ARTICLE pubs.acs.org/crt
Rapid Halogen Substitution and Dibenzodioxin Formation during Tyrosinase-Catalyzed Oxidation of 4-Halocatechols Michael R. L. Stratford,† Patrick A. Riley,‡ and Christopher A. Ramsden*,§ †
Gray Institute for Radiation Oncology & Biology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K. ‡ Totteridge Institute for Advanced Studies, The Grange, Grange Avenue, London N20 8AB, U.K. § Lennard-Jones Laboratories, School of Physical and Geographical Sciences, Keele University, Staffordshire ST5 5BG, U.K.
bS Supporting Information ABSTRACT: 4-Fluoro-1,2-benzoquinone, generated by tyrosinase oxidation of 4-fluorocatechol in aqueous buffer, rapidly undergoes substitution by O-nucleophiles (water or catechols) with release of fluoride. 4-Chloroand 4-bromocatechol behave similarly. The reactions, which have toxicological implications, have been monitored by spectrophotometry and HPLC/MS, and intermediate and final products, including dibenzodioxins, identified.
’ INTRODUCTION Quinones represent a class of toxicological intermediates that can cause a variety of hazardous effects in vivo, including acute cytotoxicity, immunotoxicity, and carcinogenesis.1 Quinones possess several reactivities, and the mechanisms by which they cause toxic effects may involve complex pathways. We have had a long-standing interest in the toxicity of ortho-quinones generated by tyrosinase [E.C.1.14.18.1] activity, in particular because of the possibility of utilizing the action of quinones in selective toxicity to pigment cells in targeted chemotherapy for disseminated malignant melanoma. It was shown that the major cytotoxic product of the noncyclizing tyrosinase substrate 4-hydroxyanisole is the corresponding ortho-quinone,2,3 and the use of melanogenesis as a targeting strategy in melanoma was proposed.4 A series of investigations showed that the acute cytotoxicity is due to rapid reaction of the tyrosinase-generated ortho-quinones with cellular thiols,5-8 and, because the cellular thiol concentration is high, the efficiency of various noncyclizing tyrosine analogues as antimelanoma agents is limited. Alternative cytotoxic strategies based on the pigment cell specificity of tyrosinase have been studied, including utilizing the spontaneous cyclization of ortho-quinones with appropriate side chain structure9-11 to release cytotoxic agents from prodrugs,12 and a variety of other targeting mechanisms.13,14 In this article, as part of our continuing interest in ortho-quinone toxicity, we describe the results of an investigation of the tyrosinase-catalyzed oxidation of 4-fluoro-, 4-chloro-, and 4-bromocatechols and the potentially toxic consequences of 4-halo-ortho-quinone formation. In a previous study, related to the suicide inhibition of tyrosinase by catechols,15-17 we demonstrated the clean rearrangement of 4-nalkyl-ortho-quinones 2 to para-quinomethanes 3 over a period of 1020 min in aqueous buffer.18 Fresh tyrosinase rapidly oxidizes 4-nalkylcatechols 1 to the corresponding ortho-quinones 2 (Scheme 1). Monitoring the UV-vis spectrum of the reaction solution at 30 s r 2011 American Chemical Society
intervals shows a steady decay of the initially formed orthoquinone 2 (λmax 400 nm) with well-defined isosbestic points corresponding to the formation of the para-quinone isomer 3. When 4-fluorocatechol 4a was used as substrate, similar behavior was observed, with an ortho-quinone chromophore (λmax 410 nm) decaying over 10 min. Since 4-fluoro-ortho-quinone 5a cannot rearrange to a quinomethane, another transformation must account for the relatively fast decay of the ortho-quinone 5a. Scheme 1
Received: September 14, 2010 Published: February 09, 2011 350
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Tyrosinase is unique in that it oxidizes both phenols (oxygenase activity) and catechols (oxidase activity) to ortho-quinones, via subtly different mechanisms.19-24 The natural substrate for the enzyme is tyrosine 6g. The resulting ortho-quinone 5g rapidly cyclizes leading in several steps to melanin pigments. Since tyrosinase, and related enzymes, can oxidize a wide variety of phenol and catechol substrates, the formation of reactive orthoquinones, which cannot rapidly cyclize, from xenobiotic substrates has toxicological implications. 4-Benzyloxyphenol 6d is a potent skin-depigmenting agent which induces permanent loss of melanocytes and onset of occupational vitiligo. This condition can also be induced by other industrial chemicals such as 4-hydroxyanisole 6e and 4-tert-butylphenol 6f. These phenols are all oxidized by tyrosinase, and a possible mechanism leading to vitiligo is covalent bonding of the ortho-quinones 5d-f to a protein, possibly tyrosinase, resulting in the formation of a neo-antigen.25 Since haloquinones are bifunctional species that could similarly lead to antigen formation or take part in other potentially deleterious reactions, we have undertaken an investigation of the products of halocatechol decay. The results, which we now describe, demonstrate extremely fast displacement of halide from the ortho-quinones 5a-c by oxygen nucleophiles, including water, followed by nucleophilic addition to the resulting orthoquinones 7 to give products that include potentially toxic dibenzodioxins (Scheme 2).
