Link 19-The Pigment Cell and its Biogenesis

www.tightrope.it/nicolaus/index.htm 

 

This year sees the 100th anniversary of the birth of A.Quilico (Milan 1901-2001).

This note reports an admirable text on Raper's melanogenesis which he wrote in 1937.

 

Sommario

Lo scheletro carbonioso fondamentale della melanina , costituito da un pseudo poliene coniugato, alloggia sia elettroni disaccoppiati che buche cariche positivamente bilanciate da contranioni, in un equilibrio dinamico tipico dei semiconduttori e definito con il termine di radical-polarone.Lo scheletro può essere assimilato a quello del nero di acetilene.

Si ritiene che la struttura della DHI-melanina sia fondamentalmente costituita da catene formate da unità ripetitive, 5-gem-diolo-indol-6-one, legate nelle posizioni 4 e 7 (VII). Queste catene possono avere due conformazioni a bassa energia, un’elica ed una sheet. La posizione 2 dell’indolo può dar luogo a legami tra i filamenti formando piani e gabbie. La formazione di gabbie spiega il comportamento tipico delle melanine capaci di legare, ioni, acqua, gas. Dati sperimentali e teorici indicano che la melanina è un semiconduttore con un gap di 1,4 eV. Il colore nero è dall'altra parte la manifestazione di un piccolo gap fra banda di valenza e banda di conduzione del semicoduttore.

Queste strutture suggeriscono un nuovo ruolo della materia nera nei sistemi biologici.

Lo schema di Raper è stato criticamente esaminato.Lo studio della melanogenesi con isotopi radioattivi, l'analisi elementare, l'esame dei prodotti di degradazione della sepiomelanina, il comportamento chimico, indicano che il precursore del pigmento cellulare è la ciclodopa od il dopacromo almeno nel caso della sepiomelanina .

E' probabile che le eumelanine si formino dalla ciclodopa e non dal 5,6-diidrossindolo (DHI) e dal 5,6-diidrossindolo-2-carbonico (DHICA) che sono solo degli artefatti.

 

Summary

Melanin is a conductor and  a stable free radical, holding positive charges, balanced by counter anions and exhibiting a small gap, 1.4 eV. Experimental data, molecular modeling studies and molecular quantum mechanics calculations agree describing DHI-melanin, the best known synthetic melanin , as a structure fundamentally constituted by repetitive units of 5-gem-diol-indol-6-one linked at 4,7 positions (VII). Electric and conformational properties explain different roles of melanins. As a UV filter and a radical scavenger black it has a protective function in epithelial tissue; in dopaminergic neurons of substantia nigra, an area of the brain involved in fine motor control, it can act as a conductor allowing rapid electron passage which, generating a magnetic field, can sense the Earth's field and control movements. Melanin destroys, in Parkinson’s disease it can participate to the symptoms and development of the neuro-degenerative process.Finally their may be considered a cell assembling factor .

The Raper scheme revisited. Analytical data show that the precursor of the pigment cell is cyclodopa. DHI and DHICA are, as was well known at Raper's time, artefacts. Experimental data of sepiomelanin, the melanin from cephalopod ink sack, suggest for the pigment a fundamentally cyclodopa derived polymer structure.

 

Chemistry

 

5,6-Dihydroxyindole (DHI), reported by Raper (1) in 1927 is believed to be the precursor of the pigment cell, formed along the dopa ® dopachrome ® DHI® melanin reaction pathway (2).

Besides melanin, carbon dioxide and H2O2 are also produced. The role played by H2O2 in melanogenesis and the production of carbon dioxide in melanin heating are still not understood. . For carbon dioxide many possibilities exist :

  1. CO2 arises from DHICA or dopachrome units.
  2. CO2 arises from COOH groups formed from the breakdown of catecholic units.
  3. CO2 arises from gem-diols groups.
  4. CO2 arises from the cationic center of the polymer.

 

More recently (3) It was found that the melanin precursor in Sepia is DHI. In other words DHI-melanin would be a perfect model for sepiomelanin structure studies . Analytical results showed by different authors are variable depending to the melanin extraction and purification method adopted. The more frequently occurring reactions during extraction and purification methods adopted are:

  1. oxidative degradations
  2. decarboxylation
  3. condensation
  4. copolymerization
  5. modification of the counteranion

Numerous papers, which report experimental proofs of the biosynthetic pathway to sepiomelanin, fail in describing the elemental composition of the pigment . All the proposed structures have, for each indole unit, one oxygen less than the requirement of analytical results. Numerous adjustments have been claimed, including the presence of water molecules, DIHCA or dopachrome units, benzene moiety breakdown forming COOH groups, etc., to justify the differences between found and calculated values of the elemental composition.

