Link 5-Melanins,Cosmids, Fullerenes.

http://www.tightrope.it/nicolaus/index.htm

 

In memory of the mathematician Donato Greco (1923-1995)

It is not the only objective of organic chemistryto elucidate the structure of organic natural
products but also to develop the nanochemistry of Nature.
The black particles constitute an universal component of great interest
for the planet evolution and living system.

 

Key words: melanins, conducting polymers, charge transfer complexes, fullerene, graphite, cosmids.

 

Introduction

Black materials are present in the Biosphere (eumelanin, phaeomelanin, allomelanin), in the Lithosphere (minerals, graphite, fullerenes), in the Atmosphere (primary and secondary pollutants, smokes), in the Hydrosphere (seas, lakes, rivers) and in the Cosmos (fullerenes, cosmids) (1, 2).All black materials belong to the solid state, following its laws.The intra or extracellular black materials of the biosphere are called melanins, which are classified in to three groups, the Eumelanins (produced by the polymerization of a nitrogenous melanogen), the pheomelanins (obtained from the polymerization of a sulphurated melanogen), the allomelanins (generated by the polymerization of polyphenols).Hybrids of the abovementioned groups can be easily formed through copolymerization or by the presence of foreign material.The polymerization of melanogens is of radical type. Attempts to isolate intermediates of relevant interest for the particles structure (melanins) have failed completely.

The black pigments are solid materials with band structure having a gap less than 2eV.They are formed from melanogens which are organized in to natural structures called melanosomes, having the soccer or rugby ball shape of fullerenes.The colours of melanins are those of a pure semiconductor.The natural materials show the properties of amorphous semiconductors with low electric activity at the borderline with an insulator state.As with graphite and fullerenes, this state is subject to changes by doping.All black pigments, melanin included, show a remarkable affinity for metals (presence of -COOH) as well as for organic electron donors.The biological properties of the melanin/chloropromazine and melanin/quinine complexes have been well investigated.On the contrary, the ability of melanins to form charge transfer complexes has been poorly treated and limited data are available on their conductivity.Melanins after decarboxylation, or those arising from neutral melanogens, are able to bind acids and metalloids.In other words, there are melanins bound to counteranions (like pyrrole-black) as well as to countercations.The conductivity of those materials is poorly understood.The oxidative cleavage of eumelanins yields nitrogenous polycarboxylic acids (like 2, 3, 4, 5, -pyrroletetracarboxylic acid), that of allomelanins graphitic acids (like mellitic acid), that of pheomelanins has not yet been sufficiently investigated. The presence of a graphitic core in melanins has been confirmed by X-ray analysis.A fullerene cage structure is proposed for black materials.A new approach to eumelanin research starts from an intact biological entity : the melanosome. In this way, physics and chemistry are referred to melanosomes for the first time.The laser beam represents in this connection a useful tool for their chemical investigation.It is worth mentioning that, under the action of a laser beam, both graphite and melanosomes are fragmented (typical for graphite is the formation of the red C60 fragment).The fragments, which are formed through a reaction comparable to an explosion, can be easily detected and identified with MALDI-TOF, LDI mass spectrometry.In the common conditions which are used in the MALDI-TOF, LDI tecnique, melanins, as expected, are not volatile.

Black materials are found in biosphere, lithosphere, atmosphere, and the cosmos. Among the natural pigments melanins occupy a unique position thanks to their physical, biological and chemical properties.The most important physical properties are electrical conductivity in amorphous semiconductors (2), the display of ''threshold switching'', the existence of ''planar stacks of monomer units", the absorption of ultrasound in the region of 1 MHz, colour attributable more to the electronic transition in band materials than to orbital transitions and, finally, the capacity to form'' charge transfer complexes '' (drugs and metals affinity). In the biological field it is to be noted that mammals melanogenesis is a rare known radical process occuring in vivo. In the field of structural chemistry the research has given poor and confused results.

 

Biosphere

Melanins(1) have been the subject of numerous investigations, and a number have been isolated and subjected to chemical examination. A satisfying method for isolation and purification has not yet been found until today. The most satisfactory preparative method is carried out on the melanosomal fraction. The situation is less satisfactory among the lower animals where there is much reliance on inadequate histochemical tests for the purpose of identification, and much of the older work does little more than record the existence of melanins (black pigments, black materials). Search is usually made for an accompanying tyrosinase system, although this too, is of uncertain value, as these enzymes are not specific.In consequence the results are ambiguous.Nevertheless it seems likely that the innumerable shades of black and brown frequently observed in vertebrates and invertebrates are produced mainly by melanins occurring in different states of oxidation and aggregation, accompanied sometimes by other pigments. As may be seen from the literature these pigments are of general occurrence in many phyla.

The most convincing description of melanin in a primitive animal is the plumose anemone Metridium senile. Coloured varieties were found to contain a black granular pigment in the endoderm which responded to the usual tests and a more diffuse brown melanin in the ectoderm; both white and coloured animals possess a complete tyrosinase system. Tyrosinase is present in the tissue fluids of sponges (e.g., Suberites domuncula, Tethya lyncurium), but its relevance to the black pigments occasionally observed in the Porifera is unknown. It is of interest that a magenta chromo-protein found in the jellyfish, Pelagia noctiluca contains a brown chromogen which appears to be an indole derivate. However, no tyrosinase could be detected, and its relationship to melanin, if any, is doubtful. The flatworms Planaria lugubris and Polycelis nigra vary in color from white to black.The histochemical tests show, that the characteristic black granular pigment in the large epidermal melanophores of Diadema antillarum and Thyone briareus is melanin. The amebocytes of these animals are associated with a phenolase complex, and if the coelomic fluid is exposed to air, rapidly become red and then gradually darken to brown or black, producing a pigment very similar to that in the skin. A similar enzyme system is present in the amebocytes of the holothurioid echinoderm Holothuria forskali, which contains an abundance of melanin in the body wall. Integumentary black pigment is frequently visible in gastropods and bivalves, and a copious supply is present in the specialized anal gland or ink sac of cephalopod molluscs. The sack of Sepia officinalis contains up to 10 ml of an intensely black suspension of melanin granules. Tyrosinase activity in both Sepia gland and dried Sepia sack has been reported. There is a recognizable pattern in the colours of deep water fishes.

The animals that inhabit the surface down to about 150 m are either transparent or blue;below this depth and down to about 500 m the inhabitants are mainly silvery and greyish fish;below this depth again the population consists of dark coloured fish (Fig.1) and red prawns(Taonius megalops, Calliteuthis reversa, Taonius megalope, Gnatheuphausis, Pentacrinus, Astronesthes, Melanocetus). Many of the deep sea cephalopods such as Mastiogteithesis and Calliteuthis are also red.The development of the colour may have a concealing effect since at depths where there is no red light, these creatures will appear black.This colouration is peculiar to salt-water animals that dwell in dark regions whereas cave-dwelling animals are usually a pallid white. The colours of deep sea animals may, of course, have no ecological significance and they could act as stores of waste products or of metabolically useful intermediates. It is known that starvation causes the depletion of carotenoids from the ink and liver of Octopus bimaculatus.In animals such as the euphausiids their red colour renders them invisible in the twilight zone of mid-ocean waters where red light does not penetrate, and they are not rendered visible by the light usually emitted by predators.But some of the fishes that feed upon them emit red light from large luminous organs.It may be that the colours of some fishes have been influenced not only by incident light from above but also by the need to avoid reflections from light emitted by predators. The melanin structure of these fish is interesting because, as is known, pigment formation is influenced by incident light, oxygen, and pressure.Unfortunately no research on the conductivity of these interesting pigments has been hitherto carried out.

