Bioorganic & Medicinal Chemistry Letters 24 (2014) 2463–2464

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Mechanistic aspects of the tyrosinase oxidation of hydroquinone Christopher A. Ramsden a,⇑, Patrick A. Riley b a b

Lennard-Jones Laboratories, School of Physical and Geographical Sciences, Keele University, Staffordshire ST5 5BG, UK Totteridge Institute for Advanced Studies, The Grange, Grange Avenue, London N20 8AB, UK

a r t i c l e

i n f o

a b s t r a c t

Article history: Received 21 March 2014 Revised 3 April 2014 Accepted 6 April 2014 Available online 13 April 2014

Contradictory reports on the behaviour of hydroquinone as a tyrosinase substrate are reconciled in terms of the ability of the initially formed ortho-quinone to tautomerise to the thermodynamically more stable para-quinone isomer. Oxidation of phenols by native tyrosinase requires activation by in situ formation of a catechol formed via an enzyme generated ortho-quinone. In the special case of hydroquinone, catechol formation is precluded by rapid tautomerisation of the ortho-quinone precursor to catechol formation. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Hydroquinone Tyrosinase ortho-Quinones para-Quinones Tautomerism

Tyrosinase is an important enzyme in the early stages of the biosynthesis of the melanin pigments.1–3 Hydroquinone is used clinically as a depigmenting agent and because of its effect on melanin pigmentation there is interest in tyrosinase oxidation of hydroquinone 1 (R = OH) and its mechanism of depigmentation.4,5 Tyrosinase, which can function as either a monooxygenase or an oxidase, occurs in three active forms, met-, deoxy- and oxytyrosinase, and in a deactivated form deact-tyrosinase.6,7 The inter-relationships of these forms are summarised in Scheme 1. We have previously shown that hydroquinone is not a substrate for native tyrosinase,8 which occurs mainly in the met form. Recent studies9,10 suggest that under different conditions hydroquinone is a substrate for tyrosinase and this has prompted us to re-evaluate the interactions between hydroquinone and tyrosinase. The purpose of this Letter is to provide a mechanistic rationalisation of all the data on tyrosinase oxidation of hydroquinone and thus remove any confusion from the literature. OH

O Tyr

R

R

1

2

O

OH NuH Nu

OH

In addition to the natural substrate L-tyrosine, tyrosinase oxidises a wide variety of monophenols 1 to ortho-quinones 2.11,12 This transformation is catalysed by oxy-tyrosinase which is depleted of oxygen forming deoxy-tyrosinase. Addition of one molecule of oxygen to the copper atoms in deoxy-tyrosinase reforms oxy-tyrosinase and the catalytic cycle continues (Scheme 1). When monophenols are oxidised by native tyrosinase there is a lag period during which the oxidation rate slowly accelerates finally reaching the maximum rate. This lag period arises because native tyrosinase occurs mainly as met-tyrosinase which can oxidise catechols to

orthoquinones

catechols

deoxy-Tyr orthoquinones

met-Tyr orthoquinones

O2

R 3

phenols

oxy-Tyr catechols or resorcinols

⇑ Corresponding author. Tel.: +44 149 488 0043. E-mail address: [email protected] (C.A. Ramsden). http://dx.doi.org/10.1016/j.bmcl.2014.04.009 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

deact-Tyr Scheme 1.

catechols

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C. A. Ramsden, P. A. Riley / Bioorg. Med. Chem. Lett. 24 (2014) 2463–2464