In this paper the 5-gem-diol-indol-6-one is proposed as repetitive unit of the DHI-melanin to explain its analytical data and chemical-physical behaviour. DHI-melanin is put forward as a model for eumelanin.

Figure 1 shows some units compounding melanin compared with the 5-gem-diol-indol-6-one polymeric unit VII ( indolequinone monohydrate )

The polymerization via 4-7 showed in the polymers does not exclude other reactive center.

 

Figure 1

 

 

 

 

Actually, elemental analysis of DHI-melanin and sepiomelanin (Table 1) are an agreement with polymers V and VII (Figure 1, ).

 

 

Table 1-Elemental Composition of DHI-melanin, Sepiomelanin and of its Derivatives

Samples

Found %

DHI-melanin 1

DHI-melanin 2,1

Sepiomelanin3,4,5

DHI-melanin methyl ether5

Sepiomelanin methyl ether5

C, 58.8; H, 3.3; N, 9.3

C, 55.3; H, 3.2; N, 8.3

C, 59.9; H, 3.4; N, 8.2

C, 61.6; H, 5.7; N, 8.6;OCH3, 21.5

C, 64.1; H, 5.5; N, 7.4;OCH3, 18.5

Calculated %

(C8H3O2N)n, polyindolequinone, (DHI-quinone)

C, 66.2; H, 2.1; N, 9.6

(C9H3O4N)n, polyindolquinone-2-carboxyl,(DHICA-quinone)

C, 57.1; H, 1.6; N, 7.4

(C8H5O3N)n, polyindolequinone monohydrate

(C9H5O5N)n polycyclodopaquinone monohydrate

C, 58.9; H, 3.0; N, 8.5

C,52;H, 2.9;N, 6.7

(C8H7O4N) n, polyindolequinone dihydrate

C, 53.0 H, 3.8 N, 7.7

(C9H7 O3N) n, polyindolequinone monohydrate methyl ether

C, 61.0; H, 4.0; N, 7.9 OCH3, 17.5

(C10H10O4N)n, polyindolequinone monohydrate dimethyl ether

C, 57.7 H, 4.8 N, 6.7 OCH3, 29.8

(C10H504N) n, polyindolequinone-2-carboxilic ester

C, 59.4 H, 2.4 N, 6.9 OCH3, 15.3

 

1. Beer, R.J.S., Broadhurst, T., Robertson, A. The Chemistry of Melanins. Part V. The Autoxidation of 5,6-Dihydroxyindoles. J. Chem. Soc. 1947-1953 (1954)

2. Ito, S., Reexamination of the Structure of Eumelanin. BBA 883, 155-161 (1986)

3. Panizzi, L., Nicolaus,R., Ricerche sulle Melanine, Gazz. Chim. Ital. 82,435-460, (1952).

4. Piattelli, M.& Nicolaus, R.A., The structure of Melanins and Melanogenesis. I. The Structure of Melanin in Sepia. Tetrahedron 15, 66-76 (1961).

5. Piattelli, M., Fattorusso, E., Magno, S. & Nicolaus, R.A. The Strucure of Melanins and Melanogenesis. II. Sepiomelanin and Synthetic Pigments. Tetrahedron 18, 941-950 (1962).

Methylation reaction, by diazomethane, suggests the presence of a methoxy group on each indole, in contrast with V and VII structures. This contrast may be resolved by conformational study and chemical analysis on oligomers V and VII formed by units linked in 4, 7, repeatedly reported as the preferential sites of polymerization reaction.

While no hydrogen bond stabilizes V oligomers making hydroxyl groups available to methylation, VII oligomers stabilized by hydrogen bonds assume two low energy conformations: an alternate sheet and the more stable helix. A 60° dihedral angle between two indole planes is formed.

Torsional angles and formation heats may be calculated. Constrained by steric hindrance, the alternated pleated sheet and the helix are held in their shapes by hydrogen bonds between a hydroxyl group and the heterocyclic NH. The hydroxyl group over (or under) the indolic plane and the heterocyclic NH generates a helix, whereas a bond between the hydroxyl group alternatively over and under the indole plane and the heterocyclic NH determines the alternate pleated sheet

In this feature, a hydroxyl group in each unit seems to be available to O-methylation reaction, according to the experimental results.

Currently, it is not possible to establish when the gem-diol formation takes place, if after or before polymerization. Nevertheless, ab initio quantum-mechanical calculations indicate that the 5-gem-diol is more, about 13 Kcal, stable than the corresponding 6-gem-diol and that the orbital coefficients (LUMO) of indole-5,6-dione make the 5-carbon the more suitable to water nucleophilic attack.

Hydration of quinone carbonyl groups of indole 5,6-dione, resulting on the 5-gem-diol is a less studied process and may be similar to hydration of ketone compounds. This reaction is favoured by electron-withdrawing groups like the a -b unsaturated system and the condensed pyrrole ring of indol-5,6-dione. The hydrogen bond formation between the pyrrole NH of an indole unit and a 5-hydroxyl group on the next indole unit can further stabilize the 5-gem-diol formation.

