insight into this problem. The electrochemical oxidation of OTA in MeCN (Figure 2A) occurred at a very high potential (Ep/2 ) 1.81V vs SCE). Ep/2 was dependent on ν, which is characteristic of a homogeneous chemical reaction following electron transfer; the linear dependence of iAp on ν1/2 and the Cottrell plot were consistent with a diffusion-limited oxidation of solution species (35). The smaller peak current observed for the cathodic wave and the large separation between the cathodic and anodic peaks was consistent with a chemical step or series of steps following oxidation. This generated a substrate that redox-cycled at lower potentials (secondary oxidation peak) than Ep/2 of OTA. Consistent with this interpretation was the observation that, when the initial forward scan was truncated before the onset of OTA oxidation, no secondary peaks were noted on subsequent scans at lower potentials. The cyclic voltammetric behavior of 4-chlorophenol (4ClPhOH) was nearly identical to that observed for OTA (Figure 2B). Both 4-ClPhOH and OTA exhibited primary oxidation peaks at high potentials followed by the

Calcutt et al.

development of secondary redox peaks at lower potentials. The electrochemical oxidation of substituted phenols generally involves a “scheme of squares” mechanism (Scheme 2) that is made up of individual proton and electron transfers (36-38). The products are typically unstable, and immediately couple, react with supporting electrolyte, solvent, or solvent impurities to yield irreversible voltammograms (37, 38) as observed for OTA and 4-ClPhOH. Additions of OTHQ (2) and BHQ to the OTA and 4-ClPhOH solutions, respectively, resulted in an increase in the anodic and cathodic peak currents for the secondary redox waves. This demonstrated that the electrochemical oxidation of both species resulted in the production of their respective quinones. Quinone/hydroquinone couples have been identified as products of electrolysis for many phenols in nonaqueous systems (Scheme 2), and it is generally accepted that their formation involves a 2e oxidation of the phenol to a phenoxonium cation (37, 38) that reacts with traces of water in the solvent, which exist even after stringent purification procedures (39). The UV-vis difference spectra taken after the onset of electrolysis of OTA (Figure 3A) and OTHQ (Figure 3B) also indicated that the electrochemical oxidation products of OTA and OTHQ were similar. Both species exhibited an increase in absorption at ca. 260 and 375 nm, which was analogous to the observed spectral changes upon treatment of OTHQ with the chemical oxidant NOBF4 (21). Since quinones are known to exhibit strong absorption maxima at λ ) 240-300 nm and weaker absorptions at λ ) 285-440 nm (40), these findings in MeCN were fully consistent with the electrochemical oxidation of OTA to the quinone species OTQ (3). The addition of 1 equiv of base to the OTA/MeCN solution produced a notable change in the oxidative mechanism; Ep/2 was lowered by 0.8 V and the anodic peak current was approximately half of the value observed in the absence of added base (Figure 4A). This was consistent with a change in the oxidative mechanism from a 2e to a 1e process, and was further evidence that the chlorophenolic constituent of OTA was the electrochemically active site. Phenolates generally exhibit lower oxidation potentials than their parent phenols and, unlike phenols, tend to undergo 1e oxidations to radical species (Scheme 2) (37, 38). For OTA- no secondary redox peaks that could be ascribed to the OTHQ/OTQ couple were evident in the CV (Figure 4A). Addition of base to 4-ClPhOH also resulted in a significant decrease in the oxidation peak current in the same fashion (Figure 4B) and the disappearance of secondary peaks resulting from the BHQ/BQ couple. The electrochemical oxidation of OTA in aqueous buffer resembled the oxidative behavior of OTA- in MeCN. Throughout the pH range of 6-8, a single irreversible anodic peak at ca. Ep/2 ) 0.8 V was noted with no secondary redox peaks upon successive scans (Figure 5A). These results are in accordance with the fact that the phenolic group of OTA (pKa ) 7) is predominantly deprotonated at physiological pH, and that phenolates typically undergo oxidation at potentials considerably lower than phenols (37, 38). According to Scheme 2 for the mechanism(s) of phenolic oxidation, it is also conceivable that the phenolic radical would undergo a second 1e oxidation to generate the phenoxonium cation. However, similar to the results obtained for OTA- in MeCN, following the initial oxidation at ca. 0.8 V, no further

