The "Benzothiazine" Chromophore of Pheomelanins: A Reassessment[Dagger]
Posted on: Friday, 16 May 2008, 06:00 CDT
By Napolitano, Alessandra De Lucia, Maria; Panzella, Lucia; d'Ischia, Marco
ABSTRACT The characteristic absorption and photochemical properties of pheomelanins are generally attributed to "benzothiazine" structural units derived biogenetically from 5-S- cysteinyldopa. This notion, however, conveys little or no information about the structural chromophores responsible for the photoreactivity of pheomelanins. At pH 7.4, natural and synthetic pheomelanins show a defined maximum around 305 nm, which is not affected by reductive treatment with sodium borohydride, and a monotonic decrease in the absorption in the range 350-550 nm. These features are not compatible with a significant proportion of structural units related to 2H-1,4-benzothiazine and 2H-1,4- benzothiazine-3-carboxylic acid, the early borohydride-reducible pheomelanin precursors featuring absorption maxima above 340 nm. Rather, these features would better accommodate a contribution by the nonreducible 3-oxo-3,4-dihydrobenzothiazine (lambda^sub max^ 299 nm) and benzothiazole (lambda^sub max^ 303 nm) structural motifs, which are generated in the later stages of pheomelanogenesis in vitro. This conclusion is supported by a detailed liquid chromatography/UV and mass spectrometry monitoring of the species formed in the oxidative conversion of 5-S-cysteinyldopa to pheomelanin, and would point to a critical reassessment of the commonly reported "benzothiazine" chromophore in terms of more specific and substantiated structural units, like those formed during the later stages of pheomelanin synthesis in vitro.
INTRODUCTION
Current concepts of skin melanin photobiology are dominated by the notion that the black insoluble eumelanins are photoprotective whereas pheomelanins, the reddish-brown, sulfur-containing variants found in red-haired individuals, are phototoxic (1,2). However, in spite of extensive clinical and epidemiological data which have shown that red haired, fairskinned people share increased UV- susceptibility with higher propensity for skin cancer (3-5), and a number of biochemical data supporting a photosensitizing action of pheomelanins (6,7), there is so far no clear-cut evidence demonstrating a direct causal link between the photochemical properties of pheomelanins and skin cancer. In fact, mounting genetic evidence points to Melanocortin 1 receptor (MC1R) mutations as the primary underlying factor in UV susceptibility (8,9), whereby pheomelanin formation would only be the visible outcome of a metabolic switch and the consequent failure of active melanocytes to produce the "normal" eumelanin pigment pathway.
In this setting, a detailed understanding of the structural properties and mechanism of assembly of the pheomelanin pigment appears essential if the above and other pending issues concerning pheomelanin photochemistry and photobiology are to be settled. The persisting uncertainties that dominate the field are a direct consequence of the current lack of knowledge about the basic architecture of pheomelanins and their key structural features.
Recently, the first ultrafast absorption spectroscopy measurements for synthetic pheomelanin have been reported, and have highlighted the fast generation of a transient species with an absorption maximum centered at 780 nm (10). This species has been attributed to a photoexcitation product whose action spectrum peaks in the range between 350 and 360 nm, thus resembling the reported absorption spectrum of benzothiazines. It was argued that the reactive chromophore of pheomelanins is of low molecular weight but is present and exhibits similar photophysics in the aggregated state, and may be adequately described in terms of "benzothiazine" structural motifs which are biogenetically derived from the key pigment precursor, 5-S-cysteinyldopa (11).
It must be noted, however, that the occurrence of benzothiazine units in the pheomelanin backbone is largely a matter of surmise, and has so far lacked direct and unambiguous experimental support. Moreover, the term "benzothiazine" is commonly associated with a broad range of structural motifs which exhibit however different chromophoric features. Nonetheless, this notion has become a central axiom in pheomelanin research and has in part been built upon the identification many decades ago in pheomelanin-containing tissues of a peculiar group of low molecular weight compounds, the trichochromes, featuring the peculiar Delta^sup 2,2'^-bi(2H-1,4- benzothiazine) skeleton (12). The configuration of the double bond in these pigments and the associated chromophoric features have recently been reexamined by computational analysis (13). Similar to pheomelanins, trichochromes exhibit a marked photoreactivity and the possibility to access to multiple electronic states upon UV and visible photoexcitation (14).