Fig.1    Adapted from P.N.Dilly ''The enigma of colouration and light emission in deep-sea animals'' Endeavour XXXII, n° 115 (1973).

The black pigment in the hypodermis of certain crustaceans (e.g., Cancer pagurus, Crangoon vulgaris) seems to be associated with tyrosinase and shows the general characteristics of melanin.There is good evidence that melanin formation is responsible for the underesirable "black spot" darkening of fresh shrimp. The blood of many insects, of which tyrosine is a common constituent, rapidly darkens on exposure to air; likewise, many pale newly hatched larvae darken in a short time after emergence.This is associated to a polyphenol system which is very widely distributed in insects and melanin formation is clearly implicated, although the pigment is not usually granular. The position is complicated, however, by the presence of protocatechuic acid and other polyphenols, and it has been difficult to disentangle the process of melanization from cuticular darkening due to sclerotisation. However, the two processes are to some extent independent. In experiments with the cuticle of the desert locust (Schistocerca gregaria) it was found that blackening could occur unaccompanied by tanning. The melanin formation in the larvae of the blowfly (Calliphora vomitoria) was completely inhibited by injection of phenylthiourea, whilst pupal hardening continued normally. There is much evidence that tyrosinase is distributed generally throughout insect tissues, but a clear relationship between pigments and the presence of the so called tyrosinase has not yet been well established. The chordate melanins are frequently prominent in skin, hair, feathers, and scales and occur also in choroid, peritoneum, pia mater, and other membranous tissues in mammals, birds, reptiles, amphibians, and fishes. Melanotic tumours are not uncommon among vertebrates.The type of melanins and the size and shape of melanosome is genetically determined, but the radical process which forms melanin may be influenced by UV light, pH, O2, pressure and other factors.Unknown biological factors cause pigmentary disorders like vitiligo, albinism, and melanoma (1e).Melanoma is not limited to humans but develops in horses and other groups of verterbrates including fish of the Xiphophorus genus(1f). The available evidence (EPR, ''colour'', electroactivity, etc.) suggests that mammalian melanins are probably all similar in nanochemical character. Less is known concerning the nature of other vertebrate melanins although their distribution in birds and amphibia has received much attention by geneticists, and some remarkable patterns of melanization have been described. The adaptive colouration of axolotls (Ambystoma mexicanum), frogs (Rana temporaria), and certain fish (e.g., Lebistes reticulatus) arises from their ability to control melanin distribution in response to background illumination. In dark surroundings the dispersion of melanophores increases, but the opposite effect is produced by a light background.What happens in abyssal fish is unknown. Integumentary melanins are indirectly responsible for many structural colours displayed by animals, notably the bluesdue to Tyndall scattering seen in certain skin areas of numerous fishes, reptiles, and mammals and in the feathers of birds. Pigmentary and structural colours are sometimes combined, this can be observed in the green feathers of birds which change to Tyndall blue on extraction of the concomitant yellow carotenoids. Melanin also plays an important background role in the display of iridescent (interference) colours frequently seen, for example, in the skin of reptiles and fish. The coexistence of black, blue, and green areas in many fishes, together with changeable iridescent colours, is not uncommon, and all these can be attributed, directly or indirectly, tothe relative distribution of melanosomal pigments in melanophores.

Neuromelanin is a granular pigment of the nigrostrial neurons in the brain stem of humans which in many respects is different from the melanin formed in epidermal melanocytes.The highest levels of neuromelanin are found in the neurons of the Substantia nigra and Locus coeruleus which are known to contain relatively

high concentrations of dopamine and norepinephrine respectively.Neuromelanin is present in Homonidea, Cercopithecoidea, Ceboidea, Lemuroidea. Small amounts of neuromelanin have been found in horses, carnivores, guinea pigs, rabbits, rats, mice and amphibians.

Neuromelanin occurs almost exclusively in catecholaminergic neurons (1d).Probably the most important property, from a biological point of view, of these pigments is their electroactivity.

The formation of dark-brown or black pigments during the normal development of plants is a well-known phenomenon commonly observed as markings on petals and leaves, in the spores and hyphae of the higher fungi, in senescent leaves and seedpods, and in the dead cells of bark, seedcoats, and pericarps. Furthermore, many plant tissues darken rapidly on injury. It is likely that many of these pigments arise by the oxidation of phenols followed by conversion into complex black products by polymerization and interaction with proteins and aminoacids.

The Japanese lacquers are the oxidation products of polyphenols exuded by many Anacardiaceae, and the black phytomelanes elaborated in the fruits of certain Compositae. It may be noted that the essential oils of many Compositae contain polyacetylenic compounds (which recall acetyleneblack) which also occur in higher fungi. These highly unsaturated substances are frequently unstable and blacken on exposure to light. The glucoside, aucubin, is responsible for the blackening of the leaves, fruits, and other parts of the Japanese variegated laurel Aucuba japonica and is found in the leaves and seeds of many other plants. The black material is an oxidation product of the aglycon aucubigenin.

Another example of an allomelanin is seen in the fungus Daldinia concentrica formed from a naphthalene polyphenol which is oxidized to a black material.

Black material is obtained from Dahlia tubers and can be detected in the broom Sarothamnus scoparius.

The blackening of bananas, which occurs frequently on storage, is due to oxidation of dopamine present in the skin and pulp. A number of bacteria (e.g., Bacillus niger) produce black pigments. The melanin (containing nitrogen) formed by one of these (B. salmonicida or a close relative) forms a dark brown solution in aqueous sodium hydroxide which is reprecipitated by acid. Little comparable work appears to have been done on the presumed melanins of fungi or higher plants, although the blackening of potato tubers has received some attention. Much more work on the isolation and purification of plant melanins is required, as only in this way can they be properly identified and compared with the animal pigments. From Vicia faba dopa may be extracted ;a hint as to the possible course of melanogenesis is given by the co-occurrence of tyrosine and dopa in a number of melanin-producing plants and by the observation that the blackening of plant tissue sometimes proceeds via a red phase, as, for example, when potato slices are ground up or exposed to chloroform vapor. Very interesting is the mould Aspergillus niger which produces Aspergillin, a graphite- like material (2) (8).

Fig.2.    The photo was a gift of Professor A.Quilico (Milan 1960). Aspergillus niger an organism that generates a rare graphite-like pigment.

Moreover, black pigmentation is often highly localized in individual plants in contrast to the more general distribution of the enzyme ''tyrosinase''. This may be attributed either to the presence of inhibitors (ascorbic acid is of common occurrence) or to circumstances which, under normal conditions, prevent the enzyme and substrate having simultaneous access to oxygen and more probably to the fact that the final stages of melanogenesis do not require the presence of an enzyme. This situation breaks down on disruption of the cell structure and melanin formation can then proceed; alternatively injury to the tissue may result in oxidation of the inhibitor with consequent loss of inhibitory capacity. An example of ascorbic acid inhibition is seen in Stizolobium hassjoo; blackening takes place most rapidly in the younger leaves, which have the lowest concentration of ascorbic acid. A study of melanin formation by a black mutant of the fungus Neurospora crassa it was possible to correlate increased pigmentation with increased tyrosinase activity, but the study was unable to demonstrate whether this was due to the presence of a larger amount of enzyme or to a partial block in the synthesis of a tyrosinase inhibitor. The inhibitor in this case may be a thiol, as it was found that tyrosinase activity in Neurospora is dependent upon sulphur nutrition.The fungal pigments are in general wallbound and extracellular in nature and are formed by a radical process starting from diphenols (catechol, 1, 8-dihydroxynaphthalene, glutamyl-3, 4, -dihydroxybenzene). To date the only pigments fully characterized by chemists are those of the large ascomycetes Daldinia concentrica, Ustilago maydis, and Aspergillus niger.Ustilago maydis, a well- known corn parasite(Fig. 3), was found to have a pigment which is a polymerization product of catechol or its derivatives (1c), (1d).