Thermogravimetric analysis of dried natural sepiomelanin and synthetic DHI-melanin showed that water is removed at transition temperature of 96.4° (about 10%), according to a quinone monohydrate structure.

MALDI and MALDI-TOF investigations of protein-free sepiomelanin, with and without the matrix which interacts with its own fragments creating artifact peaks, recorded small fragments arising from melanin breakdown at m/z 39, [C3H3+ ], 41 [HCº NH+.] . No peak at mass higher than 1500 m/z was detected according to the known melanin and heterocyclic instability to laser desorptionionization mass spectrometry (4) Occasionally, peaks corresponding to dimer, trimer, tetramer ((DHI)n+H), formally composed by DHI or by pyrrolidin-5,6-benzo-dione, by indol-5,6-dione and its hydrated forms were recorded.

Mass spectra showed increasing oligomers separated from each other by 206 mass units

, probably corresponding to the 5,6-indolyl acetic acid residue, better describing a situation which is typical of a synthetic route rather than of a fragmentation process.

Under MALDI conditions, quinones used as model, yielded peaks at mass (M+2) (10%), (M+2+H) (PP), (M+4) (20%) and at (M+H2O+H) (5%) instead of at the expected molecular peaks. These results suggest that the DHI-oligomers detected in the MALDI spectra reported in literature could be artifact compounds, arising from the corresponding oxidized and hydrated oligomers.

Actually, if DHI-melanin held a polyindolquinone structure, according to the widespread hypothesis reported in literature, a very easy reduction reaction and high stability to oxidation should be expected, whereas melanin is a substance sensible to oxygen, oxidizing agents and halogens and is not reducible with quinone typical reducing agents and catalytic hydrogen.

Reactivity of other positions of DHI can be responsible of a further link between the polymeric chains to form a sheaf stratum or a cage structure This structure explains and inorganic products,  high binding affinity for melanins the water, gas, ions, organic.

It is commonly accepted both natural as synthetic melanins are stable free radicals and exhibit typical broad IR and EPR spectra of black materials. Figure 4 shows the melanin structure which can be considered a substituted acetylene-black.

In all melanins, the unpaired electrons (one per two hundreds indole units calculated on the EPR signal) and the positive charges (one per eight units calculated on the counteranion) are distributed along the pseudo conjugated skeleton, the red line (the spine), and are responsible for their conductivity and colour.Number of charges and unpaired electrons are unknown. Biological electrical fields generated by the spine may change superficial properties of the pigment which could act as, easily removable by H2O2, an ideal basis for cell assembly and movement.

Investigation on pigment cell recovered from human tumors and on synthetic DHI-melanin, demonstrated that the black pigment, acting as a semiconductor, responds to a critical applied field by changing its conductivity and that the nature of the response depends on ''hydration'' and temperature of the melanin sample and on external circuity (www.organicsemiconductors.com). Drying-hydration equilibrium determinates the switching properties of melanins, suggesting that ‘strongly linked’ water is involved in melanin conductivity. The electronic characteristics persist in intact melanosomes. (5), (6)

This result and the discovery that melanin responds dynamically to electric field, behaving as a semiconductor, and that this property is connected to hydration are in agreement with the description of sepiomelanin as cyclodopapolyquinone monohydrated VIII as reported in the Biogenesis section.

When the helix or the alternate sheet behaves as an organic conductor, according to reported studies on acetylene-black and to the results described above, the C-2 link between the chains allows a highly ordered arrangement, yielding a metallic-looking colour of the polymer.

The helix, rolling around the axes of 4 and 7 positions of indole, constitutes a solenoid which, crossed by a current, could generate a magnetic field at far end.

Though unsaturated carbon backbone is not completely planar as in the acetylene black, ab initio calculations, performed on a single helix of three indole units, obtained by Monte Carlo-like conformational search, showed a HOMO-LUMO gap value of 1.42 eV as for to good semiconductor materials.

Moreover, black color, interpreted on the basis of band theory indicates a gap value about of 1.5 eV, as in the black crystalline semiconductor SeS.

Acetylene, pyrrole, benzene blacks and sepiomelanin seem to share common properties; their structural peculiarities may identify a new class of organic semiconductor substances (electrochromophores).

DOPA, cyclodopa, adrenaline, serotonine, tryptamine, dopamine, isoquinoline (7)

blacks may be present in nature and can play interesting but still unknown biological roles.