Though commonly cited among the primary determinants of the red colorations of hair in the true "pheomelanic" phenotype, the trichochromes themselves have been a subject of controversy and even their actual occurrence in human hair and tissues has been questioned. In fact, trichochrome pformation by oxidation of 5-S- cysteinyldopa has been reported to be a minor process which is dramatically enhanced under strongly acidic, nonnatural conditions, suggesting a possible artifactual generation under the harsh acidic conditions used for pheomelanin extraction from red hair (12,15).
With this background, a critical assessment of the spectrophotometric properties of pheomelanins and the role of putative precursors prepared under biomimetic conditions is mandatory if the above uncertainties are to be tackled through an unambiguous experimental approach.
The aim of this study was to provide a comparative description of the UV absorption properties of the main putative pheomelanin- related compounds that are generated in the oxidation of 5-S- cysteinyldopa, with a view of determining whether and to what extent each of them can be included among the primary determinants of the pheomelanin "chromophore."
MATERIALS AND METHODS
Horseradish peroxidase (EC 1.11.1.7), hydrogen peroxide (30% vol/ vol), sodium borohydride and zinc sulfate heptahydrate were purchased from Sigma-Aldrich. 5-S-cysteinyldopa was prepared from L- dopa and L-cysteine (16). 7-(2-Amino-2-carboxyethyl)-5-hydroxy-3,4- dihydro-2H-1,4-benzothiazine (2) (17), 7-(2-amino-2-carboxyethyl)-5- hydroxy-3,4-dmydro-2H-1,4-benzothiazine-3-one (3) (18), 2,2'-bi[7- (2-amino-2-carboxyethyl)-3-carboxy- 5-hydroxy-2H-1,4-benzothiazine] (4) (15), trichochrome C (6) (15) and 6-(2-amino-2-carboxyethyl)-4- hydroxybenzothiazole (9) (19) were obtained as reported. Pheomelanin from red human hair was isolated as previously described (15,20).
HPLC analyses were performed on an Agilent 1100 series instrument equipped with a UV detector set at 280, 340 or 460 nm. An octadecylsilane-coated column, 150 mm x 4.6 mm, 5 [mu]m particle size (Eclipse XDB-C18; Zorbax) at 0.4 mL min^sup -1^ was used. The following eluant systems were used: 0.1% TFA, solvent A; methanol, solvent B: from 10% to 15% B, 0-15 min; from 15% to 60% B, 15-55 min; from 60% to 80% B, 55-65 min (eluant I); 0.5% TFA, solvent A; methanol, solvent B: from 35% to 45% B, 0-25 min; from 45% to 80% B, 25-45 min (eluant II).
Liquid chromatography/mass spectrometry (LC/MS) analyses were run on an Agilent 1100 series instrument with an electrospray ionization source in positive ion mode (ESI +). The same column and the same elutographic conditions as above were used.
Preparation of synthetic pheomelanin. To a solution of 5-S- cysteinyldopa (100 mg) in 0.1 M phosphate buffer (pH 6.8) (25 mL) horseradish peroxidase (16.7 U mL^sup -1^ final concentration) and hydrogen peroxide (38 [mu]L) were added. The mixture was allowed to stand at room temperature under vigorous stirring for 2 h and then acidified to pH 3. The melanin precipitate was collected by centrifugation and washed three times with 1% acetic acid, once with water and then lyophilized. In other experiments the reaction was run in the presence of ZnSO^sub 4^ x 7H^sub 2^O (111 mg, 1.2 molar equivalents), with 100 U mL^sup -1^ of peroxidase and 228 [mu]L of hydrogen peroxide, added in two portions at 5 h intervals. The mixture was taken under stirring for 24 h and then acidified to pH 3, and the melanin precipitate was collected as above.