Fig.3.The corn parasite Ustilago maydis which causes serious damage to agriculture(Institute of Organic Chemistry, University of Naples, 1962).

 

Black materials

Black materials are found not only in the biosphere but also in the lithosphere (minerals, humic acids, graphite, black shale), in the hydrosphere (black particles in seas, lakes, rivers, originating probably from the biosphere), in the atmosphere (polluttants, soot), and the cosmos (cosmids, fullerenes).

Many synthetic pigments (materials) are also known, some of them related to melanin such as:

pyrroleblack, indoleblack, benzeneblack, quinolineblack, DHIblack, adrenalinblack, tryptophaneblack, serotoninblack, DOPAblack, acetyleneblack, dihydroxyquinolineblack catecholblack, polyphenyleneblack.

In the last fifty years melanins have been intensively studied from a chemical and physical point of view without great success. Some precursors (melanogens) have been discovered but the process of melanogenesis and its biological significance remain to a large degree obscure. Although in recent years, many organic compounds and many black polymers were found to be conducting materials, the electroactivity of melanins has never seriously been taken into consideration. Consequently the biological aspects of the problem have remained confused.

 

Chemistry and Physics

The black pigments are band structure materials with a small gap (eV = 0.1 – 1.7) value, as expected from theoretical calculations.

Generally they are amorphous semiconductors but black crystalline charge transfer complexes are also known. Melanins behave like amorphous semiconductors their colours being generated more by band transitions than by electronic intramolecular transitions as usually reported for natural and synthetic pigments (2), (3).

Melanins, the natural black pigments, belong to the solid state, or to the nanostructure area which comprises supramolecular chemistry which studies the chemical properties of aggregates, particles, objects of 10-100 nm. dimension (1 nm = 10 –9 m; 1 nm = 10 A°). This concept of supramolecular or nanostructural chemistry has never been applied to melanins. Nanochemistry requires, particles having almost one characteristic dimension lying between 1 and 50 nm. An approch to melanin nanophysics, nanobiophysics, nanochemistry is tentatively described.

Melanogens are compounds which in different chemical and physical conditions produce black materials usually in aggregates or particle form. In nature these particles are organized into structures called melanosomes with the characteristic shape of a succer or rugby ball.

The colours of "melanins" correspond to the colours of the pure semiconductors of Fermi’s prohibited band. (Fig.4) Many black materials are found in nature and are called eumelanin, pheomelanin, allomelanin.

Fig.4     Cadmium sulphide has extension of prohibited Fermi's band of 2.6 eV and for this reason appears yellow, whereas cadmium selenide with a value of 1.7 eV is black.The reddish colours are intermediates.Adapted from F.Celentano ''Luce colore e materia'', Le Scienze, quaderno 21, Febbraio 1985.

They are electroactive materials (2, 9). The conductivity depends on:

  1. the type of counteranion or countercation
  2. the method and conditions of preparation
  3. the type (orientation) of conductivity measurement adapted: parallel or orthogonal to the plane of basal cleavage
  4. the number of unpaired electrons.

Synthetic or biosynthetic melanins for conductivity experiments may be prepared by electro – oxidation of melanogens. Alternatively the preparation of a melanin can be carried out with very small quantities of peroxidase/ H2O2. The yield of melanin is a good criterion for evaluating whether the oxidative method chosen is suitable. The preparation which brought about the discovery of the pyrroleblack conductor (3) is still valid to day. The polymerization is carried out by electrodes (duration 2h) with a costant current of 100 mA° of a melanogen solution (2g) in 100 ml 0.1 N H2SO4. The laminar deposit which forms on the platinum electrode is washed out with distilled water and allowed to dry. The conductivity is measured both on the plaque and on pills obtained from the powdered plaque. The blacks give an EPR signal in the range of 2, 0025 – 2, 0045 g values. The blacks have the electrolyte of the medium incorporated as a counteranion. The observed values are reported in s= W -1 cm-1. The films only form if the monomer is oxidised from above its oxidation potential. If the film which is deposited is a conductor the film continues to grow and the current continues to pass. The pigments are described by shade colours as black, blue-black, matt-black, brilliant-black, pitch-black, brown-black, copper-black, brilliant-black, golden-black, and metallic-black. Melanins and black pigments show an affinity for metals dependent to a large extent on presence of -COOH and for organic electron donors.Biologically the complex chloropromazine/melanin and quinine/melanin is well studied. The possibility that melanins could form charge transfer complexes has been poorly considered. In consequence there is little information available on the conductivity of these complexes.If melanin is decarboxylated or formed from a neutral precursor the polymer may bind to acids and metalloids.

Black pigments have the characteristic properties of amorphous semiconductors;

that is weak electrical activity (10-11 – 10-7 W -1 cm-1) at the limit of the insulating state, the conductivity being increased to interesting values by doping.

Actually the chemistry of melanins is still obscure. The causes are due to :

  1. having considered the transformation of the melanogen into melanin a  process under enzymatic control.
  2. having worked with altered or not purified materials.
  3. having not understood the very nature and electroactivity of black materials and of melanins.

 

The re- examination of old papers and the extension of nanochemistry to the study of black materials has led us to a new aspect of melanins (2).

The pigments are classified as from three basic structures based on pyrrole, indole, benzene.

 

The formulae above reported are called cetoplasmatic (2).They show the presence of unpaired electrons and cationic(anionic) centers with counterion.All the positions of the ring may be involved in polymerization.Oxidative level and polymerization degree are not known.

Pyrrole-black, indole-black, benzene-black and derivatives give, on oxidation, a mixture of polycarboxylic acids (4). The formation of these acids means that polycondensed or polysubstituted structures are present in those black pigments. Recently [ see (2) ''Coloured organic semiconductors : melanins '' pag.34 ] a structure for an indole oligomer which can explain the formation of pyrroletetracarboxylic acid by oxidative degradation was reported. Pyrrole –2, 3, 4, 5 – tetracarboxylic and tricarboxylic acids were extracted from the mixture of degradation products obtained on oxidation of melanins prepared from rat melanoma, human hair, dog hair, horse hair, ox hair, ox choroide, sepia ink(7), squid ink, octopus ink, chicken feathers, pigeon feathers, amphiuma liver, axolotl liver (4), sepia tapetum lucidum (5). The same tetracarboxylic acid was obtained from oxidation of pyrrole black and DHI (5, 6 - dihydroxyindole) black. (5, 5b)

2, 3, 4, 5 – Pyrroletetracarboxylic acid is a characteristic product which is found in the oxidative degradation mixture of eumelanin. It was first prepared in 1954 (7) From a structural point of view this acid is the most significant product yet found.It shows the presence of a "graphitic core" in the eumelanins.

Pyrrole – 2, 3, 4, 5 – tetracarboxylic acid combines with the ion K+ to form a monopotassium salt which is insoluble in strong acids, in water and in organic solvents. It is believed to be a "clathrate". In order to readily isolate and identify the acid it is necessary to oxidize the pigment with peracetic acid (1b) avoiding any contact with sodium or potassium. In these conditions the yield is close to that of 2, 3, 5 –pyrrole-tricarboxylic acid and considerably higher to that of the dicarboxylic acids. Curiously, some authors who have been unable to isolate or identify the acid claim that it is not a genuine product, i.e. it is an artefact (5). Recently an interesting polysubstituted indole structure has been proposed for an indole tetramer which could be the terminal part of indole-black (the simplest eumelanin known). The structure presented (6) justifies the formation of the acid pyrroletetracarboxylic in the oxidative degradation processes.

