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Feb 20, 2017 - The exposure of human skin to 4-(4-hydroxyphenyl)-2-butanone (raspberry ketone, RK) is known to cause chemical/occupational leukoderma...
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Tyrosinase-Catalyzed Oxidation of the Leukoderma-Inducing Agent Raspberry Ketone Produces (E)‑4-(3-Oxo-1-butenyl)-1,2benzoquinone: Implications for Melanocyte Toxicity Shosuke Ito,*,† Maki Hinoshita,† Erina Suzuki,† Makoto Ojika,‡ and Kazumasa Wakamatsu† †

Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi 470-1192, Japan Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan



S Supporting Information *

ABSTRACT: The exposure of human skin to 4-(4-hydroxyphenyl)-2-butanone (raspberry ketone, RK) is known to cause chemical/occupational leukoderma. RK has a structure closely related to 4-(4-hydroxyphenyl)-2-butanol (rhododendrol), a skin whitening agent that was found to cause leukoderma in the skin of consumers in 2013. Rhododendrol is a good substrate for tyrosinase and causes a tyrosinase-dependent cytotoxicity to melanocytes, cells that are responsible for skin pigmentation. Therefore, it is expected that RK exerts its cytotoxicity to melanocytes through the tyrosinase-catalyzed oxidation to cytotoxic oquinones. The results of this study demonstrate that the oxidation of RK by mushroom tyrosinase rapidly produces 4-(3oxobutyl)-1,2-benzoquinone (RK-quinone), which is converted within 10−20 min to (E)-4-(3-oxo-1-butenyl)-1,2-benzoquinone (DBL-quinone). These quinones were identified as their corresponding catechols after reduction by ascorbic acid. RK-quinone and DBL-quinone quantitatively bind to the small thiol N-acetyl-L-cysteine to form thiol adducts and can also bind to the thiol protein bovine serum albumin through its cysteinyl residue. DBL-quinone is more reactive than RK-quinone, as judged by their half-lives (6.2 min vs 10.5 min, respectively), and decays rapidly to form an oligomeric pigment (RK-oligomer). The RKoligomer can oxidize GSH to GSSG with a concomitant production of hydrogen peroxide, indicating its pro-oxidant activity, similar to that of the RD-oligomer. These results suggest that RK is cytotoxic to melanocytes through the binding of RK-derived quinones to thiol proteins and the pro-oxidant activity of the RK-oligomer.



INTRODUCTION Tyrosinase is the key enzyme produced in melanocytes that catalyzes the oxidation of L-tyrosine to dopaquinone leading to the production of melanin pigments, eumelanin and pheomelanin.1,2 Tyrosinase is able to oxidize a large number of phenols and catechols to produce o-quinones. o-Quinones are highly reactive compounds that exert cytotoxicity by binding to thiol enzymes and by producing reactive oxygen species (ROS).3,4 o-Quinones can also act as haptens that covalently bind to melanosomal proteins to generate neoantigens, thus triggering immunological responses.5 Because of the high toxicity of o-quinones, phenolic compounds have the potential risk to become toxic to melanocytes through tyrosinase-catalyzed conversion to o-quinones. Contact/occupational leukoderma is caused by the loss of skin pigmentation (melanin) due to (occupational) contact with certain chemicals. A number of phenolic (and catecholic) compounds are known © XXXX American Chemical Society

to induce contact/occupational leukoderma in human skin through their activation to o-quinones. Those phenols (or catechols) include 4-methoxyphenol,6,7 monobenzone (4benzyloxyphenol),8,9 4-tert-butylphenol,10 4-tert-butylcatechol,11 and hydroquinone.12 Raspberry ketone [4-(4-hydroxyphenyl)-2-butanone, RK, 1; Figure 1], one of the major aromatic compounds of raspberries, is widely used in perfumery, in cosmetics, and as a flavoring agent in foodstuffs.13 RK (1) was reported to induce occupational leukoderma in 1998,14 when 3 workers developed leukoderma in their hands among 13 workers engaged in manufacturing RK in Japan. However, it appears that this report of the adverse effect of RK (1) had been underestimated, and a report showing the potential use of this phenol as a skin Received: January 10, 2017 Published: February 20, 2017 A