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Scheme 2. Pathways for the Oxidation of Chlorinated Phenols

oxidation was noted. If the OTHQ/OTQ couple was produced, then it should have been detected as it exhibited two distinct oxidation (Ep ) 0.34 and 0.65 V) peaks (Figure 5B) at potentials lower than the parent OTA. In fact, that OTHQ (2) showed two oxidation peaks was interesting and suggested that the oxidation (2H+/2e) may involve two distinct 1H+/1e processes. The electrochemical oxidation of OTA in aqueous media exhibited a pH dependence similar to that predicted by the Nernst equation for a 1H+/1e process (Figure 6). A Levich plot (iAL vs ω1/2) demonstrated that the oxidation of OTA in buffer was diffusion-limited and E1/2 vs log(ω-1) was linear with a slope of -32 mV, which is comparable to the calculated slope of -29.6 mV for an EC mechanism involving a 1e oxidation (n ) 1) (41). The peaked response at the very highest rotation rates and, hence, highest current densities, is consistent with the anodic deposition of an insulating film which is a common problem encountered in the anodic oxidation of chlorinated phenols (42). Attempts to simultaneously determine n and D (the diffusion coefficient) by employing electrochemical techniques governed by two different equations (43) were frustrated by the formation of this insulating film. Thus, D (0.48 × 10-5 cm2 s-1) was determined independently using CE. From this value and the chronoamperometric results, n was calculated to be ca. 1, as anticipated for a 1e oxidation to a radical species. The above information is important for understanding the metabolic fate of OTA. Labat et al. (44) have utilized methoxylated arenes to rank and compare the redox abilities of peroxidases and CYP450s. These studies have revealed that horseradish peroxidase (HRP) is unable to oxidize organic molecules with E1/2 above 1.2 V vs Ag/ AgCl (1.16 V vs SCE), while ligninase is active up to 1.4 V (Ag/AgCl). These potentials are significantly lower than the values required to electrochemically oxidize the protonated forms of OTA (Ep/2 ) 1.81 V) and 4-ClPhOH (Ep/2 ) 1.59 V) (SCE). However, based on redox potentials, they can oxidize OTA- (Ep/2 ) 1.0 V) to generate the phenolic radical. The FeTPPS system that we previously utilized to study the oxidation of OTA (21) is active up to ca. 1.7 V (Ag/AgCl), and the iron-oxo entity in CYP450 is estimated to have a redox potential between 1.7 and 2.0 V (Ag/AgCl) (45). Thus, the redox potentials of CYP450s and biomimetic iron-oxo systems are high enough to convert protonated OTA, and other chlorinated

phenols, to quinone species. Therefore, OTQ (3) production, as observed in the chemical oxidation of OTA by FeTPPS (21), is fully consistent with the electrochemical experiments and the redox potentials of the various species involved. While it remains to be determined whether CYP450s facilitate the oxidative transformation of OTA into OTQ (3), the redox potentials of the species show that it is possible. In conclusion, the electrochemical oxidation of OTA in acetonitrile solution results in the removal of 1H+/2e from the phenolic moiety to form a cationic intermediate with subsequent formation of a quinone species, OTQ (3). The phenolate of OTA (OTA-) undergoes a 1e oxidation to generate the phenolic radical, whose fate is uncertain. In aqueous buffer, the electrochemistry of OTA mimics the behavior observed for OTA- in MeCN. The electrochemical experiments predict that peroxidases would be unable to oxidize protonated OTA, but would be able to oxidize OTA- to the phenolic radical. CYP450s may oxidize OTA into OTQ (3), although detection of this reactive intermediate may not be easy.

Acknowledgment. R.A.M. acknowledges the National Cancer Institute (Grant CA080787) for support of this research. M.W.C. acknowledges Mr. P. J. Viskari and Dr. C. L. Colyer for their gracious assistance with capillary electrophoresis experiments. Helpful comments from the reviewers are also acknowledged.

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