A representation of melanin whose backbone arranges in alternate-sheets and helix giving rise to cages is possible. Water plays a crucial role stabilizing secondary and tertiary structures and promotes a cooperative phenomenon where hydrogen-bonding is a cohesive interaction. Hydrated indole units may determine the self-assembly of melanin and its pH dependent aggregation, organizing water, or squeezing it out from the internal cages. Folding of the helix backbone upon itself could form spheres, stretched-out zigzag chains and build strata.

As radical-polarone material, melanins are radical scavengers acting as a UV radical-filter in epithelial tissue and, as bio-organic conductors, which allow a rapid electron passage through the pseudo polyconjugated skeleton, the red spine. The properties of these structures suggest new functions of the pigment cell in Biology. An electric conductor in a crucial area of the brain, could also induce magnetic fields for sensing the Earth's magnetic field and to control movements,orientation and migration.

Neuromelanin, the granular black pigment, present in the dopaminergic neurons of substantia nigra and locus ceruleus of human brain, can be associated with bio-electrical activity of neurons and with the degenerative results of Parkinson’s disease. In fact, biological reactions involving radicals (OH. in Fenton reaction) and nucleophilic attacking agents can easily react on the radical and cationic sites of melanin producing different occurrences iron depletion, local carbon hybrid changes, variations of the conformational structures and a fall of conductivity deteriorating the switching functions.

 

Biogenesis

In 1895 Bourquelot and Bertrand observed that the toadstool Russula nigricans contains a colourless substance which blackens by exposition to air.In 1896 Bertrand recognized the substance as tyrosine. When tyrosine was allowed to oxidise in the presence of a mushroom extract a black precipitate was formed.. This polyphenoloxidase was given the name of tyrosinase .

This enzyme was subsequently found in extracts from many other plants and from the tissues of various invertebrates and vertebrates, including mammals.Although some properties of the black precipitate resulting from the action of tyrosinase on tyrosine were not in agreement with the properties of natural pigments ( hair, eye, melanomas, sepia ink), nevertheless it is believed that natural pigments are produced by the tyrosine/tyrosinase reaction.

Tyrosinases were found to be essential for the initial conversion of tyrosine to dopa , whereas the subsequent steps proceed spontane-ously without any specific enzymatic assistance.

Small amounts of two products, that is the 5,6-dihydroxyindole (DHI) and a related compound the 5,6-dihydroxyindole-2-carboxylic acid (DHICA), were identified by stopping the oxidation of tyrosine (dopachrome stage ) and allowing the red solution to decolorize under aneorobic conditions and in a SO2 atmosphere The indoles were recovered as dimethyl ethers.

The most important studies on melanogenesis were started out by H.S.Raper in the period 1920-1935 (Raper was Professor of Physiology and collegue of Sir Robert Robinson at the Manchester University) (8)

 

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The most relevant part of Raper's work has been reported in the elegant review : A.Quilico, I pigmenti neri animali e vegetali , Ed. FUSI, Pavia 1937. Here a text (pag.154-160) is reported.

 

 

...........All'inizio della sue ricerche , Raper ebbe ad osservare quale notevole influenza avesse sull' andamento della reazione il pH del mezzo in cui I'enzima agisce, dal quale dipende non solo la velocita con cui appare la colorazione, ma anche I' aspetto e Ia natura dei prodotti che si ottengono. A questo proposito, dobbiamo ricordare anche i risultati di H. Pribram e collaboratori ,secondo i quali appare possibile di influire sul colore ottenibile con una data tirosinasi; nelle prove eseguite su crisalidi di lepidottero, si è potuto, mediante una cauta attenuazione dell ' enzima per riscaldamento, trasformare una tirosinasi capace di colorare in violetto, in una colorante in rosa. Questo può anche dare una spiegazione della grande varietà di aspetto delle melanine naturali; è probabile ad es. che la colorazione rossa dei capelli umani o dei crini dei cavalli a mantello fulvo sia da attribuire ad un arresto o ad una deviazione dal solito modo di formazione delle melanine, e non è da escludere che il verde che domina in alcune classi di insetti (come gli ortotteri) si possa originare in modo analogo.

 

 

 

I risultati ottenuti dai precedenti A A. nello studio della reazione tirosina-tirosinasi, ottenuti senza garantirsi con I' aggiunta di opportuni tamponi, contro le variazioni del pH, sono quindi da ritenere malsicuri. Variando le condizioni di pH, e con I' impiego di un adatto materiale enzimatico, Raper e Wormall hanno potuto riconoscere che I' azione della tirosinasi sulla tirosina si compie in tre fasi distinte:

 

1) Trasformazione della tirosina in una sostanza rossa.

2) Passaggio del prodotto rosso ad una sostanza incolora.