DOI: 10.1021/acs.chemrestox.7b00006 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology

Figure 1. Tyrosinase-catalyzed oxidation of RK (1) in the absence or presence of a thiol. Oxidation of RK (1) gives RK-quinone (2) as an immediate product, which is rapidly transformed to DBL-quinone (3) via a transient quinone methide intermediate formed by base-catalyzed tautomerization. These quinones were isolated after reduction by ascorbic acid (AA) as RK-catechol (4) and DBL (5), respectively, and were identified by spectroscopic analyses. Tyrosinase-catalyzed oxidation of RK (1) in the presence of the thiol NAC affords the monoadduct, NAC-RK-catechol (6), while the oxidation of DBL (5) affords the monoadduct NAC-DBL (7) and the diadduct Di-NAC-DBL (8). These thiol adducts were isolated and identified as the NAC adducts. RK-quinone (2) and DBL-quinone (3) are also able to bind to BSA through its cysteine residue.



whitening agent was reported in 2011.15 Rhododendrol (or rhododenol, RD) has a structure closely related to RK (1), with the carbonyl group in 1 being reduced to a hydroxy group in RD. RD was added to cosmetics as a skin whitening ingredient by a cosmetic company in Japan. In July 2013, cosmetics containing RD were recalled because a considerable number of consumers developed leukoderma on their face, neck, and hands.16 RD was recently shown to exert melanocyte toxicity via a tyrosinase-dependent mechanism.17,18 In independent studies, we reported that the oxidation of RD by mushroom tyrosinase rapidly produces RD-quinone, which is quickly converted to RD-cyclic quinone through an intramolecular addition of the hydroxy group19 and that human tyrosinase is able to oxidize both enantiomers of RD.20 We proposed that those quinones exert their toxicity toward melanocytes by binding to cellular thiol enzymes and by producing ROS.21 In fact, B16F1 mouse melanoma cells exposed to RD produced a high level of RD-quinone conjugated to thiol proteins formed through their cysteinyl residues.22 It is likely that RK (1) also exerts melanocyte toxicity through o-quinone(s) produced by tyrosinase-catalyzed oxidation (Figure 1). In fact, previous studies have shown that RK (1) serves as a substrate for mushroom tyrosinase.23,24 However, those studies did not identify the products of the tyrosinase-catalyzed oxidation in detail and did not examine the chemical reactivities of the o-quinone products. In this study, we aimed to identify the o-quinone products of the tyrosinasecatalyzed oxidation of RK (1) under biomimetic conditions and to explore their reactions with thiol compounds including the thiol protein bovine serum albumin (BSA). The pro-oxidant activity of the RK oxidation product (RK-oligomer) was also examined.