ortho-quinones and is itself reduced to deoxy-tyrosinase, but it cannot oxidise monophenols. Native tyrosinase contains a small amount of oxy-tyrosinase which produces a small amount of ortho-quinone. This reactive product can then add a nucleophile, which in the case of L-tyrosine is the sidechain,13 to give a catechol derivative 3. This catechol then reduces met-tyrosinase leading to more oxy-tyrosinase and the oxidation accelerates until all the met-tyrosinase has been reduced. Oxidation with a preliminary lag period has been observed for many phenols but hydroquinone is an exception. In multiple experiments we have shown that hydroquinone is never oxidised by native mushroom tyrosinase and this led to our conclusion that hydroquinone is not a tyrosinase substrate.8 We now provide a mechanistic rationalisation of the experimental results. In a previous study we have shown that the ortho-quinone 5a, formed by chemical or enzymatic oxidation of (3,4-dihydroxyphenyl)acetonitrile 4a, rapidly isomerises to the thermodynamically more stable para-quinomethane derivative 6a.14 This transformation is facilitated by the acidity of the methylene protons. It is well known that 2-hydroxy-1,4-benzoquinone 6b is much more stable than the prototropic tautomer 4-hydroxy-1,2benzoquinone 5b.15 It follows that the structurally related orthoquinone 5b formed by traces of oxy-tyrosinase in native tyrosinase will also rapidly tautomerise in aqueous buffer (pH 6.8) to the more stable isomer 6b, which is less reactive and does not readily form catecholic products. Consequently, catechol formation by addition of a nucleophile is precluded and the enzyme remains unactivated. The tautomerism 5 ? 6 therefore explains why both (4-hydoxyphenyl)acetonitrile 1 (R = CH2CN) and hydroquinone 1 (R = OH) do not appear to be tyrosinase substrates when exposed to native tyrosinase. OH

OH

O

O

O

XH

XH

X

4

5

6

stoichiometry observed in the presence of a mixture of catechol and hydroquinone8 and explains the long-standing observation of Gregg and Nelson16 that hydroquinone inhibits catechol-induced tyrosinase inactivation since conversion of oxy- to deact-tyrosinase (Scheme 1) is disfavoured by competing phenolic substrates.17 It is also significant to note that the tautomerism 5 ? 6 provides further evidence that tyrosinase oxidation of phenols 1 to ortho-quinones 2 occurs in one step7,13 It is often claimed in the literature that tyrosinase oxidises phenols to catechols and that a second discrete step oxidises the catechol to an ortho-quinone.18,19 If this were the case, native tyrosinase would initially oxidise hydroquinone to 1,2,4-trihydroxybenzene which as a catechol derivative could reduce met-tyrosinase to deoxy-tyrosinase and the tautomerism of the second oxidation product (the ortho-quinone) would be irrelevant. The observations that neither of the phenols 1 (R = OH, CH2CN) are oxidised by native tyrosinase8,14 are entirely consistent with a single step formation of the orthoquinone product 2, which in the special cases of the ortho-quinones 5a,b are removed by tautomerism. In conclusion, contradictory reports on the behaviour of hydroquinone 1 (R = OH) as a tyrosinase substrate are reconciled in terms of the ability of the initially formed ortho-quinone 5b to tautomerise to the thermodynamically more stable para-quinone isomer 6b. In the absence of a reducing agent that can convert met-tyrosinase to deoxy-tyrosinase (e.g., a catechol or H2O2) hydroquinone is not oxidised by native tyrosinase. However, using activated enzyme hydroquinone is a substrate for tyrosinase. The reaction products include 2-hydroxy-1,4-benzoquinone 6b formed by a one-step oxy-tyrosinase oxidation of hydroquinone 1 (R = OH) to 4-hydroxy-1,2-benzoquinone 5b followed by prototropic tautomerism to the product 6b. References and notes

OH

In structure 4-6; (a) X = CHCN, (b) X = O

The tautomerism of initially formed 4-hydroxy-1,2-benzoquinone (5b ? 6b) is entirely consistent with transformations of hydroquinone, observed by ourselves8 and others,9 when carried out under conditions in which tyrosinase activation is achieved by an alternative mechanism. We have shown that in the presence of catechol, L-3,4-dihydoxyphenylalanine or 4-ethylphenol, hydroquinone forms secondary adducts together with 2-hydroxy-1,4-benzoquinone 6b,8 which is stable under the conditions of the experiment. Previously we interpreted the formation of the product 6b by addition of water to 1,4-benzoquinone, formed by redox exchange with an ortho-quinone. We now recognise that this is largely formed via oxidation of hydroquinone by oxy-tyrosinase preactivated by the catecholic components of the reaction mixture. Similarly, Garcia-Cánovas and co-workers have shown that in the presence of hydrogen peroxide,9 which like catechols reduces met-tyrosinase to deoxy-tyrosinase, hydroquinone is a tyrosinase substrate. This interpretation is consistent with the oxygen