3) Ossidazione e condensazione della sostanza incolora a melanina

 

Il primo di questi passaggi, è un processo enzimatico, dovuto all' azione specifica del fermento, ed avviene solamente in presenza dell' ossigeno molecolare, atmosferico. Il secondo, è verosimilmente una trasposizione molecolare che ha luogo spontaneamente, rapidamente per riscaldamento, ed indipendentemente dalla presenza o meno dell' enzima. Il terzo passaggio, sembra essere una ossidazione e condensazione puramente chimica, che si realizza anch' essa in assenza dell' enzima, richiede la presenza dell' ossigeno atmosferico, e la cui velocità appare essere notevolmente influenzata dalla reazione del mezzo, avvenendo rapidamente in soluzione alcalina, piu lentamente in ambiente acido. I limiti azione della tirosinasi di patata giacciono intorno a 5 e 10 I' optimum, determinato per via colorimetrica dalla intensità di colorazione dei liquidi, si trova tra 6 e 8. L' influenza del pH si fa sentire in modo differente sulle tre fasi della reazione; cosi a pH 6 si hanno le condizioni migliori per la formazione della sostanza rossa, mentre che a pH 8 si hanno le condizioni piu adatte per il suo passaggio al corpo incoloro. Di questa circostanza si approfitta per arrestare la reazione all' ottenimento del prodotto desiderato; cosi ad es., nel caso in cui si impieghi la tirosinasi di Tenebrio, per procurarsi la sostanza rossa, basta, dopo di aver fatto agire per un tempo sufficiente I' enzima ad un pH=6, rendere nettamente acido il liquido oppure, come ha fatto Raper per la tirosinasi di patata, si puo allontanare dal liquido l' enzima che ha agito. facendola adsorbire da idrato ferrico colloidale. La soluzione rossa, formatasi per azione della tirosinasi, quando venga conservata nel vuoto, o portata all' ebollizione, o più rapidamente ancora, abbandonandola a temperatura ambiente in presenza di SO2, si decolora e da essa è possibile isolare una sostanza incolora che cristallizza dall' acqua in aghi o prismi p. f. 279° con decomposizione, che è risultata identica alla diossifenilalanina naturale, levogira, che si può estrarre dalla Vicia faba. La resa, operando almeno in queste condizioni, non supera pero il 3% sulla tirosina impiegata. Alla sostanza rossa, il Raper aveva attribuito, almeno in un primo tempo , la struttura indicata a lato , la quale come diremo, è stata in seguito da lui modificata. La diossifenilalanina si puo isolare piu convenientemente sotto forma di sale di piombo, precipitando la soluzione acida con acetato di Pb. Data la grande ossidabilita dei prodotti che si formano per decolorazione della sostanza rossa, Raper è ricorso all' artificio di renderli stabili mediante metilazione dei gruppi ossidrilici fenolici presenti.

La soluzione rossa ottenuta per azione della tirosinasi di Tenebrio su una soluzione di tirosina,viene decolorata per azione del calore e della SO2 , concentrata in atmosfera di CO2 , alcalinizzata e sottoposta a metilazione con solfato di metile in ambiente di idrogeno. In questo modo è possibile di isolare altri due prodotti, uno dei quali ha carattere debolmente basico, I' altro è invece nettamente acido. La prima di queste sostanze è il 5,6 dimetossiindolo p.f. 154°-155°, la seconda I'acido 5,6 dimetossindolcarbonico; questo dimostra che nella reazione tirosinasi- tirosina, oltre alla diossifenilanina, si formano anche il 5,6 diossindolo (DHI) e I'acido 5,6 diossindolcarbonico(DHICA). Le quantità relative di questi prodotti dipendono dal modo in cui si esegue la decolorazione del prodotto rosso primario.

Se questa viene realizzata per ebollizione prolungata, senza I' intervento di SO2 , si ottiene in prevalenza il diossiindolo. se invece si opera a freddo, con SO2 , si ha maggiore quantita del secondo. Questo rende assai probabile che il diossindolo non rappresenti che un prodotto secondario di decarbossilazione delI' acido diossindolcarbonico.

In quanto alla diossifenilalanina, essa rappresenta un prodotto primario della reazione, che non è in immediata relazione con la sostanza rossa come i due derivati indolici sopra descritti.La struttura di queste sostanze è stata dimostrata rigorosamente dalla sintesi fatta da Oxford e Raper .

Circa la struttura del prodotto rosso primario il quale si forma anche per azione della tirosinasi sulla DOPA è molto verosimile si tratti dell'ortochinone corrispondente all'acido diossidrindolcarbonico(leucodopacromo o ciclodopa n.d.r).

In base a questi risultati Raper ha potuto dare uno schema del processo secondo il quale queste sostanze che sono i precursori della melanina si formano a partira dalla tirosina.