In conclusion, every analytic and structural approach to the study of eumelanins which does not consider the presence of the pyrroletetracarboxylic acid among the degradation products must be considered invalid.

In the MALDI study of melanin, pyrrolic acids are element of great interest(9).

The planar graphitic core present in melanins may show remarkable anisotropic properties.The value of physical quantity depends on the considered direction since both electric and thermic conductivity are higher if measured parallelly to the cleavage plane (9).

For physical and chemical properties graphite may be considered the most simple allomelanin. The formation of mellitic acid on oxidation is a feature common to some allomelanin in particular Aspergillin (8).

Furthermore graphite and allomelanins have in common the colour, the EPR signal the X-ray diffraction pattern, and electroactivity. No vaporization of melanin occurs in the usual condition of the MALDI-TOF (matrix assisted laser desorption ionization-time of flight) or LDI techniques, but an explosion is produced by the action of laser on melanosomes of Sepia ink (9).This is a confirmation of what occurs with epidermal melanosomes (15) and the result probably may possibly be extended to all melanins.

The allomelanins which probably incorporated graphite structures must include Daldinia-melanin, Ustilago-melanin, Humic acids, and Aspergillin (10) (11) (12) (13).

This latter material is particulary interesting because oxidative degradation produces mellitic acid the same acid which can be obtained from graphite. In other words it may be suggested that aspergillin is a " substituted graphite" synthesized by a living organism.

Aspergillin (8) is a pigment which gives to Aspergillus niger its characteristic dark colour. (Fig.2) The typical colouration of the conidia is one fundamental distinctive character of numerous species. The appearance of the pigment in the spores is at first yellow-ish, becoming green-yellow, green-grey, and finally brown-black.

These "colours" are typical of amorphous semiconductors. The oxygen in this synthesis plays a special role next to the iron in that the quantization of the chemical elements can lead to a gold-yellow pigment. The IR spectrum in the molecular phase is very similar to that of humic acids. Band theory may be extended to these amorphous semiconductors with polycondensed or polysubstituted nuclei.

The pigment is purified by dissolving it in NH4OH 5% (solubility l g per 1000 ml) and reprecipitating with 2N HCl and washing with H2O. The dissolving in NH4OH and the precipitation with HCl are carried out several times. The insoluble fractions are discarded. The final product is washed with H2O, ethanol, acetone, H2O. The wet product is dissolved in water and the solution is used for measuring the conductivity (difference between conductivity of the solution and of distilled water) or is studied in the solid state.

The elementary analysis (Sephadex purification) of aspergillin gives C%=52, 7 H%=4, O% 36. Nitrogen which is present may be part of a protein (9). Aspergillin oxidised with H2O2 10% yields mellitic acid (1g from 4 g pigment) and oxalic acid; on reduction with Raney Ni it yields perylene next to hydrogenated derivatives of phenanthrene and napthalene.

These degradation products are precious elements for MALDI-TOF, LDI, and in general, mass spectrometry studies on melanins.

Aspergillin can be dissolved both for measurement of the electrical conductivity (perylene itself is a good conductor if doped) and for spectrometry studies.

Further indications of a polysubstituted or polycondensed structure of melanin derives from X- ray studies.

A number of '' purified ''natural and synthetic melanins have been examined by X- ray diffraction (14). A diffuse ring, centered at a Bragg spacing of 3, 4 A° was consistently found in samples of melanin from animal sources, and a similar ring at 4, 2 A° in all melanins obtained from plants. Models for these two polymer types, based upon the current concept that they primarly involve indole and catechol monomeric units respectively, were then evaluated by a Montecarlo method. From the comparison of the observed spacings with the calculated ones it was concluded that the 4.2 A° spacing in catechol melanins is probably related to the average interaction between adjacent monomeric units with mutually random orientations. The 3.4 A° spacing observed in indole melanins appears to derive from the tendency of indole monomers (probably of adjacent chains) to aggregate in near parallel stacks. Some randomness in the form of translations and relations parallel to the planar groups is consistent with diffraction patterns. An interesting finding was that the diffraction pattern of synthetic of catechol melanins gives the 3.4 A° spacing found in the indole melanins of animal origin. The nanochemistry of these interesting materials has not yet been further developed.

The X- ray diffraction data allow concluding that there is a short range order at the molecular level in all melanins. A graphitic core is always present in eumelanin and allomelanin. A consistent finding with all samples was the lack of structure in their diffraction patterns of elements showing any significant crystallinity.

Fig.5.    Electron micrograph of melanosome fraction.Taken from V.J Hearing, M.A Lutzner '' Mammalian Melanosomal Proteins : Characterization by polyacrylamide Gel Electrophoresis '' Yale J.Biol.Med. 46, 553 (1973)

The separation between adjacent planar groups in the plant and fungal melanins seems to be much larger than in the case of the animal and synthetic melanins studied. Density measurements on the melanin pellets seem to support this conclusion. The adjacent planar groups in the synthetic and animal melanins are probably parallel and they may also have order extending to longer ranges. The electronic properties of these melanins, including their black colour, may be related to the possible presence of "graphitic like structures" and to the shape and size of granules of the pigment as seen at the electron microscope level. The high density of these melanins may be responsible for the observed contrast in the electron micrographs of melanin – containing tissues as depicted in (Fig. 5).

The melanosomes show soccer and rugby ball shapes like those seen in and electron micrographs of fullerenes. How the fullerenes are built up is depicted in Fig.6. In the case of melanosomes the cage may firmly enclose proteins, lipids, metals melanogens, oligomers, water, gas.

Fullerenes are formed from fine aggregates of even numbers of carbon atoms and are obtained after evaporation and cooling of graphite (laser method).

It is possible to obtain red and black fullerenes (C60 magenta, C70 red, C120 black). The C60 which can be obtained in relative good yields, form a spheroid structure (icosahedron trunk). The sphere would be formed by the collapse and curling up of graphite. Fullerenes show interesting optical properties like non – linear variation of transparency, as also shown in melanin, as a function of intensity of incident light.In order to understand the dynamic aspects of fullerene formation a new model, the Wakabayashi-Achiba model, was suggested for the growth mechanism of fullerene (17).The model is based on the criteria that the fullerene is formed by a sequential stacking with numbers and combinations of even-numbered carbon rings (atoms) and both intermediates and the final product consist of only pentagons (cyclopentane, pyrroles) and hexagons (benzene) or a combined system (benzocyclopentane, benzofurane, benzopyrrole, indene). Although the process which leads to cage formation seems to require high temperature, a similar process occuring for in vivo melanosome formation cannot be excluded, expecially for substituted hydroxyl hexagons. Recently formation of fullerenes by pulsed laser irradation of gaseous benzene was observed (34).

