Eumelanins oxidation

Melanin biosynthesis in animals is a complex process starting with the L-tyrosine amino acid. In the first step, L-tyrosine is converted first into DOPA and then into dopaquinone, a process catalyzed by tyrosinase. In the biosynthesis of eumelanins, dopaquinone undergoes a cyclization to form dopachrome and subsequently a tau-tomerization into 5,6-dihydroxyindole-2-carboxylic acid (DHICA). DHICA is further oxidized to indole-5,6-quinone2-carboxylic acid, the precnrsor of DHICA eumelanins. Tyrosinase-related proteins TRP-2 and TRP-1, respectively, are responsible for the last two steps, and they are under the control of the tyrosinase promoter. 

The oxidative polymerization of 5,6-dihydroxyindole (1) and related tyrosine-derived metabolites is a central, most elusive process in the biosynthesis of eumelanins, which are the characteristic pigments responsible for the dark color of human skin, hair, and eyes. Despite the intense experimental research for more than a century,36 the eumelanin structure remains uncharacterized because of the lack of defined physicochemical properties and the low solubility, which often prevents successful investigations by modem spectroscopic techniques. The starting step of the oxidative process is a one-electron oxidation of 5,6-dihydroxyindole generating the semiquinone 1-SQ (Scheme 2.7). 

Melanin granules are secreted by melanocytes in the hair papilla and distributed to keratin in the hair cortex and inner layers of the hair sheath during normal development. Melanogenesis is subject to hormonal control and has been the focus of intensive genetic studies. Two main forms of melanin exist in human skin—eumelanin and phaeomelanin, both of which are derived from tyrosine through the action of tyrosinase (a cupro-enzyme) and possibly other key enzymes (with nickel, chromium, iron, and manganese as cofactors). Tyrosine is converted to dihydroxyphenylalanine and, via a series of intermediate steps, to indole-5,6-quinone, which polymerizes to eumelanin. Phaeomelanins are produced by a similar mechanism but with the incorporation of sulfur (as cysteine) by a nonenzymatic step in the oxidation process. 

Enzymes present in melanosomes synthesize two types of melanin, eumelanin and pheomelanin. Figure 2 illustrates the proposed biosynthetic pathways of eumelanin and pheomelanin. The synthesis of eumelanin requires tyrosinase, an enzyme located in melanosomes. Tyrosinase catalyzes the conversion of tyrosine to dopa, which is further oxidized to dopaquinone. Through a series of enzymatic and nonenzymatic reactions, dopaquinone is converted to 5,6-indole quinone and then to eumelanin, a polymer. This polymer is always found attached to proteins in mammalian tissues, although the specific linkage site between proteins and polymers is unknown. Polymers affixed to protein constitute eumelanin, but the exact molecular structure of this complex has not been elucidated. Pheomelanin is also synthesized in melanosomes. The initial steps in pheomelanin synthesis parallel eumelanin synthesis, since tyrosinase and tyrosine are required to produce dopaquinone. Dopaquinone then combines with cysteine to form cysteinyldopa, which is oxidized and polymerized to pheomelanin. The exact molecular structure of pheomelanin also has not been determined. 

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>. 

Ty initiates melanin synthesis by the hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (Dopa) and the oxidation of dopa to dopaquinone. In the presence of L-cysteine, dopaquinone rapidly combines with the thiol group to form cysteinyldopas, which undergo nonen-zymatic conversion and polymerization to pheomelanin via benzothiazine intermediates. In the absence of thiol groups, dopaquinone very rapidly undergoes conversion to dopachrome, which is transformed to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) by dopachrome tautomerase. Alternatively, dopachrome is converted nonenzymatically to 5,6-dihydroxyindole (DHI). Oxidation of DHICA and DHI to the corresponding quinones and subsequent polymerization leads to eumelanins. It is still questionable if Ty is involved in this step. 

The natural colour of animal fibre is closely related to the character of environment in which the animal lives [19]. Wool lots completely free of dark fibres do not exist [20]. In animal (and human) hair two kinds of pigments occur, namely eumelanin (responsible for black, dark brown and grey colours and commonly referred to as melanin) and pheomelanin (present in yellow, reddish-brown and red hair). Both are thought to be formed by different mechanisms and chemically differed [21]. Eumelanin is formed by enzymatic (tyrosinase) oxidation of tyrosine and polymerisation of several oxidation product [22]. Pheomelanin occurs in form of discrete grannules. Melanin grannules can occur in the cortex or in the cuticle. 

Permanganate oxidation of eumelanins yields pyrrole-2,3,5-tricarboxylic acid, while HI hydrolysis of III... 

Study of the oxidation mechanism of S—S and S-H bonds by X-ray photoelectron spectroscopy Enzymatic eumelanin reduction after exposure to 4-rcrt-butyl catechol Mechanism of decolorization study... 