linear gradient to 75% over 5 min. Bromide was separated on a Dionex AS4ASC column (250 4 mm) and AS4ASC guard column (50 4 mm) using eluents A, water, and B, 70 mM Na2B4O7. Starting conditions were 7% B, followed by a linear gradient to 40% B over 8 min. The flow rate was 1.5 mL min-1. HPLC/MS. Separations were carried out using a Waters 2695 separations module (Waters, Elstree, UK) with an Atlantis dC18, 150 3.0 mm HPLC column (Waters) maintained at 35 °C. The HPLC eluents were as follows: A, 10 mM formic acid; B, acetonitrile, with gradients of 10-65% B (F), 10-70% B (Cl), and 20-70% B (Br) over 5 min. The flow rate was 0.5 mL min-1, which was split after diode array detection to give a flow rate of ∼200 μL min-1 to the mass spectrometer. The eluent was monitored using a Waters 2996 diode array detector (215-450 nm, 2.4 nm resolution) and a Waters Micromass ZQ mass detector (mass range 115500 Da) in negative ion mode using electrospray ionization, and using Waters Empower 2 software. The mass detector employed the following conditions: capillary voltage, 2.1 kV; cone voltage, 20 V; source temperature, 120 °C; desolvation temperature, 425 °C; desolvation gas flow, 450 L h-1; cone gas flow, 100 L h-1. The major product 16 (X = Cl) from the oxidation of 4-chlorocatechol by tyrosinase was isolated as follows: 4-chlorocatechol (43 mg) was dissolved in 50 mM sodium phosphate buffer, pH 6.8 (500 mL), and tyrosinase (1 mg/ mL) (1 mL) was added with stirring. This mixture was incubated at room temperature (24 h) and the product isolated in 35% acetonitrile and 10 mM formic acid by semipreparative HPLC. After the removal of acetonitrile in a centrifugal evaporator, the aqueous sample was applied to a C18 solid phase extraction cartridge and eluted with acetonitrile which was removed in a centrifugal evaporator to give an amorphous solid (25 mg).