EXPERIMENTAL PROCEDURES

Materials and Instruments. Raspberry ketone [4-(4-hydroxyphenyl)-2-butanone; RK; 1], 3,4-dihydroxybenzalacetone [(E)-4(3,4-dihydroxyphenyl)-3-buten-2-one; DBL; 5], L-ascorbic acid, and BSA were purchased from Wako Pure Chemicals (Osaka, Japan). Tyrosinase (from mushrooms, specific activity 1715 U/mg), N-acetylL-cysteine (NAC), reduced glutathione (GSH), oxidized glutathione (GSSG), and Ampliflu Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of the highest purity commercially available. An HPLC system was used to follow the course of tyrosinase oxidation and consisted of a JASCO 880-PU pump (JASCO Co., Tokyo, Japan), a Shiseido C18 column (Capcell Pak MG; 4.6 × 250 mm; 5 μm particle size, Shiseido, Tokyo, Japan), and a JASCO UV detector (JASCO Co., Tokyo, Japan) at 280 nm unless otherwise described. The mobile phase was 0.4 M HCOOH/methanol, 60:40 (v/v). Analyses were performed at 45 °C and at a flow rate of 0.7 mL/ min. For preparative separation of RK-catechol (4), DBL (5), NACRK-catechol (6), NAC-DBL (7), and Di-NAC-DBL (8), a Shiseido C18 preparative column (Capcell Pak MG; 20 × 250 mm; 5 μm particle size) was used at 45 °C and at a flow rate of 7.0 mL/min with the same mobile phase used for the analytical separation. Sizeexclusion (gel-filtration) HPLC was performed with a Shodex OHpack SB-802.5 HQ column (8.0 × 300 mm, 6 μm particle size, Showa Denko, Kawasaki, Kanagawa, Japan) as previously described.21 UV−visible spectra were analyzed with a JASCO V-630 UV−vis spectrophotometer (JASCO CO., Tokyo, Japan). 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were obtained using a Bruker AVANCE 400 spectrometer (Billerica, MA, USA), and mass spectra were obtained using a Biospectrometry Workstation Mariner (mode: electrospray ionization−time-of-flight, positive; ESI(+)-TOF) (Applied Biosystems, Foster City, CA, USA). Oxidation of RK (1) or DBL (5) by Tyrosinase in the Absence or Presence of NAC. A solution (2 mL) of 100 μM RK (1) or DBL (5) was oxidized by mushroom tyrosinase (50 U/mL) at 37 °C in 50 B