In a similar study the metal-catalyzed oxidative polymerization of DICA (190,191) in which position 2 is occupied produced dimers and trimers 13-15 coupled at positions 4 and 7. The observation is interesting considering that natural eumelanins appear to contain a substantial proportion of DICA-derived units (7,37,192). However the presence of... 

Pyrrolecarboxylic acids are the final products of oxidative degradation of eumelanins. The origin, reaction conditions, as well as the isolation and identification techniques used are the factors responsible for the different ratios of di-, tri-, and tetracarboxylic acids formed (7). Thus, untreated sepiomelanin and a number of synthetic melanins oxidized via KMNO4 showed the following trend in the relative ratios of pyrrolecarboxylic acids 2,3,5 2,3 = 2,3,4,5. The same samples after decarboxylation at 200°C followed the sequence 2,3,5 > 2,3 > 2,5 2,4 = 2,3,4,5. The decrease in 2,3,5 triacids and the increase in 2,3 diacids are attributed to the loss of carboxyl groups owing to the thermal treatment (7). Resistance to further oxidative degradation u der specific experimental conditions may substantially influence the ratio of the individual pyrrolecarboxylic acids formed (315). 

Hydrogen peroxide, which oxidatively degrades eumelanins, undergoes disproportionation with catechol melanin to produce oxygen and water 205). 

The process of photoinduced interaction of melanin with molecular oxygen is accompanied by photobleaching (301,304,331). Using ESR measurements the rates of photooxidation of eumelanin and phaeomelanin have been found to be comparable. In other reports, however (337), phaeomelanin has been found to be more resistant toward photobleaching than eumelanin. A similar pattern of reactivity has been noticed in photo- and chemical oxidation of these two pigments in their native milieu (hair) (338). 

The first two steps in the synthesis of melanin are catalyzed by tyrosinase, a copper-containing oxidase, which converts tyrosine to dopaquinone. All subsequent reactions presumably occur through nonenzymatic auto-oxidation, in the presence of zinc, with formation of the black to brown pigment eumelanin. The yellow to reddish brown, high-molecular-weight polymer known as pheomelanin and the low-molecular-weight trichromes result from addition of cysteine to dopaquinone and further modification of the products. Pheome-lanins and trichromes are primarily present in hair and feathers. 

Other evaluated serological markers from the melanogenesis pathway include the phaeomelanin metabohte S-S-CD (see Ref. 5 for an review of 5-S-CD in melanoma with 2648 samples taken from 218 patients) and to a lesser extent the eumelanin metabohte 6-hydroxy-5-methoxyindole-2-carboxyhc acid. Since 5-S-CD is very sensitive to light and oxidation, analysis requires immediate centrifugation, freezing of the samples after blood collection and exclusion of light and air-oxygen. 

Figure 10. ESR spectra ( — 196°C) from different forms of melanin, a. Melanin from oxidation of dopa (a eumelanin) b, melanin from oxidation of cysteinyldopa (a pheomelanin) c, melanin from cooxidation of dopa and cysteinyldopa. From [164], with permission.

In that scheme, tyrosine is first oxidized to dopaquinone, the key intermediate for the formation of all melanins. If virtually no cysteine is present, then eumelanins are the primary products that arise through cychzation and oxidative polymerization, as described in the section on eumelanins. 

An intense black eumelanin-type pigment is produced by reacting DOPA with controlled amounts of potassium ferricyanide. Simple analogs of intermediates of the melanic pathways such as 8-hydroxy-l,4-dihydrobenzoth-iazine or thio-substituted catechols when oxidized with different oxidants at specific pH conditions and sometimes in the presence of oxidation dye nucloephiles provide different colors similar to those of permanent hair dyes [10]. These systems are still developmental and have not been commercialized to date, but they do appear to offer promise for the future. 

Although the exact structure of eumelanin has still not been determined, there is strong evidence that it has a polymeric structure built up of 5,6-dihydroxyindole 1, 5,6-indolequinone 6 and semiquinone 7 monomers. Eumelanin has exceptionally good photoprotective properties and an absorption spectrum spanning a large part of the solar spectrum. These special properties have prompted a number of theoretical studies of 5,6-dihydroxyindoles, their oxidation products and dimers, and oligomeric models of eumelanin. 

The most significant 2,3-dihydro-5,6-dihydroxyindole is L-cyclodopa 78, also known as leucodopachrome, which is an intermediate in the biosynthesis of eumelanin and is formed by the spontaneous cyclization of dopaquinone 79 (92MI2) (04MI1). In turn, cyclodopa is readily oxidized to dopachrome 80 but this oxidation can be reversed chemically using sodium dithionite. Wyler and Chiovini have described the preparation of both enantiomers of cyclodopa using transformations... 

Dopachrome 80 is a particularly significant aminochrome because of its formation as an early intermediate in the biosynthesis of eumelanin. In vivo the dihydroindole 136 (R = = H, = CO2H) is oxidized by its precursor (dopaquinone) to... 

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