Come da esso si vede il primo passo della azione della tirosinasi consiste nella introduzione di un nuovo ossidrile fenolico in orto rispetto a quello gia esistente nella tirosina con formazione di diossifenilalanina;ha luogo poi una deidrogenazione a dopachinone il quale per trasposizione molecolare si trasforma per chiusura dell'anello in acido diossiidrindolcarbonico.Questo per una nuova deidrogenazione dà origine alla sostanza rossa ( chinone dell'acido diossiidrindolcarbonico ) che per una nuova trasposizione passa all'acido diossiindolcarbonico DHICA (n.d.r.) incolore dal quale per eliminazione di CO2 si arriva finalmente al 5,6 diossindolo DHI (n.d.r ).

 

 

 

 

 

Come si è accennato di questi corpi sono stati isolati DOPA, DHI, DHICA gli altri sono ipotetici Le ultime due sostanze rappresentano gli ultimi precursori di nota costituzione delle melanine le quali si formano da essi per processi di condensazione e ossidazione ancora oscuri.

L'azione della tirosinasi sarebbe necessaria solo fino ad ottenere la sostanza rossa dopo entrerebbero in gioco fattori puramente chimici.............

 

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The synthesis of DHI and DHICA was reported in 1948. It was found that on exposure to air, especially in alkaline solution DHI rapidly undergoes oxidative polymerization to give a dark insoluble melanin, whereas under similar conditions the transformation of DHICA into melanin is much slower yielding a brown solution with no precipitation of melanin.

From these experiments it was inferred that of the two indole precursors only DHI was involved in melanogenesis. It was later confirmed that oxygen consumption and carbon dioxide evolution during the enzymatic oxidation of dopa were consistent with DHI being the major ultimate precursor of the melanin polymer.

 

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Schweizerische Chemische Gesellschaft, Herbstversammlung in Bern ,19. Oktober 1983,

Universitaet Bern (Organische chemie ) : Sur la chimie des melanines:transformation du dopachrome en dihydroxy-5, 6- indole carboxylate -2.

Luisa Stravs - Mombelli , Hugo Wyler, Universite de Lausanne

 

DHI and DHICA are obtained artificially and are not physiological melanin intermediates.

 

Selon le schema de la melanogenese de Raper , le dopachrome se transforme en dihydroxy- 5,6-indole(DHI) et dihydroxy-5,6-indole-carboxylate-2(DHICA), composes, qui par oxidation et polymerisation conduisent aux melanines(R.A.Nicolaus,Melanins,Hermann,Paris,1968.). Les conditions de formation et reactivitè de ces deux indoles sont pourtant toujours peu connues .

Nous avons reexamine les proprietes et la transformation du dopachrome par spectrophotometrie rapide. En solution a pH 6-7 , a temp . amb . ,le dopachrome est stable pendant quelques heures ; per contre, a temperature plus elevèe (60°) , la decomposition est complete en quelques minutes : le produit forme est le dihydroxy-5,6- indole (DHI). A pH èlevè ( 10) , on observe dèjà a temp . amb. la transformation rapide en dihydroxy -5,6- indole carboxylate- 2. (DHICA). A des pH intermediaires on obtient des mèlanges de 2 et 3 . La sèparation des deux chemins rèactionels coincide avec une ètape de dèprotonation du dopachrome (pK ca. 8 ) .Sur la base de ces observations, nous avons èlaborè des methodes simples pour la prèparation de 2 et de 3 . Ces composès,en particulier 3 et ses dèrivès mèthylès en position 5- et 6- , servent ègalement de substances de rèference en mèdecine . et ne pouvaient ètre obtenus jusqu' à prèsent que par des voies laborieuses.

 

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Only 50% of the Dopa is converted into melanin. DHICA is an artificial product of melanogenesis.

 

( L.Stravs-Mombelli,H.Wyler '' Reinvestigation on the formation of Dopa-melanin: New aspects of the autoxidation of Dopa '' ) presented at the XIIIth, International Pigment Cell Conference,Giessen, De,1983.

 

The autoxidative formation of dopachrome and its conversion to 5, 6- dihydroxyindole-2-carboxylate (DHICA) at high pH are shown . Under the usual autoxidation conditions, at pH 8, only half of dopa is converted to melanin : a new method is applied to separate it from the rest of the products, These ,mostly monomeric carboxylic acids , are shown to derive in part from the intermediate dopachrome by oxidative cleavage of the aromatic ring . For the main product a tentative structure is proposed and its relevance in connection with the melanin structure is discussed

 

 

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The reaction tyrosine-melanin occurs with formation of CO2 and H2O2 The H2O2 formation in the oxidative process of diphenols was a well known reaction in Raper's time.It was a surprise to find that the CO2 arises not only from the carboxyl of DOPA but also from the breakdown of the benzene nucleus In the oxidation of DOPA only 50% of DOPA is transformed into melanin the rest being formed by breakdown products of the DOPA derivatives .Morever it was found that DHI is not the only melanogen but also DOPA, and Cyclodopa may produce melanin.