’ EXPERIMENTAL PROCEDURES General. 4-Methylcatechol was supplied by TCI Europe N.V., Belgium. 3-Fluorocatechol, 4-fluorocatechol, and 4-chlorocatechol were obtained from Sigma-Aldrich, Poole, Dorset, U.K. 4-Bromocatechol was synthesized by the method of Ding and co-workers.26 Tyrosinase (ex Agaricus bisporus) was obtained from Sigma-Aldrich. This material migrates as a single electrophoretic band27 and was used without further purification. A standard solution of 300 units/mL was made up in 0.1 M phosphate buffer (pH 6.3) and stored at 4 °C prior to use. Combined Oximetry and Spectrophotometry. Experiments were conducted at room temperature (24 °C) using the apparatus previously described.28 The test catechol was dissolved in deionized water and added to 0.1 M phosphate buffer (pH 6.3) in a silica cuvette positioned in a Hewlett-Packard diode-array spectrophotometer (Model 8452A) and fitted with a Clark-type polarimetric electrode attached to a Yellow Springs Instruments oxygen meter (Model 5300). Tyrosinase was added to the mixture through a narrow orifice in a stopper and the oxygen utilization and spectral changes recorded. Unless otherwise stated, the catechol concentration was 600 μM. For kinetic investigations of halogen release, sample aliquots of 100 μL were withdrawn from the spectrophotometer cuvette at intervals and the reaction stopped by 1:1 dilution in acetonitrile. Experiments to measure total halogen release were conducted in 2 mL ampules using 1 mL of buffer containing the halocatechol substrate at a concentration of 740 μM. Tyrosinase in varying amounts (1 to 20 units, see details below) was added to the vials and samples analyzed after 5 min of incubation. Halogen Release Assay. The quantification of the halogen released was performed by ion chromatography using a Waters 616 gradient pump, a Waters 717 autosampler (Waters, Elstree, UK), and a Dionex ED40 detector and ASRS suppressor run in external water mode. Fluoride was separated on a Dionex AS12A column (250 4 mm) and an AG12A guard column (50 4 mm) using eluents A, water, and B, 50 mM Na2B4O7 and 37.5 mM NaOH. Starting conditions were 15% B held for 1 min, followed by a linear gradient to 75% B over 7 min. The flow rate was 1.5 mL min-1. Chloride was separated using the same column and eluents, but with a 40% start and 1 min hold followed by a
’ RESULTS Combined Oximetry and Spectrophotometry. Each of the halocatechols investigated, 4-fluoro-, 4-chloro-, and 4-bromocatechol, was oxidized by tyrosinase with utilization of oxygen and giving rise to ortho-quinone species absorbing in the region of 400 nm. The rates of oxygen utilization varied according to the halogen substituent in the order: F > Cl > Br. Halogen release, investigated by ion chromatography, was exhibited in each case. Catechols are known to slowly inactivate tyrosinase by a mechanism that is believed to be associated with the reduction of copper in the active site; a process known a suicide inactivation.15-17,29 Tyrosinase-catalyzed oxidation of the halocatechols exhibited the characteristic suicide-inactivation kinetics, and, in the case of 4-fluorocatechol, it was shown that this inactivation was not due to halide release since the activity of the enzyme against the standard substrate (4-methylcatechol) was unaffected by fluoride addition up to 10 times the concentration present in the substrate. Because of the suicide inactivation concomitant with catechol oxidation by tyrosinase, the total extent of oxidation can be determined by the initial amount of enzyme added; in the case of 4-fluorocatechol, the total oxygen utilized is approximately 20 nmol/unit.1 Using different amounts of tyrosinase (between 1.5 and 9 units/mL), it was shown that halogen release was linearly dependent on oxygen uptake (see Figure 1). Investigation of the initial reactions in the case of 4-fluorocatechol showed that the kinetics of fluoride release closely follows the oxygen uptake (see Figure 2). However, the absorbance at 410 nm showed different kinetics (Figure 3), and in all cases, the observed quinone spectrum was relatively stable and decayed slowly over a period of tens of minutes. The first order decay of the 410 nm peak was estimated as 8.0 10-2 AU min-1 in the case of 4-fluorocatechol and 1.9 10-2 AU min-1 in the case 351
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Figure 1. Relationship between fluoride release and oxygen utilization. The chart shows the data for fluoride released from the oxidation of 4-fluorocatechol (600 μM) as a function of the oxygen utilized in the presence of 1.5, 3, 6, and 9 units/mL tyrosinase. The calculated slope is 1.91 with a coefficient of correlation of 0.9936.
Figure 3. Time pattern of generation and decay of the quinone products of 4-fluorocatechol oxidation by tyrosinase (10 units). The inset shows the absorbance spectra recorded at 30 s. intervals and the time trace charts the change in absorbance at 410 nm.
