Catechol, tyrosinase-catalyzed oxidation

Activation mechanism for tyrosinase-catalyzed oxidation of catechol with 02... 

Generally, the regioselective formation of o-quinones has been known to be accomplished by using polyphenol oxidase in chloroform and not in water, because of rapid inactivation of the enzyme in water. However, catechol underwent mushroom tyrosinase-catalyzed oxidation in phosphate buffer (pH 6.8) containing 4-hydroxycoumarin (322) to afford 321 in 96% yield, as shown in Scheme 69. 

The results of the experiments just described provide strong, although indirect, evidence for quinone formation during the conversion of dopamine to norepinephrine and during the conversion of phenylethylamine to phenylethanolamine (in the presence of catechol). The spectral changes which occur during the latter reaction were found (Levin and Kaufman, 1961) to be virtually identical with those reported for the tyrosinase-catalyzed oxidation of pyrocatechol to o-benzoquinone (Mason, 1949) and this result provides direct evidence in favor of the view that Eq. (19) correctly describes the hydroxylation of phenylethylamine in the absence of ascorbate. 

In 1996, the first successful combination of an enzymatic with a nonenzymatic transformation within a domino process was reported by Waldmann and coworkers [6]. These authors described a reaction in which functionalized bicy-clo[2.2.2]octenediones were produced by a tyrosinase (from Agaricus bisporus) -catalyzed oxidation of para-substituted phenols, followed by a Diels-Alder reaction with an alkene or enol ether as dienophile. Hence, treatment of phenols such as 8-1 and an electron-rich alkene 8-4 in chloroform with tyrosinase in the presence of oxygen led to the bicyclic cycloadducts 8-5 and 8-6 in moderate to good yield (Scheme 8.1). It can be assumed that, in the first step, the phenol 8-1 is hydroxylated by tyrosinase, generating the catechol intermediate 8-2, which is then again oxidized enzy-... 

While only tyrosinase catalyzes the ortho-hydroxylation of phenol moieties, both tyrosinase and catechol oxidase mediate the subsequent oxidation of the resulting catechols to the corresponding quinones. Various mono- and dinu-clear copper coordination compounds have been investigated as biomimetic catalysts for catechol oxidation [21,194], in most cases using 3,5-di-tert-butylcatechol (DTBC) as the substrate (Eq. 16). The low redox potential of DTBC makes it easy to oxidize, and its bulky tert-butyl groups prevent un-... 

Tyrosinase. The oxidation of catechol with 02, catalyzed by tyrosinase, was concluded by Mason (I) in 1961 not to involve any radical species therefore, an ionic mechanism was proposed by Hamilton (2). A possible activation mechanism seems to involve the interaction between Cu(I)-protein and 02 to give an active Cu(II)—O—OH species in which Cu(II) and OH act as electron-deficient centers, withdrawing electrons from the substrate (Figure 1). 

A conducting, polymeric film of poly(indole-5-carboxylic acid) has been employed for covalent immobilization of tyrosinase, which retains catalytic activity and catalyzes oxidation of catechol to the quinone <2006MI41>. Poly(l-vinylpyrrole), polyfl-vinylindole), and some methyl-substituted compounds of poly(l-vinylindole) are of potential interest as photorefractive materials with a relatively low glass-transition temperature and requiring a lower quantity of plasticizer in the final photorefractive blend <2001MI253>. Polymers of 5,6-dihydroxyindoles fall within the peculiar class of pigments known as eumelanins and their chemistry has been reviewed <2005AHC(89)1>. 

A apart. Dioxygen binding results in the two-electron reduced peroxide in a side-on bound dicopper(II) structure (Fig. 1). Crystallographic information is (also) established for the reduced, as well as the oxidized met forms (3-5). Based on their spectroscopic similarities, a similar active-site structure is proposed for the monooxygenase tyrosinase (Tyr) X-ray structures are available for catechol oxidase (CO) depicting a similar active-site structure to He and Tyr (6). However, unlike the reversible binding reactivity of hemocyanin, tyrosinase catalyzes the aromatic hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (l-DOPA), and can further perform catechol oxidase activity which oxidizes the catechol to the quinone (Fig. 2) (7). 

Tyrosinases from various sources also catalyze the hydroxylation of phenols to catechols and the oxidation of catechols to o-quinones [139]. 

Cosnier and Innocent [135] recognized tliat one of the problems with incorporation of enzymes from solutions into CP films during electropolymerization was that the concentration of the enzyme in the CP film could only be approximately controlled. They took a derivatized pyrrole monomer containing a surfactant group (Eq. (17.211 which has a strong affinity for the enzyme tyrosinase within specific pH ranges. A film of the monomer-tyrosinase complex, with predetermined monomer-tyrosinase concentrations, was cast from solution onto a Pt electrode. The electrode was then removed from this solution and immersed in a second electrolyte for polymerization of the adsorbed monomer-tyrosinase complex, yielding a P(Py)-derivative/tyrosinase film. Tyrosinase catalyzes the oxidation of monophenols and o-diphenols to o-quinones in the presence of O2. The sensor could thus be used for detection of such analytes as catechol, as shown in Fig. 17-12. Response times were of the order of seconds. 