In both cases, aliquots of the reaction mixtures were periodically withdrawn, reduced with NaBH^sub 4^ when required, and analyzed by HPLC, LC/MS or UV-visible spectroscopy.
RESULTS AND DISCUSSION
The electronic absorption spectra of natural and synthetic pheomelanins obtained under conditions insuring minimal structural alterations have been recorded in 0.1 M phosphate buffer pH 7.4 (Fig. 1). Selected pigments included a natural sample from red human hair, that was freed from the proteic matrix by a reported enzymatic procedure (15,20) and two standard synthetic pheomelanins produced by peroxidase/H^sub 2^O^sub 2^ oxidation of 5-S-cysteinyldopa in the presence and in the absence of Zn^sup 2+^. The peroxidase/H^sub 2^O^sub 2^ couple was chosen as the oxidizing system as it insured an efficient oxidation of 5-S-cysteinyldopa with substantial and rapid consumption of the substrate as shown in previous studies (21,22). Under different reaction conditions including aerial or tyrosinase catalyzed oxidation the kinetic of the process is much lower, and the yields of the final pigment are rather poor (21). Inclusion of Zn^sup 2+^ in the oxidation mixture was suggested by the high levels of the metal in human red hair (23) and by its established stabilizing effect on the course of pheomelanogenesis in vitro (15), allowing for a more clear-cut detection of intermediate species and more defined Chromatographie profiles. Figure 1. Absorption spectra at pH 7.4 of natural pheomelanin from human hair (A) and synthetic pheomelanin from 5-S-cysteinyldopa prepared in the presence (B) and in the absence (C) of Zn^sup 2+^.
Inspection of the absorption profiles in Fig. 1 showed a gross similarity between the natural and synthetic pigment samples. A salient common feature includes a flat maximum around 305 nm with barely detectable inflections at shorter and longer wavelengths, and in all cases a low featureless absorption above 500 nm, indicating limited scattering effects compared to eumelanins. Despite such similarities, however, some differences emerged relating to the shapes of the traces, and the presence/positions of inflections.
To inquire into the nature of the species responsible for the basic chromophoric features of the pheomelanin pigments, the spectrophotometric changes accompanying the oxidative conversion of 5-S-cysteinyldopa in the presence and in the absence of Zn^sup 2+^ were investigated. Data in Fig. 2 indicate a profound effect of Zn^sup 2+^ which markedly slowed down the kinetic course of the reaction and allowed for detection of well defined chromophoric phases. Comparison of traces in Fig. 2a vs Fig. 2b indicate in the latter case the fast development within 30 s of two absorption maxima, one at 309 nm, which survived after 2 h, and the other at 339 nm, which decayed within a few minutes.
By contrast, in the presence of Zn^sup 2+^ (Fig. 2a) a well- defined chromophore with a maximum centered at 390 nm developed within the first minutes. Following addition of ethylenediaminetetraacetic acid (EDTA), this chromophore underwent ipsochromic shift to 337 nm suggesting a Zn^sup 2+^-chelate (data not shown). Gradually, the 390 nm chromophore was replaced by two bands at 302 and 356 nm but, after 24 h, only the band at 302 nm was detectable. Centrifugation of the mixture at this stage allowed separation of the pheomelanin pigment which, taken up in phosphate buffer, gave the absorption spectrum in Fig. 1. UV analysis of the supernatant revealed a chromophore with an absorption maximum at 302 nm contributing to a significant extent to the absorption properties of the whole mixture (data not shown).
Figure 2. Spectral changes during oxidation of 5-S-cysteinyldopa in the presence (a) and in the absence (b) of Zn^sup 2+^. Plot a: trace 1, 15 min; trace 2, 3 h; trace 3, 24 h. Plot b: trace 1, 30 s; trace 2, 5 min; trace 3, 2 h.
Based on these preliminary data, the species formed at the various chromophoric phases in Fig. 2 were investigated by HPLC analysis with UV and ESI+/MS detection (Fig. 3).