Fig.6

A phenomenon very similar to the graphite-fullerene laser collapse occurs with melanosomes both in vitro and in vivo in the form of the explosive vaporization of melanosome.We suggest that melanosomes may be considered substituted (H, N, OH, COOH) fullerenic cage structures.Melanosomes and fullerenes may represent evolution phases.Intermediary of particles produced by stars and on the earth.Melanosomes are subcellular particles which contains melanin and a primary protein matrix. The complex melanin+protein is sometimes not chemically bound as in the ink sack. The black material which loses CO2 by heating and by acid hydrolysis is, from a chemical point of view, very reactive. On the other hand electron micrographic studies show that melanosomes after strong acid hydrolysis preserved their size, shape and, to a great extent, their ultrastructural features of either lamellar or granular melanosomes(23).Molecular weight, chemical structure can not be determined for melanins or melanosomes. Only the techniques of nanochemistry and nanobiochemistry could reach some success.

Recently the problem of characterizing the black materials (melanins) by means of light scattering techniques, (static and dynamic) was investigated (32).The static technique allow to identication the ''macromolecular '' properties of the ink sac melanin, autoxidative dopamelanin, ''enzymatic'' dopamelanin. Natural melanin at physiological pH polymerizes and aggregates in particles of average molecular weight - 10 6 and radius of gyration Rg about 90 nm, with spherical symmetry and with an open, rigid and not largely hydrated texture. Synthetic melanin prepared by autoxidation at physiological pH appears the more reliable structural model for the natural material. Both natural and synthetic melanins grow as fractales aggregates of small units, with kinetics depending on the conditions of the medium. The black solid is considered in general a very stable material but melanin is easily disintegrated by lysosomal digestion, bland action of H 202, heating (loss of CO2)and by LASER beam.This seems in agreement with a cage or globular structure of melanins. Pulsed lasers are capable of selective photodermal destruction of melanosomes (15).

When sufficient radiant energy is delivered from a pulsed laser to the skin, the melanosomes in the stratum corneum reach a temperature that exceeds the treshold for explosive vaporization(15). The result is an explosive event which disrupts the laminated stratum corneum structure to yield an observable whitening of the skin surface. Probably the explosion begins with dilatation of the metallo protein of the cage.

 

Fullerenes, cosmids, melanins.

It was observed that some of the dust clouds of the Milky Way Galaxy were made of long carbon chain molecules (the polyynes). Among the polyynes molecules like HC5N, HC7 N, HC9 N were discovered.

On earth the atomic relationship of some of those polyyne structures corresponds to pyridineblack, and quinolineblack (two isomers).The question of the existence of indoleblack HC 8N and pyrroleblack HC 4N will be discussed later.

 

We have several different types of intermediates participating in the formation of carbon microparticles in the gas phase.Cumulene, polyyne chains, monocyclic ring have been suggested .Some of the more stable clusters are closed cage fullerenes like C60 or other more reactive ones which ultimately result in characteristic carbon microparticles very similar to melanosomes.The small carbon species C2 and C3 were identified in comets and C5 in the cool carbon star IRC+10216. In the positive ion mass spectrum of the clusters with up to 30 or so atoms the 11, 15, 19, 23 magic number sequence (@=4) has been recognized.Giant fullerenes might also exist. C540 would have a diameter three times that of C60.The icosahedral shape is quite clear at this size.The structure is really truncated but the truncation is of microscopic dimensions.The surface is that of a smoothly curving net with more or less flat triangular surfaces between the 12 cusps (18, 19)

These observations represent a surprising and important breakthrough in our knowledge of the carbon content of space and the biochemistry of the earth. Further work drew attention to red giant stars which were pumping vast quantities of carbon molecules, as well as carbon dust, into space and suggested that there might be a link between carbon chains and soot formation (17).

Interesting are the Bok globules. The photography represent the time of the Earth is formation.

The possible relationship between melanins and the structural features of fullerenes is interesting from a point of view of organic chemistry and the science of the solid state. Considering the chemical and physical relationship existing between melanins (in their ordered part) and fullerene a cage structure may be present in the black materials in nature.

X-ray powder diffraction measurements, 13C NMR, and MALDI-TOF-MS have proved invaluable, differently from melanins, in the unambigous characterization of fullerene. Nanochemistry has largely  been  applied to the study of fullerenes. It was found that crystalline C60 undergoes a phase transition to a simple cubic structure at 260 K which is accompanied by an abrupt lattice contraction.Further approach to fullerenes structure has been made with the help of geometry (16, 17)

C60 was predicted to be a possible by-product of soot formation and subsequently shown to occur in a sooty flame. Giant fullerenes were shown to possess quasi-icosahedral structure consistent with the polyhedral shapes of certain graphite microparticles (18).

Weak IR signals observed in arc-processed graphite are consistent with the presence of fullerenes.

Powerful mass spectrometric techniques for studying refractory clusters generated in a plasma by focusing a pulsed laser were developed, including a technique which offered a way of simulating the chemistry in a carbon star if a graphite target was used. The C60 molecules have a truncated icosahedral closed cage structure; the signal for a C70 structure was also prominent.

The most significant observations to emerge were:

  1. Atoms could be encapsulated in the cage
  2. Giant fullerenes possess quasi icosahedral structure consistent with the polyhedral structure and shapes of certain melanosomal microparticles.
  3. The transition temperatures are sensitive functions of both the crystallinity and the purity of the solid fullerene.

The low temperature simple cubic structure arises as a result of orientational ordering of the C60 molecules in the unit cell. The high-resolution diffraction work also revealed that there is a subtle optimisation of the packing arrangement of the C60 molecules indicating that an interaction over and above the simple van der Waals intermolecular force operates. The electron density distribution on the surface of the C60 sphere is significantly anisotropic.The question of conductivity or super conductivity of doped melanosomes has not as yet dealt with.

Recently a new type of microscopic carbon fibre has been detected which appears to consist of very small diameter graphite tubes from 10-100 A° in diameter. We have the TEM (Trasmission Electron Microscope) images of such microfibres wich appear to grow on the cathode of a fullerene arc-processor. Similar structures have also been observed in melanosome micrographs. The cylinder walls may consist of only a few layers of graphite, 2-5 or may be as many as 50 or more.

A comparison between observed transmission electron microscope (TEM) images of nano-fibers and simulated TEM images for giant tubular fullerenes and related quasi-icosahedral elongated graphite microparticles has been carried out. Apart from the cylindrical structures of the nanofibres the most striking result is the fact that the TEM images show that the ends are capped by a continuous dome of carbon (17, 18, 19).

An interesting property of the carbon atoms is that the cylindrical walls of the nanofibers are in general arrayed in a helical screw configuration. This can be readily explained if the cap of the fullerene is non-symmetrical which is almost certainly the general case. A simple example is shown in the following figure where the Schlegel

diagram for one of the simplest (and smallest) unsymmetric fullerene caps is depicted (17).

A stoichiometric mixture of La2O3 and C60 from the laser ablation of graphite in a helium buffer gas was obtained. The same experiment with melanosomes has not yet been accomplished. Very weak laser radiation has been used to create desorption of endohedral fullerene complexes from the surface where they were formed. On the contrary, it appears that up to now very little information is reported in literature on the desorption and eventual modification of the properties of fullerene, by itself or in presence of doping elements. Sincea few carbon bonds within the fullerene structure are released upon laser absorption and coalescence, we may expect that some endohedral fullerene complexes may be formed.

The properties of doped fullerenes have attracted much attention since the first detection of these molecules. At first, fullerenes with metal inside were produced by laser vaporization of metal mixed with graphite, and endohedral fullerene complexes with lanthanum, potassium and cesium were formed. Later the superconductivity of C60 doped with rubidium, cesium and other alkali metal alloys stimulated interest in the metallic and superconductive properties of fullerides with alkali or alkaline earth metals. The most surprising result is the production of CO+ and CO2+ masses, for both the undoped and doped fullerene targets when the number of laser blastings is increased. For the target containing lanthanum oxide it may be supposed that CO+ and CO2+ species are produced by dissociation of lanthanum oxide by a photochemical reaction of the uv radiation on fullerene. For the undoped fullerene target the CO+ and CO2+ production should be associated with the release of O2 adsorbed in the bulk, photochemically reacting with C60.

