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:
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 :
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:
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.
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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
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Napoli, 16 Gennaio 1999.
R.Nicolaus, Rampe Brancaccio 9,
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