The HPLC elution trace of the oxidation mixture carried out in the presence of Zn^sup 2+^ at 15 min reaction time (Fig. 3a) indicated a single major species (R^sub T^ 9.5 min) exhibiting higher absorption at 340 than at 280 nm, which gave a pseudomolecular ion peak [M + H]^sup +^ at m/z 297 (Fig. 3b). Reduction of the mixture with sodium borohydride resulted in the formation of a 1:1 mixture of two related compounds (Fig. 3c, R^sub T^ 27.3 and 27.9 min) with absorption maxima at 290 nm and pseudomolecular ion peaks [M + H]^sup +^ at m/z 299. On this basis, it was concluded that the first formed species was the benzothiazine carboxylic acid 1. This forms a stable Zn^sup 2+^ complex, giving the maximum at 390 nm, that can be reduced to give diastereoisomeric 3-carboxy dihydrobenzothiazines (2).
HPLC analysis of the mixture after 3 h (Fig. 3d) revealed complete conversion of the benzothiazine 1 into a number of species including the 3-oxo-3,4-dihydrobenzothiazine derivative 3 (lower trace at 280 nm, R^sub T^ 25.1 min,[ M + H]^sup +^ m/z 269) and the diastereoisomeric 2,2'-bi(2H-1,4-benzothiazine) dimers 4 eluted at R^sub T^ 40.6, 40.9, 43.4 min (upper trace at 340 nm, [M + H]^sup +^ m/z 591, Fig. 3e). The above structural assignments were supported by the different behavior to sodium borohydride reduction: whereas the amide 3 resisted the treatment (Fig. 3f) as confirmed also in separate experiments on standard samples, the dimers 4 gave the reduction products ([M + H]^sup +^ m/z 595) corresponding to the gross structure 5, two of which were identified as the species eluting under peaks at R^sub T^ 29.0 and 37.9 min (Fig. 3f) with absorption maxima in fairly good agreement with literature data (15). Most of the products shown in the profile taken at 340 nm were converted by the reduction treatment and were well detectable at 280 nm.
Figure 3. HPLC elution profiles and ESI + /MS spectra of the mixtures obtained by oxidation of 5-S-cysteinyldopa in the presence of Zn^sup 2+^: (a) elution profile of the mixture at 15 min reaction time (eluant II, detection at 340 nm); (b) ESI+/MS spectrum of the species eluting at 9.5 min; (c) LC/ESI + /MS elution profile of the mixture at 15 min reaction time after reduction with NaBH^sub 4^ (eluant I); (d) elution profile of the mixture at 3 h reaction time (eluant I, lower trace, detection at 280; upper trace detection at 340 nm); (e) ESI+/MS spectrum of the species eluting at 40.6, 40.9 and 43.4 min; (f) elution profile of the mixture at 3 h reaction time after reduction with NaBH^sub 4^ (eluant I, detection at 280 nm); (g) elution profiles of the mixture at 5 h reaction time (eluant I, detection at 460 nm); (h) ESI + /MS spectrum of the species eluting at 42.9 min; (i) ESI + /MS spectrum of the species eluting at 49.2 min. Elutographic peaks corresponding to identified compounds are marked with structural numbers.
After 5 h chromatographic analysis showed gradual conversion of the dimers 4 into a collection of unidentified species exhibiting intense absorption at 460 nm (Fig. 3g) among which a peak eluting at R^sub T^ 42.9 min ([M + H]^sup +^ at m/z 561, Fig. 3h) was identified as trichochrome C (6) (24) by comparison of the chromatographic behavior and chromophoric features with those of an authentic sample (15). Figure 3i shows the mass spectrum of the species eluting at R^sub T^ 49.2 min with a pseudomeolecular ion peak [M + H]^sup +^ at m/z 575, suggesting a trichochrome-related structure. Structural elucidation of this species and the other major components of the oxidation mixture of 5-S-cysteinyldopa at this stage is the focus of ongoing studies. At 24 h reaction time, when the oxidation process was apparently coming to an end and no further chromophoric change was detectable, HPLC analysis indicated loss of dimers 4 and trichochromes, but persisting quantities of the amide 3 and substantial amounts of more polar species which eluded isolation and characterization.