Infrared, Raman and uv spectroscopic analyses have been performed on thin films formed on KBr and quartz substrates by the laser ablation of C60 and C60 mixed with La2O3 targets. The results of the spectroscopic investigation performed so far, are not very satisfactory because no infrared or uv bands associated to C60 could be clearly identified. Laser acts on graphite producing a series of degradation products whereas, in the case of immature melanosome, an explosion occurs both in vitro or in vivo with the formation of a series of pyrrole and indole fragments in the molecular weight range of circa 100-1000 (9).The fragmentation of the melanosomal cage resembles the breaking of crockery. Among the various fullerenes the more stable is C60. May be present in form of C+, albeit in minute amounts, in a sooty Bunsen burner flame, and among the combustion products of cars deposited on a tunnel wall.The pinkish red fraction isolated from the tunnel of Victory in Naples in 1964 may, in retrospect, be attributed to a fullerene fraction (20, 21).Recently fullerenes were found in the fossil of a dinosaur egg (22). The fullerenes have been experimentally and theoretically known for long time and a large number of papers have been devoted to this subject (25). Many attempts have been made at rationalizing the electronic structure of the fullerenes, in particular to understand how the mixing of five-and six-membered rings in a three-dimensional structure affects the properties of these molecules.The behaviour of the fullerenes in electric fields, although direct measurement of conductivity were not made, and in particular their nonlinear properties, attracted much early attention due to the potential use of this class of ''molecules'' as efficient non linear optical devices.Interesting is the first degenerated four -wave mixing measurements on C60 which later was shown to be three orders of magnitude too large (24).Later studies indicated that the potential of fullerenes as efficient materials in non linear optics can be at best be considered modest.The presence of five- and six-membered rings in the fullerenes has prompted a number of studies of the magnetic properties of these ''molecules'' as the prototype for spherical aromaticity, a concept which may be enlarged to include melanosomes and melanins.A number of ab initio calculations of the noble gas shieldings of endohedral fullerenes have been presented as well as some studies of the magnetizability of C60.The electric and magnetic properties of C60, C70, C84 were investigated (25) : fully analytical calculations of the electric polarizability, the second hyper polarizability of fullerenes C70 and C84, London atomic orbitals have been presented.

Two fullerenes C72 an C74 have been isolated and their electronic properties studied (26) with the aid of Ca encapsulation. The treatment of monoarylated azafullerenesAzC59N with iodine monochloride in CS2 (27) leads to the exclusive formation of the tetrachlorinated heterofullerenes C14ArC59N which contain a pyrrole moiety in the fullerene cage.

The synthesis of oxide such [C120]O has been described (28). The oxide generates the odd C119 by thermal decarbonilation of [ C60 ]O and addition of the resultant C59 ([C60]O-CO = C59+CO2) to C60. The dimeric fullerene oxide is the precursor for the synthesis of odd-numbered fullerenes.A new method of extraction (28) produces oxide a such [C120]O and [C130]O and several dimeric fullerene oxides were obtained in small amount with higher fullerene oxides.The generation of semiconducting polymers has been achieved by incorporation into host matrices formed by conventional polymers such as polyethylene and polystyrene (29).Similar work with melanosomes and melanins has not been carried out.The utilization of the strong photoluminiscence of conjugated polymers for light emitting devices resulted in the emphasis on research into this class of materials that combine the electronic and optical properties as well as processing properties of polymers. The characterization of C60 as an electron acceptor capable of accepting as many as six electrons candidates it an acceptor in blends with conjugated polymers as good photoexcited electrons donors.A wide class of these conjugated polymers and oligomers (the binding affinity of melanins) shows a photoinduced electron transfer from the excited state of the conjugated polymer onto the fullerene C60.The stabilization of the charge separated state in the conjugated polymer is assumed to result from the stability and delocalization of the positive polaron on the conjugated polymer backbone. A simple, new, carbon nucleation scheme has been developed which results in quasi-single crystal particles of concentric, spiral-shell internal structure and overall quasi-icosahedral shape.Intermediates consisting of curved graphitic networks and overall growth controlled by epitaxy are key factors in the scheme which also produces C60 as an inert close cage (31) The scheme which explains the occurence of polyhedral carbonaceous particles may also apply to soot and circumstellar dust formation. C60 is a fullerene which may be useful in the nanochemistry and nanophysics studies of melanins in particular and of black material in general.

Electro-oxidation of L-DOPA was recently applied to prepare melanin and the effect of the electrolyte ions on the rate of charge propagation in the polymer was investigated (33). The decrease of the rate of charge transport observed upon increasing of electrolyte concentration may be explained by the distance between adjacent electroactive sites due to a swelling of the polymer film which reduces electron hopping between sites. Spectroelectrochemical data show that in melanin electroactive quinones may be present. The charge transport properties of this heterogenous material has been shown to compare very well with those of other conducting ''polymers''. Consequently DOPAmelanin may be used as a matrix for preparing reagentless enzyme-based biosensors(33).

Taking in account all that is discussed above we conclude that the fullerene-graphite system presents a model, from the chemical and physical point of view, which may stimulate the study of melanosomes and melanins and emphasizes the value of mass spectroscopy as tool to inquire into the properties of melanosomes and black materials found in biological systems.

 

Biology

Melanins are amorphous natural semiconductors with fullerene-cage like structure.Electroactivity may also be shown in those materials like charge transfer complexes.What is the role played by melanins in nature ?
Black materials are present in all the universe and are of great interest for the evolution of our planet and living systems. The black particles of the brain and black particles of the stellar spaces are similar...

 

Appendice

Preparazione dei melanosomi dell'inchiostro di Seppia (Dr.B.Nicolaus, Istituto per le Molecole di Interesse Biologico del CNR, Via Toiano 2, I-80072 Arco Felice, Napoli.)10 borse di seppia fresche e turgide si premono gentilmente.Il liquido si centrifuga 50000 rev/min. Si lava con H2O x3.Resa del materiale seccato all'aria circa 10g.Questo materiale viene purificato secondo : V.J.Hearing, M.A.Lutzner Yale Journal of Biology and Medicine 46, 553 (1973) e viene indicato con la lettera A (sale di calcio e magnesio del melanosoma). A viene lavato in centrifuga con porzioni (10 cc) nelle seguenti successioni :H2O x3, Alcool x3, acetone x3, H2O x3, HCl 2N x6, H2O x3. Il prodotto seccato all'aria si chiama B. Sia A che B si trattano con solventi e reattivi puri il piu' possibile fuori del contatto della luce e dell'aria. B e'costituito da melanosomi carbossilati.B si puo' decarbossilare in olio di vaselina per riscaldamento a 120° ; dopo l'allontanamento della vasellina con solventi (benzene, CS2, CHCl3 etc.) si ottiene C (melanosomi con centri cationici).Studio del melanosoma1.La frammentazione, indicata dai dermatologi con il termine esplosione, dei melanosomi A, B, C viene studiata con la tecnica di spettrometria di massa MALDI, MALDI-TOF, LDI. (prof.A.Malorni, Centro Internazionale di Spettrometria di Massa del CNR, Arco Felice, I-80072 Napoli)2.Si determina la conduttivita' e la superconduttivita' di A e B e dei derivati (sali, charge transfer complexes, vapori di bromo etc.)3. Si determina la conduttivita' e la superconduttivita' di C e dei suoi derivati contranionici (vedi nero di pirrolo e relativa letteratura in (2))Figure al PC Le figure elaborate al computer sono opera di G.Nicolaus (LIGB, Via Marconi 10, I-80125 Napoli) e di M.Olivieri (Dipartimento di Chimica Generale ed Inorganica, Via Mezzocannone 4, I-80134 Napoli.)