DOI: 10.1021/acs.chemrestox.7b00006 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology mM sodium phosphate buffer (pH 6.8 or 5.3). Changes in absorption spectra were periodically followed for 60 min. The oxidation was also carried out in the presence of 200 μM NAC (for RK) or 250 μM NAC (for DBL). Aliquots of the reaction mixtures were also subjected to HPLC analysis. For reductive termination of the reaction, 100 μL aliquots were mixed with 20 μL of 5 mM ascorbic acid followed by the addition of 100 μL of 0.8 M HClO4. For experiments in the presence of NAC, the addition of ascorbic acid was omitted. The half-lives (t1/2) of the ortho-quinones produced were calculated using the equation ln(c0/c) = kt and kt1/2 = 0.693. Isolation of 4-(3,4-Dihydroxyphenyl)-2-butanone (RK-Catechol, 4) and (E)-4-(3,4-Dihydroxyphenyl)-3-buten-2-one (DBL, 5). A solution of RK (1, 16.4 mg, 100 μmol) in ethanol (1 mL) was mixed with 100 mL of 50 mM sodium phosphate buffer (pH 5.3). The mixture was vigorously shaken at 37 °C to which tyrosinase (20,000 U) in 1 mL buffer was added. After 10 min, 176 mg (1 mmol) of ascorbic acid and then 5 mL of 1 M HCl were added to stop oxidation, and the mixture was extracted three times with ethyl acetate (100 mL each). The organic layers were combined, washed once with 40 mL of saturated NaCl solution, dried over anhydrous Na2SO4, and evaporated under reduced pressure. The residue (20.3 mg) was subjected to preparative HPLC to afford, after lyophilization, 14.7 mg (82% yield) of RK-catechol (4) (HPLC purity >99%) and 1.2 mg (7% yield) of DBL (5) (HPLC purity 99%). When the oxidation was carried out in pH 6.8 buffer, 4.1 mg (23% yield) of RK-catechol (4) and 1.6 mg (9% yield) of DBL (5) were obtained. RK-Catechol (4). 1H NMR and 13C NMR data are shown in Figure S1. 1H NMR (CD3OD): δ 2.10 (3H, s, H-1), 2.70 (2H, m, H-3), 2.70 (2H, m, H-4), 6.48 (1H, dd, H-6′, J5′,6′ = 8.0 and J2′,6′ = 2.0 Hz), 6.60 (1H, d, H-2′, J2′,6′ = 2.0 Hz), and 6.64 (1H, d, H-5′, J5′,6′ = 8.0 Hz). 13C NMR (CD3OD): δ 30.0 and 30.3 (C-1 and C-3), 46.2 (C-4), 116.3 and 116.4 (C-2′ and C-5′), 120.5 (C-6′), 134.0 (C-1′), 144.5 and 146.2 (C-3′ and C-4′), and 211.5 (C-2). ESI(+) MS: m/z 179 ([M + H −2H]+). High-resolution MS 179.0714, calcd for C10H11O3, 179.0703 (+ 1.1 mDa). UV (ethanol) λmax 284 nm (ε 2430). DBL (5). 