Scheme 2
is much slower. These proposed initial reactions are consistent with the mass spectrometric data. We attribute halogen release from 4-haloquinones to rapid nucleophilic substitution either by water or by a catecholic OH (5 f 7, Scheme 2). A number of products are theoretically possible including a hydroxylated monomer 8 and several dimers 9-17. Mechanistic considerations lead us to propose four pathways leading to the major products, and these are summarized in Scheme 3. The products indicated in square brackets (9, 11, 12 and 14) are not observed, and we deduce that further reaction of these species occurs rapidly. Mass Spectrometry. The 4-halocatechol oxidation products were examined using LC/MS. The incubations were made in 1 mL of 0.1 M phosphate buffer (pH 6.3). Sufficient tyrosinase was added to limit the oxidation to the total available dissolved oxygen in the buffer (approximately 240 μM). Because of the kinetic differences both in the rate of oxidation and the subsequent reactions, a range of conditions was examined with the three halocatechols, and the optimal conditions for comparison were those in which the substrate was in slight excess of the oxidation limit, i.e., 600 μM. Under these conditions, it was possible, by scanning specifically for the molecular ion corresponding to quinone 8 (m/z 123), to demonstrate small quantities of this dehalogenated product 8 of 4-fluorocatechol (RT 2.7 min). However, the main products were identified as the peaks a-g in the chromatograms shown in Figures 4 and 5.
Figure 2. Comparative kinetics of oxygen utilization (0) and fluoride release (2). The initial 4-fluorocatechol concentration was 660 μM, and 10 units of enzyme were added at the time point indicated. Suicide inactivation of the enzyme limited the total extent of oxygen utilization to about 40 μmol, i.e., equivalent to the generation of about 80 μmol of quinone. The fluoride assays were made on 100 μL aliquots of the reaction mixture withdrawn at the time points indicated and the reaction stopped by dilution 1:1 in acetonitrile.
of 4-chlorocatechol. As the absorbance observed is due to the initial 4-halo-ortho-quinone product of oxidation and the subsequent quinone product of the halogen substitution reaction, the relatively slow oxidation of the 4-bromocatechol prevented the accurate assessment of the quinone decay constant for this substrate. These results indicate that the release of halide is consistent with rapid halogen substitution of the initial ortho-quinone product, while the decay of the resulting ortho-quinone chromophore (Scheme 2) 352
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Table 1. Characteristics of Elution Products of TyrosinaseCatalyzed Oxidation of 4-Halocatechols 4a 4-F-catechol peak
RT
a
m/z abs RT
4-Cl-catechol
4-Br-catechol
m/z
m/z
abs RT
abs structure
4.78 143/145 386 4.65 187/189 384
5
b
5.23
127 280 5.94 143/145 285 5.65 187/189 285
4
c
5.51 5.67
251 290 5.86 267/269 292 5.51 311/313 295
17
d
6.32
249 278 6.62 265/267 278 6.23 309/311 278
16
e
6.40
249 280 6.77 265/267 280 6.59 309/311 278
15
f
6.86
265 394 7.24 281/283 398 6.97 325/327 396
13
g
7.54
233 304 7.79 249/251 304 7.47 293/295 306
10
a
The principal products, identified as the peaks a-g in the absorbance chromatograms, are characterized by their retention times in minutes (RT), their mass values (m/z) in negative electrospray mode (M H), and their absorbance in nm (abs). The putative structures are indicated in the final column (see Scheme 3).
ortho-quinone 5, which was also seen, to a lesser extent, in the chromatograms of the 4-chlorocatechol incubation products but was not observed in the products of the 4-fluorocatechol incubation. Note that, while the absorbance data clearly identified these products as quinones, the mass detected was identical to the substrate. We have observed this in several instances, and it is attributable to the effect of negative electrospray, which, under conditions employed here, is able to reduce quinones with relatively low reduction potentials. The failure to observe the ortho-quinone product in the case of the 4-fluorocatechol oxidation is attributed to the greater ease of fluoride substitution. The relative stability of 4-bromoquinone is attributed to the comparatively slow substitution rate of the bromine, and this relatively slower rate of reaction was associated with the generation of a number of additional minor products not seen with the other halogen-substituted compounds. For example, a product with m/z 