 

Studi Chimici

Gli studi chimici sono stati condotti ed elaborati dal Dr.A.Bolognese (Dipartimento di Chimica Organica Biologica, Via Mezzocannone 16, I-80134 Napoli)

 

Advice

For Chemistry : Prof.R.A.Nicolaus, Rampe Brancaccio 9, I-80132 Naples e-mail : rnicolaus@tightrope.it

For Pharmacology : Prof.B.J.R.Nicolaus, Via Crescitelli 6, I-20052 Monza.

For Biology : Prof.P.A.Riley, Windeyer Istitute of Medical Science, University College London, London WIP 6DB.
e-mail, rebc@ucl.ac.uk

For Molecular Modeling : Dr. Marco Olivieri, Dipartimento di Chimica. Via Mezzocannone 4 - I-80134 Napoli. e-mail : olivieri@chemna.dichi.unina.it

Il lavoro di ricerca sara' oggetto di una serie di prossime pubblicazioni scientifiche da parte degli autori.

 

Bibliography

1.a. R.H.Thomson''Melanins''in Comparative Biochemistry Vol.III, Ed.M.Florkin, H.S.Mason, A.P.(1962).
b Nicolaus ''Melanins'' Hermann, Paris, (1968)
c. R.A.Nicolaus ''Melanine'' Quaderni della Accademia Pontaniana n°4. Ed. Giannini, Napoli (1984).

d. G Prota''Melanins and Melanogenesis'' AP, San Diego (1992)
e. P.A.Riley''Melanin''Int.J.Biochem.Cell Biol. 29, 1235, (1997)
f. F.Anders''Contributions of the Gordon-Kosswig melanoma system to the present concept of neoplasia'' Pigment Cell Res. 3, 7, (1991).

2. R.A.Nicolaus, G.Scherillo La melanina. Un riesame su struttura, proprieta', sistemi. Atti Accademia Pontaniana, Vol, XLIV, Anno Accademico 1995, DLIII dalla fondazione. Ed.Giannini, Napoli (1995) ;B.J.R. Nicolaus, R.A. Nicolaus "Speculating on the Band colours in Nature" Atti della Accademia Pontaniana Vol. XLV, Giannini ed. Napoli (1997); R.A. Nicolaus "Coloured organic semiconductors: melanins" Rend. Acc. Sci. Fis. Mat. LXIV, 325-360 (1997);R.A.Nicolaus ''Le melanine del cosmo '' Rend.Acc.Sci.Fis.Mat. LXIV, 7 (1997).

3. A. Dall’Ollio, G. Dascola, V. Varraca, V. Bocchi "Resonance paramagnetique electronique et conductivité d’un noir d’oxypyrrol electrolytique" C.R. Acad. Sci. Paris 267 433 (1968).

4. R.A. Nicolaus, M. Piattelli, E. Fattorusso "The structure of melanins and melanogenesis – IV'' Tetrahedron 20 1163 (1964).

5. a. S.Ito, J.A., Colin Nicol "Isolation of oligomers of 5, 6 – dihydroxyindole carboxylic acid from the eye of the catfish" Biochem. J., 143 207 (1974).

b. Prota, G., "The Chemistry of Melanins and Melanogenesis". Progress Chem. Nat. Prod. 64, 93-148, (1995). See pag. 111-112. SpringerVerlag, Wien (1995)

c. Ito, S., "Reexamination of the structure of eumelanin" Biochim, Biophys. Acta 883 155 (1986).

d. Ito, S., Fujita, K. "Microanalysis of Eumelanin and Pheomelanin in Hair and Melanomas by chemical degradation and Liquid chromatography" Anal. Biochem. 144, 527, (1986).

6. Berlin, A., Canavesi, A., Schiavon, G., Zacchin, S., Zotti, G. "Electrooxidation Products of Methylindoles: Mechanism and Structures" Tetrahedron 52, 7947, (1986).

7. Nicolaus, R.A., Vitale, A., Piattelli, M., "Acido 2, 3, 4, 5, -pirroltetracarbonico nell’ossidazione della melanina di seppia" Rend. Acc. Sci. Fis. Mat. della Società Nazionale di Scienze, Lettere ed Arti di Napoli, Serie 4°, Vol. XXV, (1958).

Nicolaus, R.A., Oriente, G., "Sugli acidi pirrocarbonici: acido 2, 3, 4, 5-pirroltetracarbonico – Nota II.'' Gazz. Chim. Ital. 84, 230, (1954).

Nicolaus, R.A., "Melanins" Hermann, Paris pag. 82-83 (1968)

D.M. Piattelli, E. Fattorusso, S. Magno, R.A. Nicolaus "The structure of Melanins and Melanogenesis – II" Tetrahedron 18 941 (1962).

8. A.Quilico "I pigmenti neri animali e vegetali. Esposizione riassuntiva e contributo sperimentale alla conoscenza della loro natura chimica e della loro genesi" Ed. Fusi, Pavia, (1937).

Barbetta, M., Casnati, G., Ricca, A., (1966) "Ricerche sulla Aspergillina" Acc. Naz.Lincei, 8, 450; (1967)"Aspergillina" Ist.Lombardo di Scienze 101, 85 85.

9. To be pubblished.

10. D.C. Allport, J.P. Bu’Lock "Biosynthetic pathway in Daldinia concentrica" J. Chem Soc. 654 (1958); J. Chem. Soc. 4090 (1958).

11. M. Piattelli, E. Fattorusso, R.A. Nicolaus, S. Magno "The structure of melanins and melanogenesis – Note V" Tetrahedron 21 3229 (1965); 20 1163 (1964).

12. M.M. Kononova "Soil organic matter, its nature, its role in soil formation and in Soil fertility" P.P., Oxford (1961).

13. R.D. Haworth "The Chemical Nature of Humic Acid" Soil Science 111, 7 (1971).

14. Y.T. Thathachari, M.S. Blois "Physical Studies on melanins – II. X- ray diffraction" Bioph. J. 9 77 (1969); Y.T. Thathachari "Structure of Melanins" Pigment Cell Vol. 1, 158, Karger, Basel (1973); "Spatial Structure of Melanins" Pigment Cell. Vol. 3, 64, Karger Basel (1976).

15. a) G.F. Murphy, R.S. Shepard, B.S. Paul, A. Menkes,

R.R. Anderson, J.A. Parrish "Organelle – specific injury to melanin – containing cells in human skin by pulsed laser irradiation" Lab. Invest. 49 680 (1983).

b) J.A. Parrish R.R. Anderson, T. Harrist, B. Paul, G.F. Murphy "Selective thermal effects with pulsed irradiation from lasers: from organ to organelles" J. Invest. Dermat. 80 75 (1983).