Tyrosinase is a monooxygenase which catalyzes the incorporation of one oxygen atom from dioxygen into phenols and further oxidizes the catechols formed to o-quinones (oxidase action). A comparison of spectral (EPR, electronic absorption, CD, and resonance Raman) properties of oxy-tyrosinase and its derivatives with those of oxy-Hc establishes a close similarity of the active site structures in these proteins (26-29). Thus, it seems likely that there is a close relationship between the binding of dioxygen and the ability to "activate" it for reaction and incoiporation into organic substrates. Other important copper monooxygenases which are however of lesser relevance to the model studies discussed below include dopamine p-hydroxylase (16,30) and a recently described copper-dependent phenylalanine hydroxylase (31). 

Tyrosinase, a copper-containing oxidoreductase, catalyzes the orthohydroxy-lation of monophenols and the aerobic oxidation of catechols. The enzyme activity will be assayed by monitoring the oxidation of 3,4-dihydroxyphenyl-alanine (dopa) to the red-colored dopachrome. The kinetic parameters Ku and Vmax will be evaluated using Lineweaver-Burk or direct linear plots. Inhibition of tyrosinase by thiourea and cinnamate will also be studied. Two stereoisomers, L-dopa and D-dopa, will be tested and compared as substrates. 

Figure 2.21. Mechanisms of the oxidative polymerization of catechol to melanins (humic polymers) in the presence of tyrosinase or birnessite. Reprinted with permission from Naidja, A., Huang, P. M., Dec, J., and Bollag, J.-M. (1999). Kinetics of catechol oxidation catalyzed by tyrosinase or 8-Mn02. In Effect of Mineral-Organic-Microorganism Interactions on Soil and Freshwater Environments, Berthelin, J., Huang, P. M., Bollag, J.-M., and Andreux, F., eds., Kluwer Academic/Plenum Publishers, New York, 181-188.

Catechol melanin, a black pigment of plants, is a polymeric product formed by the oxidative polymerization of catechol. The formation route of catechol melanin (Eq. 5) is described as follows [33-37] At first, 3-(3, 4 -dihydroxyphe-nyl)-L-alanine (DOPA) is derived from tyrosine. It is oxidized to dopaquinone and forms dopachrome. 5,6-Dihydroxyindole is formed, accompanied by the elimination of C02. The oxidative coupling polymerization produces a melanin polymer whose primary structure contains 4,7-conjugated indole units, which exist as a three-dimensional irregular polymer similar to lignin. Multistep oxidation reactions and coupling reactions in the formation of catechol melanin are catalyzed by a copper enzyme such as tyrosinase. Tyrosinase is an oxidase con-... 

There is evidence that quinone methides form as intermediates in the metabolic oxidation of catechol derivatives, a key step in a variety of biosynthetic processes such as melanization and sclerotization of animal cells. Tyrosinase from mushrooms catalyzes the oxidation of a-methyldopa methyl ester 54a. It has been proposed that this reaction observed in vitro is part of a metabolic pathway for the metabolism of 54a. This reaction proceeds by oxidation of ct-methyl dopa methyl ester 54a to give 54b, which cyclizes and is further oxidized to quinone methide 54c (Scheme 26).101 This quinone methide was identified by comparison to authentic 54c, which was prepared by chemical oxidation of 54a to 54c.102... 

FIGURE 57.12. Reactions catalyzed by the two activities of tyrosinase. Phenol is first oxidized to catechol and then to o-quinone. Reproduced with permission from Kohli et al. (2007a). 

The distinguishing feature of tyrosinase is that it catalyzes the oxidation of monohydric phenols, like tyrosine, to the dihydric form and dihydric phenols, like DOPA and catechol, to the corresponding quinones. The striking biological effects of this enzyme arise from quinones which polymerize to produce the darkening of various plants on injury and melanin in mammals. The relative oxidation rates of several dihydric phenols by tyrosinase are given in Table III. 

Laccase shares with tyrosinase the ability to oxidize dihydric phenols, like catechol, to the corresponding quinones. Whether the enzyme is active in the oxidation of monohydric phenols like cresol is a matter of controversy since the purity of preparations said to catalyze such oxidations has been questioned 107), Much more important is the ability of laccase to oxidize various aminophenols, like p-phenylenediamine, which is in fact the best substrate for the enzyme (Table III). The enzyme is important commercially because it oxidizes some complex... 

Tyrosinases (synonyms phenol oxidases, poly-phenolases or polyphenol oxidases) are copper-containing monooxygenases, which catalyze two consecutive reactions with molecular oxygen as cosubstrate, namely the ortho-hydroxylation of phenols and the oxidation of the resulting catechols to ortho-quinones (Fig. 16.3-4). 

Some enzymes capable of oxidizing C-H bonds contain copper ions [52]. For example, tyrosinase [53a] contains a coupled, binuclear copper active site which reversibly binds dioxygen as a peroxide that bridges between the two copper ions. This enzyme catalyzes the orthohydroxylation of phenols with further oxidation of catechol to an ortho-quimne [53b-d], The mechanism proposed for phenolase activity of tyrosinase is shown in Scheme XI. 13 [521]. 

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