1H NMR and 13C NMR data are shown in Figure S2. 1H NMR (CD3OD): δ 2.33 (3H, s, H-1), 6.54 (1H, d, H-3, J3,4 = 16.4 Hz), 6.78 (1H, d, H-5′, J5′,6′ = 8.4 Hz), 6.98 (1H, dd, H-6′, J5′,6′ = 8.4 and J2′,6′ = 2.0 Hz), 7.07 (1H, d, H-2′, J2′,6′ = 2.0 Hz), and 7.51 (1H, d, H-4, J3,4 = 16.4 Hz). 13C NMR (CD3OD): δ 27.0 (C-1), 115.3 and 116.6 (C-2′ and C-5′), 123.5 (C-6′), 124.7 (C-1′), 127.7 (C-3), 146.8 and 146.9 (C-3′ and C-4′), 150.0 (C-4), and 201.6 (C-2).UV (ethanol) λmax 252 nm (ε 3570) and 344 nm (ε 17910). Isolation of 3-S-[(N-Acetyl)cysteinyl]-5-(3-oxo-butyl)-1,2-dihydroxybenzene (NAC-RK-Catechol, 6). A solution of RK (1, 16.4 mg, 100 μmol) and NAC (32.6 mg, 200 μmol) in ethanol (1 mL) was mixed with 100 mL of 50 mM sodium phosphate buffer (pH 6.8). The mixture was vigorously shaken at 37 °C to which tyrosinase (20,000 U) in 1 mL buffer was added. After 30 min, 10 mL of 1 M HCl was added to stop oxidation, and the mixture was treated as detailed above. The residue (24.0 mg) was subjected to preparative HPLC to afford, after lyophilization, 19.4 mg (57% yield) of NAC-RK-catechol (6) (HPLC purity >99%). NAC-RK-Catechol (6). 1H NMR and 13C NMR data are shown in Figure S3. 1H NMR (CD3OD): δ 1.92 (3H, s, COCH3), 2.12 (3H, s, H-1), 2.72 (2H, m, H-3), 2.72 (2H, m, H-4), 3.06 and 3.33 (1H × 2, m, SCH2), 4.46 (1H, m, SCH2CH), 6.61 (1H, d, H-2′, J2′,6′ = 2.0 Hz), and 6.72 (1H, d, H-6′, J2′,6′ = 2.0 Hz). 13C NMR (CD3OD): δ 22.4 (COCH3), 30.0 and 30.1 (C-1 and C-3), 36.9 (SCH2), 45.9 (C-4), 54.0 (SCH2CH), 116.8 (C-2′), 120.7 (C-6′), 125.5 (C-5′), 134.2 (C1′), 145.1 and 146.5 (C-3′ and C-4′), 173.2 and 173.9 (COCH3 and COOH), and 211.3 (C-2). ESI(+) MS: m/z 342 ([M + H]+). Highresolution MS 342.0976, calcd for C15H20 NO6S, 342.1006 (− 3.0 mDa). UV (ethanol) λmax 256 nm (ε 2930) and 294 nm (2570). Isolation of 3-S-[(N-Acetyl)cysteinyl]-4-(3-oxo-1-butenyl)-1,2-dihydroxybenzene (NAC-DBL, 7) and 3,6-Di-S-[(N-acetyl)cysteinyl]-4(3-oxo-1-butenyl)-1,2-dihydroxybenzene (Di-NAC-DBL, 8). A solution of RK (1, 16.4 mg, 100 μmol) and NAC (32.6 mg, 200 μmol) in ethanol (1 mL) was mixed with 100 mL of 50 mM sodium phosphate buffer (pH 6.8). The mixture was vigorously shaken at 37 °C to which

