Pulsed irradiation studies of unstable intermediates
of melanin pigment formation

The fast reaction techniques of flash photolysis and pulse radiolysis are known mainly for their enormous contributions to knowledge of free radical and excited state chemistry, and, in particular, the involvement of such species in the chemistry of biology and medicine (1). There is however a whole "spectrum" of unstable intermediates including, for example, orthoquinones, quinone-imines and quinone methides which are neither free radicals nor excited states, yet are too short-lived to be studied by normal methods, but are amenable to study by these pulsed radiation techniques. A variety of such transient intermediates are thought to be involved in the complex processes of melanin formation, and this article attempts to call attention to the largely unexplored potential of pulsed radiation techniques to elucidate these processes.
A common mode of decay of semiquinones is disproportion to give quinones and dihydroxybenzenes. Several unstable orthoquinones, for example, dopaquinone (I) (2-4) and the false melanin precursor anisyl-3,4-quinone (II) (5), can be prepared almost instantaneously by one-electron oxidation of the corresponding dihydroxybenzenes with sub-microsecond pulses of radiation. For dopaquinone, under the conditions chosen (3), formation of dopaquinone from stable dopa was complete within ~5 ms of the pulse, and its decay, eventually to form dopachrome, followed over tens of ms to seconds, depending upon the pH. For anisyl-3,4-quinone, formation of the orthoquinone was complete within 20 ms of pulse irradiation (Aqueous solutions of the latter cannot be made by simple dissolution of solid quinone without significant polymerisation occurring during the process of dissolution). In the formation of both orthoquinones, one-electron oxidation can be per formed either photochemically, via photoionization and/or photo radical homolysis, or radiation chemically, for example, via N3 oxidation, the corresponding semiquinone being formed initially which disproportionates to form the orthoquinone (and reform the parent dihydroxybenzene).
The corresponding orthoquinone (III) of 5-S-cysteinyldopa, which can only be similarly formed radiation chemically (3,4), decays by a different and competing cyclisation path to that pertaining to dopaquinone, the cysteinyl side chain amino substituent attacking the carbonyl of the phenyl ring with loss of water to produce an unstable guinone-imine (IV), postulated by Prota et al (6). This quinone-imine then rearranges by a hydrogen shift to a more stable benzothiazine isomer.
Another important quinone which has long been postulated to be crucial to melanin pigment formation is 5,6 indolequinone (V).
One-electron oxidation of 5,6 dihydroxyindole is thought to lead to the corresponding oxygen-centred semiquinone radical which probably disproportionates to 5,6 indolequinone and reform 5,6 dihydroxyindole (7). The 5,6 indolequinone appears to be unstable, decaying by 1rst order kinetics into a substance, or substances, possessing considerable absorption around 540 nm where melanochrome has a maximum (8,9). One of these substances could be the rearrangement product (VI), reminiscent of dopachrome but with added conjugation, which may be a seed of the polymerisation which ultimately leads to melanin. Positive assignments of the above unstable species are difficult however to establish unequivocally, and it could be that the semiquinone from 5,6 dihydroxyindole, in part at least, decays by demerisation, the dimers initially formed, possibly (VII) and (VIII) for instance, being unstable and rearranging by unimolecular processes into more stable dimers of the type that have been isolated by Corradini et al (10) and d'Ischia and Prota (11).
Decarboxylations are also involved in melanin pigment formation and pulsed radiation techniques may help in elucidating such processes. For instance, tyrosinase catalyses the oxidative decarboxylation of 3,4-dihydroxymandelic acid leading to 3,4-dihydroxybenzaldehyde (12) although 4-hydroxymandelic acid and 3-methoxy-4-hydroxymandelic acid are inert. One-electron oxidation of 3,4-dihydroxymandelic acid via pulse radiolysis (13) led to the corresponding semiquinone which decayed by disproportionation into a species which subsequently decayed by 1rst order kinetics (k~2 s-1 ) into a stable product with an absorption spectrum matching that of 3,4-dihydroxybenzaldehyde. Since one-electron oxidation of 4-hydroxymandelic acid did not appear to lead to 4-hydroxybenzaldehyde, it is suggested that the intermediate resulting from the second order decay of semioxidised 3,4-dihydroxymandelic acid is the orthoquinone (IX) rather than the corresponding quinone methide (X) previously postulated (12), the equivalent of which should also have been formed from 4-hydroxymandelic acid.
It is hoped that the above gives some idea of the scope of pulsed radiation techniques for studies of unstable melanin precursors. Since the equipment in particular the sources of pulsed ionizing radiation for carrying out such experiments are expensive and comparatively rare, if any reader wishes to consider jointly pursing other ideas for investigating unstable intermediates related to melanogenesis please get in touch.