  1. L.L. Polla, R.J. Margolis, J.S. Dover, D.W. Whitaker, G.F. Murphy, S.L. Jacques, R.R. Anderson "Melanosomes are a primary target of a – switched ruby laser irradiation in guinea pig skin" J. Invest. Dermatol. 89 281 (1987).
  2. A.R. Anderson, R.J. Margolis, S. Natanabe, T. Flotte, G.J. Hruza, J.S. Dover "Selective photermolysis of cutaneous pigmentation by Q –switched Nd: YAG laser pulses at 1064, 532, and 355 nm" Invest. Dermatol. 93 28 (1989).
  3. K.A. Sherwood, S. Murray, A.K. Kurban, O.T. TAN "Effects of wanvelength on cutaneous pigment using pulsed irradiation" J. Invest. Dermatol. 92 717 (1989).
  4. S. Ara, R.R. Anderson, K.G. Mandel, M. Ottesen, A.R. Oseroff "Irradiation of pigmented melanin – cells with high intensity pulsed radiation generates acoustic waves and kills cells" Lasers Surg. Med. 10 52 (1990).
  5. S.L. Jacques, D.J. McAuliffe "The Melanosome: treshold temperature for explosive vaporization and in termal absorption coefficient during pulsed laser irradation" Photochem. Photobiol. 53 769 (1991).

16. K. Kondo "Three Phases of Epistemological Penetration to Nature" Quaderni Accademia Pontaniana n° 20 Ed. Giannini, Napoli (1997).

17. L. Taliani, G. Ruani, R. Zamboni "Fullerenes: status and perspectives", WS, Singapore (1993).

The name fullerene is derived from Richard Buckminster Fuller american Architect (B. Milton 1895 Died. Los Angeles 1983) who tested a new type of linkage between geometrical elements like tetrahedron and octahedron.

In the book you can find precious articles (and literature until 1990)like:

Fullerenes: physics and astrophysics studies
H.W. Kroto, K. Prassides, M. Endo and M. Jura
Higher Fullerenes: Isolation, characterization and growth mechanism
Y. Achiba, K. Kikuchi, T. Wakabayashi, N. Nakahara and S. Suzuki
Fullerene Nanowires
Changming Jin, Ting Guo, Yan Chai, Ade Lee and R.E. Smalley
Laser Ablation and deposition of graphite, lanthanum and lanthanum-doped fullerene
M. Allegrini, E. Arimondo, C. Callegari, F. Fuso, A. Iembo,
G. Masciarelli, V. Berardi, N. Spinelli, S. Rossini and R. Danieli
Solved and unsolved problems in fullerene systematics

P.W. Fowler

Cosmoids, fullerenes and continuous polygons

L. Saffaro

Spectroscopic evidence of phase transition in fullerene. Importance of librational (rotational) modes in the superconducting properties of M3C60

V.N. Denisov, B.N. Mavrin, G. Ruani, C. Taliani,
R. Zamboni and G.N. Zhizhin
Wave dispersed nonlinear spectroscopy in C60 thin films
F. Kajzar, C. Taliani, R. Zamboni, S. Rossini and R. Danieli
Analysis of the vibrational structure of emission and absorption spectra of C60
F. Negri, G. Orlandi and F. Zerbetto
Theoretical calculations on the MCD spectrum of C60
G. Marconi and P.R. Salvi
Theoretical determination of the electric and magnetic properties of fullerenes C60 and C70
P.W. Fowler, P. Lazzeretti, M. Malagoli and R. Zanasi
Thomas-Fermi model for the C60 molecule
F. Siringo, G. Piccitto and R. Pucci
Spectroscopic investigation of C60 interaction GaAs and Bi
U. del Pennino, S. Gozzi, P. Rudolf, P. Lazzeretti and R. Zanasi
Interface formation and charge transfer of ultrathin C60 films deposited on Cu(100) and Cu(III)
J.E. Rowe, P. Rudolf, L.H. Tjeng, R.A. Malic,
G. Meigs, C.T. Chen, J. Chen and E.W. Plummer
Conductivity and superconductivity in alkali-metal doped C60
R.C. Haddon
Neutron scattering studies of fullerenes and alkali-metal doped fullerides
K. Prassides, C. Christides, J. Tomkinson, M.J. Rosseinsky, D.W. Murphy, R.C. Haddon, T.J.S. Dennis, J.P. Hare, H.W. Kroto, R. Taylor and D.R.M. Walton

Electronic structure studies of undoped and doped fullerenes

J. Dink, E. Sohmen, M. Merkel, A. Masaki, H. Romberg, M. Alexander, M. Knupfer, M.S. Golden, P. Adelmann and B. Renker

Low energy electronic excitations in K doped C60 from Raman scattering excited at 1.16 eV

V.N. Denisov, R. Danieli, C. Taliani, R. Zamboni, G. Ruani, A.A. Zakhidov, K. Yakushi and Y. Achiba

Dynamics of fcc-C60 fullerite

G. Onida and Benedek

Properties of doped fullerenes: Ab-initio molecular dynamics studies

W. Andreoni and M. Parrinello

Vibrational frequencies of C60 hexaanion

F. Negri, G. Orlandi and F. Zerbetto

Itinerant electrons on the icosahedral Group: Meeting the challenge of superconductivity of the fullerene

M. Rasetti and R. Zecchina

Non-adiabatic superconductivity in the fullerene compounds,

L. Pietronero and Strässler

18. H.W. Kroto, K. Prassides, N. Endo, N. Jura "Fullerenes: Physics and Astrophysics studies" in C. Taliani, G. Ruani, R. Zamboni"Fullerenes: Status and perspectives" WS, Singapore 1992.

19. H.W.Kroto '' Fullerene cage clusters '' J.Chem.Soc.Faraday Trans. 86, 2465 (1990)

20 V.Dovinola, F.Imperato '' Inquinamento atmosferico-Nota II-Studio analitico della frazione organica '' Rend.Acc.Sci.Fis.Mat. XXXI, 338. Ed.Genovese Napoli 1964.

21. F.Imperato ''Sugli inquinanti atmosferici '' Rend.Acc.Sci.Fis.Mat. XXXII, 156.Ed.Genovese, Napoli 1965.

22. Z.X.Wang, X.P.Li, W.M.Wang, X.J.Xu, C.T.Zi, R.B.Huang, L.S.Zheng''Fullerenes in the fossil of dinosaur egg'' Fullerenes Science and Tecnology 6, 715, (1998).

23. J.Borovansky, P.Hach, J.Duchon '' An Melanosome : unusually resistant subcellular particle '' Cell Biology International Reports 1, 549, (1997).

24. J.Cioslowski "Electronic Structure Calculations on fullerenes and their derivatives'' Oxford Press (1995)

25. D.Jonsson, P.Norman, K.Ruud, H.Agren, T.Helgaker '' Electric and magnetic properties of fullerenes '' J.Chem.Phys. 109, 579, (1992)

26. T.S.M.Wan, Hong-Wei Zhang ''Production, Isolation and Electronic Properties of Missing Fullerenes :Ca C72 and Ca C74 '' J.Am.Chem.Soc. 120, 6806 (1998)

27. U.Reuther, A.Hirsch, '' Pyrrole-embedded [C60] fullerenes '' Chem.Comm.1401, (1998)

28. H.Takahashi, K.Tohji, I.Matsuoka, B.Jeyadevan, A.Kasuya, S.Ito, YNishina, T.Nirasawa ''Extraction and Purification of dimeric fullerene Oxides from Fullerene Soot '' J. Phys Chem.B 102, 5438, (1998).

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Napoli, 16 Gennaio 1999.


R.Nicolaus, Rampe Brancaccio 9, I-80132, Naples, Italy.
Accademia delle Scienze Fisiche e Matematiche, Via Mezzocannone 8, I-80134-Naples.
Tel +39 +815527549
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rnicolaus@tightrope.it
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Giancarlo Nicolaus

Biologia Molecolare,I-00040,Pomezia

Giancarlo_nicolaus@merk.com