1. Prota, G. Melanins and Melanogenesis; Academic Press: San Diego, 1992. 2. The Pigmentary System: Physiology and Pathophysiology; Norlund, J. J., Boissy, R. E., Hearing, V. J., King, R. A., Oetting, W. S., Ortonne, J.-P., Eds., 2nd ed.; Blackwell Publishing: Oxford, 2006. 3. García-Borrón, J. C.; Sánchez, C. O. In Melanins and Melanosomes: Biosynthesis, Biogenesis, Physiological and Pathological Functions; Borovansky, J., Riley, P. A., Eds.; Wiley-VCH GmbH: Weinheim, 2011; p 87. 4. Palumbo, A.; d’Ischia, M.; Misuraca, G.; Prota, G. Biochim. Biophys. Acta 1991, 1073, 85. 5. Palumbo, A.; d’Ischia, M.; Misuraca, G.; Prota, G. Pigm. Cell Res. Suppl. 1992, 2, 299. 6. Land, E. J.; Ramsden, C. A.; Riley, P. A. Acc. Chem. Res. 2003, 36, 300. 7. Ramsden, C. A.; Riley, P. A. Bioorg. Med. Chem. 2014, 22, 2388. 8. Stratford, M. R. L.; Ramsden, C. A.; Riley, P. A. Bioorg. Med. Chem. 2012, 20, 4364. 9. Garcia-Molina, M.; Muñoz-Muñoz, J. L.; Berna, J.; Rodriguez-López, J. N.; Varón, R.; Garcia-Cánovas, F. Biosci. Biotechnol. Biochem. 2013, 77, 2383. 10. Chen, K.; Zhang, Z.-L.; Liang, Y.-M.; Liu, W. Sensors 2013, 13, 6204. 11. Naish-Byfield, S.; Cooksey, C. J.; Latter, A. M.; Johnson, C. I.; Riley, P. A. Melanoma Res. 1991, 1, 273. 12. Selinheimo, E.; Gasparetti, C.; Mattinen, M.-L.; Steffensen, C. L.; Buchert, J.; Kruus, K. Enzyme Microb. Technol. 2009, 44, 1. 13. Cooksey, C. J.; Garratt, P. J.; Land, E. J.; Pavel, S.; Ramsden, C. A.; Riley, P. A.; Smit, N. P. M. J. Biol. Chem. 1997, 272, 26226. 14. Land, E. J.; Ramsden, C. A.; Riley, P. A.; Yoganathan, G. Tetrahedron 2003, 59, 9547. 15. The Chemistry of the Quinonoid Compounds; Patai, S., Ed.; Wiley & Sons Ltd: London, 1974. 16. Gregg, D. C.; Nelson, J. M. J. Am. Chem. Soc. 1940, 62, 2510. 17. Land, E. J.; Ramsden, C. A.; Riley, P. A. Tohoku J. Exp. Med. 2007, 212, 263. 18. Citek, C.; Lyons, C. T.; Wasinger, E. C.; Stack, T. D. P. Nat. Chem. 2012, 4, 317. 19. Fujieda, N.; Yabuta, S.; Ikeda, T.; Oyama, T.; Muraki, N.; Kurisu, G.; Itoh, S. J. Biol. Chem. 2013, 288, 22128.

Mechanistic aspects of the tyrosinase oxidation of hydroquinone.

Contradictory reports on the behaviour of hydroquinone as a tyrosinase substrate are reconciled in terms of the ability of the initially formed ortho-...
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