In auto or enzymatic DOPA oxidation, using labelled precursors, melanins composed of dopachrome , cyclodopa, uncyclised dopa units, indole units, pyrrole units were obtained in disagreement with the Raper's scheme.

In conclusion while the difference between natural and synthetic melanins was not fully appreciated tyrosinase and DHI were generally accepted as the enzyme and the melanogen of the pigment cell.

The most updated Raper's scheme (see) reports DHICA as an important intermediate of melanogenesis this being a process which produces both DHI and DHICA (9). Nevertheless it has been suggested that melanin is a polyindolequinone with the of formula C8H5O2N

 

Raper's scheme which is considered to operate also in vivo is an arbitrary chemical representation of melanogenesis.All the compounds which are considered intermediates may give a melanin or may contribute to the melanin formation Another unexplained finding of Raper's scheme is that polymer has at least one oxygen and two hydrogen atoms more than the monomer (DHI or DHICA ).This is a genaral rule : oxidation of polyphenols leads to polymers which have one oxygen more with to the monomer (precursor) As a consequence some melanins are derived from a precursor which does not appear in Raper's scheme. In other words eumelanin has been considered until now polymer of artefacts (DHI and DHICA). Furthermore it was found that DHI shows potent cytotoxicity (10) As observed, on the basis of old laboratory data, some melanins are hydrated polyindolequinones characterized by the radical-polarone system of acetylene-black, having one oxygen atom more with respect to the precursor ( DHI-melanin, adrenalin-melanin , catechol-melanin).

 

The study of dopa-melanin , the chemical analysis of sepiomelanin, the study of degradation products, always show the presence of cyclodopa or dopachrome units in the natural polymer. (11)

Typical are the results obtained in experiments conducted on sepiomelanin in comparison to DHI-melanin:

a. oxidation of methylated pigments shows the presence of NH3 and CH3 NH2 in the breakdown products of the pigment (absence of DOPA units)

  1. in alkali fusion methylcatechol is obtained from the sepiomelanin and not from DHI-melanin.

 

It is surprising that all these results were systematically ignored by chemists of Pigment Cell Society (PCS) in the last past century. The paper (12) which reports the absence of dopa, dopachrome, leucodopachrome, DHICA units in dopa-melanin is in contrast with the presence of carboxylic groups in the pigment.

Cyclodopa (CDO ) or leucodopachrome was synthetized in 1968 and was found to be a very reactive melanin-producing substance, which is not converted under physiological conditions into DHI or DHICA. Nevertheless melanin has been considered a polyindolequinone (13).

 

The evidence against Raper's scheme and its enzyme control is impressive.

 

a. The presence of H2O2 and its function have not been considered.

b. The amounts of O2 consumed and CO2 developed in melanogenesis do not correspond to the equations of the Raper's scheme.

c. Elemental analysis of the melanin in vitro and in vivo do not correspond to the theoretical value of the proposed structure.

d. The DHI-melanin have one more oxygen and two fewer hydrogens per unit than the monomer,the polymer being (C8H5O3N)X rather (C8H3O2N)X.

e. The precursors DHI and DHICA are powerful cytotoxic products unsuitable in a cellular environment.

f. The pigment cell do not correspond chemically and physically to DHI-melanin and DHICA-melanin.

  1. The presence of ''pyrrolidine'' rings may be deduced by the isolation of degradation products like succinic acid and methylindole (Panizzi et al.,Gazz.Chim.Ital.,82,435-460,1952)

h. The presence of ''pyrrolidine'' rings is deduced from experiments of MALDI and FAB (Seraglia et al. Biol.Mass.Spectr. 22, 687, 1993 ), (private communication of AB ), Collisional spectroscopy (Allegri et al. Pigment Cell Research, 1, 87-93, 1987 ), IR ( Bonner et al., Nature,194, 1078, 1962 ), 13C solid state NMR ( Herve' et al.,BBA, 1204, 9, 1994) ; Crescenzi et al., Liebigs Ann.Chem., 563, 1994.

  1. Retention of the hydrogen of lateral chain of DOPA is well known by isotopic studies ( G.Prota, Melanins and melanogenesis,AP,San Diego, 1992).

 

To sum up :

 

Raper's scheme of melanogenesis which put forward DHI and DHICA as biological precursors of the eumelanins seems to be erroneous . A polymer derived from the aminoacid CYCLODOPA at different oxidative levels could reasonably represent the pigment cell biogenesis.