HPLC analysis of the reaction mixture obtained in the absence of Zn^sup 2+^ at 5 min showed a poorly defined chromatographic pattern. However, after reduction with sodium borohydride, distinct species were apparent, which could be identified as the dihydrobenzothiazine 7 (R^sub T^ 12.0 min, [M + H]^sup +^ m/z 255) and the diastereoisomeric dihydrobenzothiazine-3-carboxylic acids 2 at a 3:1 ratio (Fig. 4a,b). This indicated the formation in the very early phases of the reaction of decarboxylated derivative 8 along with very small amounts of the benzothiazine carboxylic acid 1. The ratio of the diastereoisomeric dihydrobenzothiazines 2 different from the 1:1 expected by sodium borohydride reduction of 1 suggests that the isomer of 2 having configuration at C-3 related to L-cysteine was present in the reaction mixture prior to reduction, as a result of a redox exchange process between the o-quinonimine generated by cyclization of the cysteine chain of 5-S-cysteinyldopa with 5-S- cysteinyldopa itself (25). After 1 h the dihydrobenzothiazine 7 had decayed (Fig. 4c) and the major components of the reduced mixture were the 3-oxo-3,4-dihydrobenzothiazine 3 and a compound at R^sub T^ 20.3 min ([M + H]^sup +^ m/z 239, Fig. 4d), identified as the benzothiazole 9 by comparison with an authentic sample (19). In separate experiments it was shown that, similar to 3, the benzothiazole 9 resisted sodium borohydride reduction.
Given the different behavior of the various benzothiazine intermediates to sodium borohydride reduction, it was reasoned that the treatment of pheomelanins with sodium borohydride might yield important clues as to the nature of the main structural units. Thus, in a final experiment we assessed whether exposure to sodium borohydride caused any detectable change in the pheomelanin chromophore, and it was found that under the conditions in which the benzothiazine carboxylic acid 1 is efficiently reduced, the pheomelanin chromophore (both natural and synthetic samples) is unaffected (data not shown).
Figure 4. LC/ESI + /MS elution profiles and ESI + /MS spectra of the mixtures obtained by oxidation of 5-S-cysteinyldopa in the absence of Zn^sup 2+^ + : (a) LC/ESI + /MS elution profile of the mixture at 5 min reaction time after reduction with NaBH^sub 4^ (eluant I); (b) ESI + /MS spectrum of the species eluting at 12.0 min; (c) LC/ESI + /MS elution profile of the mixture at 2 h reaction time after reduction with NaBH^sub 4^ (eluant I); (d) ESI + /MS spectrum of the species eluting at 20.3 min. Elutographic peaks corresponding to identified compounds are marked with structural numbers.
From the experiments so far described, a number of conclusions can be drawn. First, it can be argued that Zn^sup 2+^ affects in part the kinetic, chemical and spectrophotometric course of 5-S- cysteinyldopa oxidation favoring in particular retention of the carboxylic group in the early benzothiazine intermediates, but does not seem to modify to any appreciable extent the general features of the final pigment. This would be supported by the observation in Fig. 1 indicating similar spectra for synthetic pheomelanins prepared in the presence and in the absence of Zn^sup 2+^. Another central point emerging from the present results is that not all the "benzothiazine" units supposed to contribute to the pheomelanin chromophore exhibit absorption spectra entirely consistent with the observed features of the final pigments (26). In particular, those structural components based on the 2H-1,4-benzothiazine ring system or the trichochrome skeleton would exhibit absorption maxima at relatively higher wavelengths than 305 nm, where the most characteristic feature of the pheomelanin chromophore occurs. Moreover, such 2H-1,4-benzothiazine units would be reducible by borohydride with a detectable ipsochromic shift, an effect that was not observed on pheomelanin pigments. On the other hand, the 3-oxo- 3,4-dihydrobenzothiazine structure, which is found both in a monomer intermediate and as a structural moiety in the trichochrome skeleton, displays absorption and chemical properties that suggest a major presence in the pheomelanin backbone. Indeed, the significance of 3-oxo-3,4-dihydrobenzothiazine moieties as a final evolution product of 3-carboxy-2H-1,4-benzothiazine moieties, their characteristic absorption at 305 nm and their insensitivity to sodium borohydride treatment would concur to support this conclusion.