tyrosinase (20,000 U) in 1 mL of buffer was added. After 5 min, 10 mL of 1 M HCl was added to stop oxidation, and the mixture was treated as detailed above. The residue (23.2 mg) was subjected to preparative HPLC to afford, after lyophilization, 11.9 mg (35% yield) of NACDBL (7) and 8.6 mg (17% yield) of DiNAC-DBL (8) (HPLC purity >99%). NAC-DBL (7). 1H NMR and 13C NMR data are shown in Figure S4. 1 H NMR (CD3OD): δ 1.88 (3H, s, COCH3), 2.38 (3H, s, H-1), 3.05 and 3.41 (2H, m, SCH2), 4.34 (1H, m, SCH2CH), 6.59 (1H, d, H-3, J3,4 = 16.4 Hz), 6.86 (1H, d, H-5′, J5′,6′ = 8.4 Hz), 7.25 (1H, d, H-6′, J5′,6′ = 8.4 Hz), and 8.36 (1H, d, H-4, J3,4 = 16.4 Hz). 13C NMR (CD3OD): δ 22.4 (COCH3), 27.3 (C-1), 37.1 (SCH2), 54.1 (SCH2CH), 117.1 (C-5′), 120.2 (C-6′), 122.2 (C-1′), 126.8 (C-2′), 130.7 (C-3), 144.9 (C-3′), 148.5 and 149.1 (C-4 and C-4′), 173.2 and 173.8 (COCH3 and COOH), and 211.3 (C-2). ESI(+) MS: m/z 340 ([M + H]+). High-resolution MS 340.0816, calcd for C15H18NO6S, 340.0849 (− 3.3 mDa). UV (ethanol) λmax 260 nm (ε 8500) and 342 nm (11850). Di-NAC-DBL (8). 1H NMR data are shown in Figure S5. 1H NMR (CD3OD): δ 1.90 (3H, s, COCH3), 1.93 (3H, s, COCH3) 2.38 (3H, s, H-1), 3.04, 3.18, 3.33, and 3.44 (1H × 4, m, SCH2), 4.36 and 4.54 (1H × 2, m, SCH2CH), 6.70 (1H, d, H-3, J3,4 = 16.0 Hz), 7.43 (1H, s, H6′), and 8.28 (1H, d, H-4, J3,4 = 16.0 Hz). ESI(+) MS: m/z 501 ([M + H]+). High-resolution MS 501.0978, calcd for C20H25N2O9S2, 501.0996 (− 1.8 mDa). UV (ethanol) λmax 276 nm (ε 14720) and 340 nm (11690). The Di-NAC-DBL preparation was found to contain ca. 10% (judged by 1H NMR) of impurity, possibly the 2-S isomer of NAC-DBL (m/z 340). Adduct Formation of RK-quinone (2) and DBL-quinone (3) with BSA. RK (1; 100 μM) or DBL (5; 100 μM) was oxidized by mushroom tyrosinase (50 U/mL) in the presence of BSA (200 μM) in 50 mM sodium phosphate buffer (pH 6.8, 2 mL) at 37 °C. The reaction was monitored with UV/vis spectrophotometry. The reaction products of RK-quinone (2) or DBL-quinone (3) with BSA were analyzed after acid hydrolysis followed by HPLC analysis of the resulting cysteinylcatechol derivatives.22 For this, oxidation in a 1 mL scale was performed in a screw-capped conical glass tube for 2 min and was terminated by adding 10% NaBH4 (100 μL) followed by 10% trichloroacetic acid (1.5 mL). RD was subjected to oxidation following hydrolysis as a positive control, and RK without tyrosinase added was used as a negative control. After cooling at 4 °C for 1 h, the resulting protein precipitate was collected by centrifugation and was washed twice with 5% trichloroacetic acid (1 mL). To the protein precipitate, 6 M HCl containing 1% phenol and 5% thioglycolic acid (1 mL) was added and heated at 110 °C under an argon atmosphere. After 20 h, the reaction mixture was cooled and subjected to alumina extraction. A standard of 100 nmol NAC-RD-catechol22 or NAC-DBL (7) in the presence of BSA (13.2 mg, 200 nmol) was also subjected to hydrolysis following alumina treatment. The acid-washed alumina (50 mg), 1% Na2S2O5−1% Na2EDTA (200 μL) and the hydrolysate (100 μL) were placed in conical plastic tubes. Catechols were adsorbed onto the alumina by adding 2.7 M Tris containing 2% Na2EDTA (pH 9.0, 700 μL) and immediately shaking for 5 min. The alumina was washed three times with water (1 mL), and then catechols were eluted with 0.4 M HClO4 (200 μL). After centrifugation, the supernatant (80 μL) was injected into the HPLC-UVD (see above) and was eluted with a mobile phase of 0.4 M HCOOH/methanol, 60:40 (v/v) and a column temperature at 45 °C monitored with a UV detector at 292 nm for cysteinyl-RD-catechol chloride.22 Pro-oxidant Activity of the RK Oxidation Product, RKOligomer. RK-oligomer and RD-oligomer were prepared in 50 mM sodium phosphate buffer (pH 7.4) from the precursors RK (1 mM) or RD (1 mM) as previously described.21 Tyrosinase (100 U) was added to 1 mL of each precursor solution, and the mixture was incubated at 37 °C for 60 min. Tyrosinase alone (100 U/mL) was used as a control. The oligomer solutions (500 μL) were mixed with 10 mM GSH (50 μL) and were incubated at 37 °C. Periodically, 100 μL aliquots were withdrawn and mixed with 0.4 M HClO4 (800 μL) to terminate oxidation. GSH and GSSG in the oxidation mixtures were analyzed using our HPLC method.21,25 H2O2 was analyzed spectrophotometriC