New and old experimental data suggest for sepiomelanin, the well known pigment of ink sack, a structure based on CYCLODOPA and DOPACHROME and their decarboxy derivatives The occurence of natural dopa-melanin as well cyclodopa-melanin can not be excluded. Some synthetic melanins ( DHI-melanin, adrenalin-melanin, catechol-melanin) studied until now are characterised by the radical-polarone system of acetylene black and by having one oxygen more than the starting monomer ( formation of gem-diol groups).We suggest the same chemistry for eumelanin

Some differences between the natural and the synthetic melanins could be related to the presence and distribution of gem-diol groups, pyrrolidine rings, and carboxylic groups in the polymeric material.

Melanins belong to a new class of pigments which are characterized by the electrochromophore of acetylene-black..

Cysdopa through the 5-oxy-7-alanyl-benzothiazinone forms pheomelanins and pheochromes.

 

MELANOGENESIS AND PHEOMELANOGENESIS IN VIVO

DOPA----------melanin

DOPA----------cyclodopa-------------------------------------melanin

DOPA----------cyclodopa-----decarboxycyclodopa--------melanin

DOPA----cysteine------cysdopa------------------pheomelanin, pheochromes.

 

 

 

.

 

Number of cationic centres and unpaired electrons to be determined .

Elemental analysis of CDO and DCDO and related polymeric structure are to be calculated and compared with the analytical value of the eumelanins.

CDO = Cyclodopa

DCDO = Decarboxycyclodopa

 

 

Naples, September 2001

 

Accademia Pontaniana

Via Mezzocannone 8

I-80134 Napoli.

accponta@tin.it

pontaniana.unina.it.

 

rnicolaus@tightrope.it

 

bologne@unina.it

 

bnicolaus@icmib.na.cnr.it

 

 

www.tightrope.it/nicolaus/index.htm

 

www.organicsemiconductors.com

 

 

 

Bibliography

 

 

 

  1. Raper, H.S. The Tyrosinase-Tyrosine Reaction. VI. Production from Tyrosine of 5,6-Dihydroxyindole and 5,6-Dihydroxyindole-2-Carboxylic Acid, the Precursor of Melanin. Biochem. J. 21, 86-96 (1927).

 

(2) Nicolaus, R.A., Melanins, Hermann, Paris (1968).

(3) Palumbo, A., Di Cosmo, A., Gesualdo, I., Hearing, V.J., Subcellular Localization and Function of Melanogenic Enzymes in the Ink Gland of Sepia Officinalis. Biochem. J. 323, 749-756, (1997).

(4) A literature reference reports that melanin weight, deduced from mass experiments, ranges between 500 and 1500 m/z; Napolitano, A., et al., Rapid Comm. Mass Spectr. 10, 204-8 , (1996) We propose to assign the ignoble prize 2002 for the discovery of melanin molecular weight. Candidate names will be communicated later.

(5) McGinness, J.E., Corry, P.M., Proctor, P.H. Amorphous Semiconductor Switching in Melanins. Science 183, 853-4 (1974)

 

(6) Kirkpatrick, D.S., McGinness, J.E., Moorhead, W.D., Corry, P.M., Proctor, P.H. Melanin-Water-Ion Dielectric Interactions. Pigment Cell 4, 257-262, Karger, Basel (1979)

 

(7) Mosca, L., Blarzino, C., Coccia, R., Foppoli, C. & Rosei, M.A. Melanins from Tetrahydroisoquinolines: Spectroscopic Characteristics, Scavenging Activity and Redox Transfer Properties. Free Radical Biology & Medicine 24, 161-7, (1998).

 

(8) L.Linnel, H.S.Raper (1935) Biochem. J. 29, 75-85 ; R.D. Hoskin-Heard, H.S.Raper, Biochem. J. 27, 36 - 53 (1933); W.L. Duliere, H. S. Raper Biochem . J. 24 , 239- 249,(1930) ; H. S. Raper , Physiol. Rev. 8 , 245- 282, (1928) ; H. S. Raper , Biochem. J. 21 , 89- 95, (1927) ; H. S. Raper Biochem. J. 20, 735- 745, (1926) ; H. S: Raper , A. Wormall 19, 84- 91, (1925).

All the papers cited were made with funds of Government Grant Committee of the Royal Society.

(9) Prota, Pigment Cell Res. 13, 283-293, (2000).

(10) J.M.Pawelek, A.B.Lerner, Nature, 278,627, (1978).

(11)M.Piattelli,R.A.Nicolaus,Tetrahedron,15,66-75, (1961), pag.66,71;M.Piattelli, E.Fattorusso, S.Magno,R.A.Nicolaus,Tetrahedron,19,2061-2072, (1963) ,(pag.2061,2064,2066); R.A.Nicolaus, M.Piattelli, J. Polym. Sci.,58, 1133-1139, (1962).

(12) V.J.Hearing,T.Ekel, P.M.Montague, J.M.Nicholson, BBA, 611, 261-268,(1980).

(13) H.Wyler, J.Chiovini, Helvetica Chimica Acta, 51, 1476-1494, (1968).