Acknowledgements

I am grateful to J.M. Bruce, J.N. Chacon, M.R. Chedekel, C., Lambert, P.A. Riley, T. Sarna, A. Thompson and T.G. Truscott for collaboration, G. Prota for comments, and the Cancer Research Campaign and Medical Research Council for support.

References

(1) Bensasson RV, Land EJ, Truscott TG. Flash photolysis and pulse radiolysis : contribution to the chemistry of biology and medicine. Pergamon Press 1983.
(2) Chedekel MR, Land EJ, Thompson A, Truscott TG. Early steps in the free radical polymerisation of 3,4-dihydroxyphenylalanine (Dopa) into melanin. J Chem Soc Chem Comm 1170-1172, 1984.
(3) Thompson A, Land EJ, Chedekel MR, Subbarao KV, Truscott TG. A pulse radiolysis investigation of the oxidation of the melanin precursors 3,4-dihydroxyphenylalanine (dopa ) and the cysteinyldopas. Biochim Biophys Acta 843:49-57, 1985.
(4) Land EJ, Thompson A, Truscott TG, Subbarao KV, Chedekel MR. Photochemistry of melanin precursors : dopa, 5-S-cysteinyldopa and 2,5-S,S'-dicysteinyldopa. Photochem Photobiol 44:697-702, 1986.
(5) Cooksey CJ, Land EJ, Riley PA, Sarna T, Truscott TG. On the interaction of anisyl-3,4-semiquinone with oxygen. Free Rad Res Comm 4:131-138, 1987.
(6) Prota G, Crescenzi S, Misuraca G, Nicolaus RA. New intermediates in phaeomelanogenesis in vitro. Experientia 26:1058-1059, 1970.
(7) Lambert C, Chacon JN, Chedekel MR, Land EJ, Riley PA, Thompson A, Truscott TG. To be published
(8) Napolitana A, Corradini MG, Prota G. A reinvestigation of the structure of melanochrome. Tetrahedron Lett 26:2805-2808, 1985.
(9) Vachtenheim J, Duchon J, Matous B. A spectrophotometric assay for mammalian tyrosinase utilizing the formation of melanochrome from L-dopa. Anal Biochem 146:405-410, 1985.
(10) Corradini MG, Napolitano A, Prota G. A biosynthetic approach to the structure of eumelanins. The isolation of oligomers from 5,6-dihydroxy-1-methylindole. Tetrahedron 42:2083-2088, 1986.
(11) d'Ischia M, Prota G. Photooxidation of 5,6-dihydroxy-1-methyl-indole. Tetrahedron 43:431-434, 1987.
(12) Sugumaran M. Tyrosinase catalyses an unusual oxidative decarboxylation of 3,4-dihydroxymandelate. Biochem 25:4489-4492, 1986.
(13) Bouheroum M, Bruce JM, Land EJ To be published.

 

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E.J. LAND
Paterson Institute for Cancer Center
Christie Hospital & Holt Radium Institute
Manchester M20 9BX
England, U.K.
Phone : 061 445 8123 ext 215