DOI: 10.1021/acs.chemrestox.7b00006 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology

Figure 2. Time course of tyrosinase-catalyzed oxidation of RK (1). (A) UV/visible spectral changes at pH 6.8. (B) UV/visible spectral changes at pH 5.3. (C) HPLC analysis following the oxidation at pH 6.8, the reaction being stopped by the addition of ascorbic acid followed by HClO4. (D) HPLC analysis following the oxidation at pH 5.3, the reaction being stopped by the addition of ascorbic acid followed by HClO4. Panels A and B were obtained from single experiments, but reproducibility was confirmed for each experiment. Panels C and D were obtained from averages of two independent experiments. cally after reaction with the chromogen Ampliflu Red to form a red pigment having an absorption maximum at 568 nm26 closely following the manual (Invitrogen).

confirm the structure, this compound was prepared by tyrosinase oxidation of RK (1, 1 mM) at pH 5.3, isolated by preparative HPLC, and then identified as 4-(3,4-dihydroxyphenyl)-2-butanone (RK-catechol, 4) by spectroscopic analyses (Figure S1). The structure of RK-catechol (4) was further supported by the identification of a product obtained when the oxidation mixture was reduced by NaBH 4 as 4-(3,4dihydroxyphenyl)-2-butanol (RD-catechol).17,19 This identification of RK-catechol (4) led to confirmation of the structure of the immediate product showing an absorption maximum around 400 nm as 4-(3-oxobutyl)-1,2-benzoquinone (RKquinone, 2). Interestingly, HPLC analysis indicated that another product that had a retention time of 8.5 min appeared at 2 min of the reaction, increased at 5 min, and then gradually decreased over 60 min. That compound (5), when isolated by preparative HPLC, showed an absorption maximum at 342 nm, suggesting an extension of π-conjugation in the catechol structure as compound 2 has an absorption maximum at 284 nm, the difference being 58 nm. This unique feature led us to suggest the structure 5, (E)-4-(3,4-dihydroxyphenyl)-3-buten-2-one, for this compound. This compound, usually called 3,4-dihydroxybenzalacetone (DBL), has been known for its antioxidant activity since its isolation from the mushroom Chaga (Inonotus obliqueus (persoon) Pilat in 2007.27 An authentic sample of DBL (5)28 was kindly provided by Professor Yutaka Nakamura (Niigata University of Pharmacy and Applied Life Sciences). The structure was confirmed by a direct comparison of the product from RK with the authentic sample using HPLC, UV, and 1H and 13C NMR spectra (Figure S2). This identification led to confirmation of the structure of the second product formed from RK-quinone (2) as DBL-quinone (3). The



RESULTS AND DISCUSSION Tyrosinase-Catalyzed Oxidation of RK (1) Produces RK-Quinone (2) and DBL-Quinone (3). The oxidation of RK (1, 100 μM) by mushroom tyrosinase (50 U/mL) was carried out at 37 °C in 50 mM sodium phosphate buffer at pH 6.8. UV/visible spectral changes were followed for 60 min, which showed the appearance of an absorbance maximum near 400 nm, typical of o-quinone, within 2 min (Figure 2A). This absorption feature was gradually replaced by a more monotonous absorption extending to a longer wavelength at 60 min, suggesting an extensive structural change of the initial o-quinone product. As the spectral changes at pH 6.8 were rather complex, we slowed the reactions by performing the oxidation at pH 5.3. As shown in Figure 2B, the changes were slowed down and showed clearer absorption spectra of the oquinone, which remained even at 60 min. As the UV/visible spectral changes were very complex, we then followed the oxidation at pH 6.8 and 5.3 by HPLC using UV detection at 280 nm to detect phenols and catechols.19As most of the products appeared to be unstable quinones, we converted them to their more stable, corresponding catechols by reduction with ascorbic acid. In previous studies, we used NaBH4 to reduce o-quinones,19,24 but in this study, we did not use NaBH4 in order to avoid reduction of the carbonyl group in the side chain. As shown in Figure 2C, RK (1) was consumed at pH 6.8 within 2 min, giving a new compound with a retention time of 7.0 min (RK appeared at 10.0 min) whose structure was assumed to be RK-catechol (4, Figure 1). To D

DOI: 10.1021/acs.chemrestox.7b00006 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology formation of DBL-quinone (3) can proceed through an unstable quinone methide structure that is transformed rapidly to a stable benzalacetone structure by virtue of the electronwithdrawing effect of the carbonyl group (Figure 1). A similar type of dehydrogenation (desaturation) reaction has been reported for the conversion of dihydrocaffeic acid derivatives and 3,4-dihydroxyphenylalanyl amide esters to their dehydro derivatives.29,30 The significance of transient quinone methide intermediates was illustrated in a recent review31 and in our study on the biomimetic transformation of o-quinones from catecholamine metabolites.32 The oxidation of RK-quinone (2) by tyrosinase was then carried out at pH 5.3 to characterize the effects of slowed oxidation. The time course of the oxidation, when stopped by ascorbic acid reduction (Figure 2D), showed that the decay of RK-quinone (2) was greatly suppressed compared to the reaction at pH 6.8 and that DBL-quinone (3) did not accumulate so much (