201/203 coeluted with peak a at later incubation times, and peak e also included coeluting products with m/z 413/415 and 495/497/499; the latter product contains two bromine atoms, deduced from the isotopic pattern, and is probably a trimer. Also, in the bromine series, a minor product with m/z 413/415 was present in peak f. In the case of the oxidation products of 4-fluorocatechol, a double peak (indicated by c in the chromatogram obtained at 140 min of incubation; see Figure 5) indicated the accumulation of isomeric products which we ascribe to the diarylethers 17 formed by the alternative minor reaction 8B þ 4 (see Scheme 3). For the chloro- and bromocatechols, the retention time of c is very close to that of the parent catechol b (Table 1), and only one peak could be seen in the absorbance or total ion chromatograms. However, extracting similarly timed chromatograms at m/z 269 (4-chlorocatechol) or m/z 313 (4-bromocatechol) revealed two closely eluting peaks in each case corresponding to the isomers of 17 (data not shown). The relative proportions of the products varied with time and the nature of the halogen, but in all cases, the ultimate major product is peak d (see Figure 5) to which we assign the dibenzodioxin structure 16 for which two isomeric forms are possible. Peak e we attribute to a relatively stable precursor of the final product 16, and, on the basis of its absorption spectrum, this is believed to be the para-quinone intermediate 15. Peak f is attributed to ethers 13, which cannot cyclize, and in all cases
Figure 4. Chromatograms of tyrosinase oxidation products. The panels show the chromatograms of (A) 4-fluorocatechol after 1 min of incubation, (B) 4-chlorocatechol after 10 min, and (C) 4-bromocatechol after 21 min. Equivalent elution fractions in the chromatograms are designated peaks a-g corresponding respectively to the putative structures 5, 4, 17, 16, 15, 13, and 10 shown in Scheme 3. Peak a was not evident in the 4-fluorocatechol products, but the existence of the corresponding ortho-quinone 5a was inferred from the other products. The properties of the products are summarized in Table 1.
Figure 5. Chromatogram of tyrosinase oxidation products of 4-fluorocatechol at 140 min of incubation.
Peak b was identified as the parent substrate 4 and the earliereluting peak a, which was clearly observed in the case of 4-bromocatechol oxidation, identified as the corresponding 353
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Scheme 3a
a
X = F, Cl, Br.
exhibited a clear absorbance spectrum, peaking near 400 nm, characteristic of ortho-quinones. In the bromocatechol incubation, a significant amount of a product with m/z 413/415 was also detected. Peak g in all cases corresponds to a compound with the structure 10, formed by cyclization of the ethers 9 (Scheme 3). Since the dibenzodioxin derivatives 16 are the major tyrosinase oxidation products from all three halocatechols, we attempted the isolation of the product 16 (X = Cl). Since there are alternative modes of nucleophilic attack of the catechol on quinone 8, and the proposed intermediate 15 may undergo a Smiles rearrangement prior to cyclization, the final product 16 is almost certainly a mixture of constitutional isomers. Semipreparative HPLC on a batch of 4-chlorocatechol (43 mg) incubated with tyrosinase for 24 h gave, after centrifugal evaporation, an amorphous solid (∼25 mg) that corresponded to band d (Figure 5). Examination of the 1H NMR spectrum of a d6DMSO solution showed a multiplet at ∼6.7 ppm corresponding to three coupled aromatic protons and two sharp singlets at ∼5 ppm together accounting for one aromatic proton. We attribute these singlets to the protons in the 3 positions of the 1,2,4trihydroxy ring of structure 16. The chemical shift is attributed to the electron-rich nature of the penta-oxy ring together with the antiaromatic character of the adjacent 1,4-dioxin ring.30 The almost equal intensities (4:5) of these singlets suggests that the constitutional isomers 16 (X = Cl) occur in almost equal amounts. The hydroxy protons are seen as a broad signal at
∼4 ppm. The sample was contaminated by a small amount of 4-chlorocatechol, which is probably formed by oxidative decomposition of the product 16. The mass spectrum of product 16 (X = Cl) at a cone voltage of 20 V showed only the molecular ion [M - H-](m/z 265/7). At 60 V, in addition to the molecular ion, a strong fragment ion at m/z 143/5 is observed, corresponding to a retro-Diels-Alder fragmentation of the molecular ion.