Interestingly, a careful scrutiny of the literature would indicate that in addition to the 3-oxo-3,4-dihydrobenzothiazine system, benzothiazole units also absorb at significantly lower wavelengths compared to 2H-1,4-benzothiazines and might likewise contribute to the chromophore of pheomelanins. In particular, benzothiazole 9 has a maximum at 303 nm. Moreover, benzothiazoles are not reducible with sodium borohydride, and their formation is another possible evolution pathway of 2H-1,4-benzothiazines (19,26,27) via ring contraction. This latter step may occur at various stages of pheomelanin build up, either before or after the pigment backbone has been assembled. In this connection, it may be worth noting that early tentative structural models of pheomelanins incorporated benzothiazole but not benzothiazine units (28-30).
In Fig. 5 the absorption spectrum of natural pheomelanin is shown together with those of putative structural units, to permit a better appreciation of the above reasonings. It is well apparent that besides the two main chromophores accounting for the absorption around 305 nm (traces B and C), nonreducible 2,2'-bi(3- carboxybenzothiazine) and trichochrome-like units may contribute to the long wavelength absorption. The origin of the main chromophoric units in pheomelanin is summarized in Scheme 1.
Clearly, this is an oversimplification of the actual process of generation of the pheomelanin chromophore. The chromophoric changes accompanying oxidative conversion of 5-S-cysteinyldopa to pheomelanin have been interpreted for the first time in terms of structurally defined intermediates and pigment precursors, which have been analyzed for their absorption properties and reduction behavior. However, beyond a guess about the involvement of 3-oxo- 3,4-di-hydrobenzothiazine and benzothiazole units it is not possible to go. It is likely that the gradual broadening of the absorption spectrum of the first formed chromophores with consequent loss of the structural features in the final pigment is due to an oxidative breakdown of dimeric trichochrome-like oligomers, but the effect of superposition of the absorptions due to different species cannot be rationalized or excluded at the present level of analysis.
Figure 5. Absorption, spectra at pH 7.4 of natural pheomelanin from human hair (trace A), 3-oxo-3,4-dihydrobenzothiazine 3 (B), benzothiazole 9 (C), dimers 4 (D) and trichochrome C 6 (E).
Scheme 1.
In conclusion, we have shown that the commonly held axiom that the pheomelanin chromophore is made up of "benzothiazine" units is ambiguous and must be revisited in light of a detailed analysis of the chemical and spectrophotometric features of the main intermediates involved in the build up of the pheomelanin chromophore. Verification of the main conclusions of this study awaits a more detailed insight into the photophysical and photochemical behavior and into the oxidation chemistry of these species, which form the subject of ongoing work in our laboratory.
Acknowledgements-This work was supported in part by a grant from "Regione Campania, legge 5/2002 anno 2006." We thank the "Centro Interdipartimentale di Metodologie Chimico-Fisiche" (CIMCF, University of Naples Federico II) for NMR facilities, and Mrs Silvana Corsani for technical assistance.
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Alessandra Napolitano*1, Maria De Lucia1, Lucia Panzella1,2 and Marco d'Ischia1
1 Department of Organic Chemistry and Biochemistry, University of Naples "Federico II" Complesso Universitario Monte S. Angelo, Naples, Italy
2 Dermatology Unit, Department of Systematic Pathology, University of Naples "Federico II", Naples, Italy
Received 27 July 2007, accepted 23 September 2007, DOI: 10.1111/ j.1751-1097.2007.00232.x
[dagger] This invited paper is part of the Symposium-in-Print: Melanins.
* Corresponding author email: alesnapo@unina.it (Alessandra Napolitano)
(c) 2007 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/08
Copyright American Society for Photobiology May/Jun 2008
(c) 2008 Photochemistry and Photobiology. Provided by ProQuest Information and Learning. All rights Reserved.
Source: Photochemistry and Photobiology
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