’ DISCUSSION We conclude that in aqueous buffer, 4-halo-ortho-quinones 5a-c, generated by tyrosinase oxidation of the corresponding catechols 4a-c, undergo very rapid substitution by O-nucleophiles with halide release (X-). Fluoride release is particularly fast suggesting that addition of the nucleophile to the orthoquinone is the rate determining step rather than subsequent cleavage of the strong C-F bond. Both of these steps are very fast, and the resulting 4-O-substituted-ortho-quinones 7 react further over a period of 10-30 min (Scheme 2). The formation of dibenzodioxins by halide substitution of 4-chloro-ortho-quinone was reported by Frejka and coworkers,31 and fluoride release from phenols, including fluorotyrosine, by the action of tyrosinase has been previously observed.32,33 Phillips and co-workers32 proposed that fluoride release from 6-fluoro-dopaquinone was the result of intramolecular nucleophilic substitution during cyclization to form cyclodopa: no fluoride release occurred from 3-fluoro-dopa when oxidized by tyrosinase. In the intermolecular reactions that we report here, the nucleophile is a catecholic hydroxyl group or water. The relative amounts of the products identified shows that, despite the great concentration of water, the predominant reactions are with the residual catechol in the incubation mixture. Our data are consistent with the results of Battaini and coworkers who studied tyrosinase oxidation of halophenols.33 We also observed fluoride release from 3-fluorocatechol with the formation of a product with m/z 233, consistent with the generation of a dimer by nucleophilic substitution. The possible therapeutic utilization of halide release by tyrosinase-catalyzed oxidation has been noted31 but, to our knowledge, has not been applied to targeted antimelanoma treatment. From the toxicological viewpoint, it is possible that the susceptibility of halogen substituted ortho-quinones to nucleophilic substitution alters the pattern of their metabolism by the action of quinone reductases and excretion through conjugation, and these pharmacokinetic implications may be of some interest. In particular, the formation of quinone derivatives gives rise to neoantigens25 capable of eliciting immune responses as well as direct quinone toxicity.1,34,35 The demonstration of fluoride release is potentially a tool for implicating ortho-quinone intermediates in synthetic or metabolic pathways,36 and the ortho-quinone activation of halogenated aromatic carbons might possibly be utilized in synthesis as a technique for site-directed nucleophilic attack. Some benzodioxins are carcinogenic: 2,3,7,8-tetrachlorobenzodioxin (TCDD) is a class 1 carcinogen. There appear to be several mechanisms whereby ortho-quinones may be involved in carcinogenesis. The facile generation of semiquinones and the formation of reactive oxygen species (ROS) may cause oxidative stress with the production of not only oxidized lipids and proteins but also oxidized nucleic acid bases, such as 8-oxodeoxyguanosine, which have been associated with mutagenesis in aging and carcinogenesis. Moreover, ROS are able to activate several signaling pathways, including protein kinase C and Ras.1 354
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Other mechanisms of cytotoxicity implicate quinone arylation which involves activation of signaling pathways such as the pancreatic endoplasmic reticulum kinase (PERK) C/EBP homologous protein (CHOP) induction.37 Interestingly, catechol estrogen 3,4-quinones have been implicated as initiators of breast, prostate, and other cancers through the formation of depurinating adducts.38,39 The relative ease with which the products of halogen-substituted catechols form adducts suggests that the generation of dibenzodioxin derivatives from polyhalocatechols may have toxicological and occupational health implications.
(11) Land, E. J., Perona, A., Ramsden, C. A., and Riley, P. A. (2009) Dopamine quinone chemistry: a study of the influence of amide, amidine and guanidine substituents [-NH-CX-Y] on the mode of reaction. Org. Biomol. Chem. 7, 944–950. (12) Jordan, A. M., Khan, T. H., Malkin, H., Osborn, H. M., Photiou, A., and Riley, P. A. (2001) Melanocyte-directed enzyme prodrug therapy (MDEPT): development of second generation prodrugs for targeted treatment of malignant melanoma. Bioorg. Med. Chem. 9, 1549– 1558. (13) Land, E. J., Ramsden, C. A., and Riley, P. A. (2006) Toxicological Aspects of Melanin and Melanogenesis, in The Pigmentary System: Physiology and Pathophysiology (Nordlund, J. J., Boissy, R. E., Hearing, V. J., King, R. A., Oetting, W. S., and Ortonne, J.-P.) 2nd ed., pp 354394, Blackwell, Oxford, U.K. (14) Riley, P. A. (2004) Melanoma and the problem of malignancy. Tohoku J. Exp. Med. 204, 1–9. (15) Land, E. J., Ramsden, C. A., and Riley, P. A. (2007) The mechanism of suicide-inactivation of tyrosinase: a substrate structure investigation. Tohoku J. Exp.Med. 212, 341–348. (16) Land, E. J., Ramsden, C. A., Riley, P. A., and Stratford, M. R. L. (2008) Evidence consistent with the requirement of cresolase activity for suicide inactivation of tyrosinase. Tohoku J. Exp.Med. 216, 231–238. (17) Ramsden, C. A., Stratford, M. R. L., and Riley, P. A. (2009) The influence of catechol structure on the suicide-inactivation of tyrosinase. Org. Biomol. Chem. 7, 3388–3390. (18) Land, E. J., Ramsden, C. A., Riley, P. A., and Stratford, M. R. L. (2008) Studies of para-quinomethane formation during the tyrosinasecatalyzed oxidation of 4-alkylcatechols. ARKIVOC (ii) 258–267. (19) Mason, H. S., Fowlks, W. L., and Peterson, E. (1955) Oxygen transfer and electron transport by the phenolase complex. J. Am. Chem. Soc. 77, 2914–2915. (20) Pomerantz, S. H. (1966) The tyrosine hydroxylase activity of mammalian tyrosinase. J. Biol. Chem. 241, 161–168. (21) Mason, H. S. (1955) In Advances in Enzymology (Nord, F. F., Ed.) Vol. 16, pp 105-184, Interscience Publishers Inc., New York. (22) Lerner, A. B., Fitzpatrick, T. B., Calkins, E., and Summerson, W. H. (1949) Mammalian tyrosinase: preparation and properties. J. Biol. Chem. 178, 185–195. (23) Lerch, K. (1981) Copper monooxygenases: tyrosinase and dopamine γ-hydroxylase. In Metal Ions in Biological Systems, Vol. 13. (Sigel, H., Ed.) pp 143-186, Marcel Decker, New York. (24) Land, E. J., Ramsden, C. A., and Riley, P. A. (2003) Tyrosinase autoactivation and the chemistry of ortho-quinone amines. Acc. Chem. Res. 36, 300–308. (25) Manini, P., Napolitano, A., Westerhof, W., Riley, P. A., and d’Ischia, M. (2009) A reactive ortho-quinone generated by tyrosinasecatalysed oxidation of the skin-depigmenting agent monobenzone: selfcoupling and thiol-conjugation reactions and possible implications for melanocyte toxicity. Chem. Res. Toxicol. 22, 1398–1405. (26) Ding, Y.-S., Shiue, C.-Y., Fowler, J. S., Wolf, A. P., and Plenevaux, A. (1990) No-carrier-added (NCA) aryl[18F]fluorides via the nucleophilic aromatic substitution of electron-rich aromatic rings. J. Fluorine Chem. 48, 189–206. (27) Naish-Byfield, S., and Riley, P. A. (1998) Tyrosinase autoactivation and the problem of the lag period. Pigm. Cell Res. 11, 127–133. (28) Cooksey, C. J., Garratt, P. J., Land, E. J., Pavel, S., Ramsden, C. A., Riley, P. A., and Smit, N. P. M. (1997) Evidence of the indirect formation of the catecholic intermediate substrate responsible for the autoactivation kinetics of tyrosinase. J. Biol. Chem. 272, 26226– 26235. (29) Ramsden, C. A., and Riley, P. A. (2010) Mechanistic studies of tyrosinase suicide inactivation. ARKIVOC (i) 260–274. (30) Katritzky, A. R., Ramsden, C. A., Joule, J. A., and Zhdankin, V. V. (2010) Handbook of Heterocyclic Chemistry 3rd ed., Elsevier, Oxford, U.K. (31) Frejka, J., Sefranek, B., and Zika, J. (1937) Nouveau mode de preparation des derives halogens du dioxyde de dioxy-di-o-phenylene. Collect. Czech. Chem. Commun. 9, 238–246.
’ ASSOCIATED CONTENT
bS
1
H NMR spectrum of isomers 16 (X = Cl). This material is available free of charge via the Internet at http://pubs.acs.org. Supporting Information.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
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