RICERCA ITALIANA     

AN INTEGRATED APPROACH TO THE SYNTHESIS, CHARACTERIZATION AND FUNCTION OF 5,6-DIHYDROXYINDOLE-DERIVED EUMELANIN BIOPOLYMERS AND THEIR BLENDING WITH CONVENTIONAL POLYMERS AND COMPOSITES

Università degli Studi di Napoli "Federico II"

Abstract

The aim of the proposed project is to combine for the first time the complementary expertises of various research groups in the fields of organic natural products and biopolymers, organic physical chemistry, mass spectrometry, electron paramagnetic resonance, electrochemistry and polymer science to address long-lasting issues concerning the structure and mechanism of synthesis of eumelanins, a characteristic group of natural pigments arising by oxidative polymerization of 5,6-dihydroxyindole (DHI), its 2-carboxylic acid (DHICA) and related metabolites, and to assess the potential of these biopolymers for technological applications. The eumelanins occupy a unique position among the numerous pigments found in nature because of their central role in human and mammalian pigmentation, where they provide the dark colourations to skin, hair, eyes and substantia nigra. The social and biomedical importance of eumelanins stems from their relevance to ethnic pigmentation, photoprotection, and certain disorders such as albinism, vitiligo, melanoma and Parkinson’s disease. Recently, eumelanins have also attracted the attention of the soft matter and functional organic materials communities because of their unique physicochemical properties, including broadband monotonic absorption in the UV-visible range; persistent and inducible free radical populations that can be detected by EPR; metal-binding and redox properties; and electrical conductivity. Eumelanins have been suggested to be particulate amorphous semiconductors and the properties of films prepared by electro-polymerization of 5,6-dihdroxyindoles have been the focus of much interest, contributing to the rapid growth of 5,6-dihydroxyindole and eumelanin research in recent years. Despite several efforts, however, eumelanin structure remains an enigma, because of the complete insolubility, chemical heterogeneity and the lack of well defined spectral properties. It is unknown: a) what is the average molecular weight and degree of polymerization of 5,6-dihydroxyindoles; b) what are the mechanisms of oligomer assemblage; c) what is the origin of the EPR signal; d) what is the basic three-dimensional structural unit forming the pigment particle. Investigation of the oxidative polymerization of 5,6-dihydroxyindoles, in combination with direct analysis of the pigments, remains the most promising approach for developing a consistent structural model of eumelanins. Interest in this process is also warranted by the prospects of mimicking nature to produce new materials, and of tailoring eumelanins to improve functionality and prepare synthetic pigments with controlled structural properties. The research plan will therefore pursue the following aims: 1) to elucidate the mode of coupling of 5,6-dihydroxyindoles and their oligomers; 2) to determine the mode of assemblage of indole oligomers and the mechanism of growth of the eumelanin particles; 3) to investigate the intrinsic and inducible free radical properties of eumelanins and to assess what are the underlying structural features; 4) to develop and apply DFT and other computational models for predicting reaction mechanisms and properties of intermediates/polymers; 5) to explore viable routes for controlling 5,6-dihydroxyindole polymerization and electropolymerization to obtain biocompatible polymers, eumelanin films and composites of potential technological interest. Material science-oriented goals involve potential applications as soft electronics, sensors, photon harvesting materials, and will expectedly depend on a better understanding of the effect of aggregation and oxidation on the mode of build-up and optical absorption of indole oligomeric sheets. The research plan is built upon a solid foundation of knowledge, gained over more than 20 years, as well as of preliminary data, and will be developed in a highly coordinated fashion through the close interaction of participating groups. <<<

Principal Investigator

Marco D'ischia Università degli Studi di NAPOLI "Federico II"

Research Objectives

The long-standing issues of 5,6-dihydroxyindole oxidation and eumelanin structural characterization provide the major focus for the research plan, which will pursue the following specific aims.

Specific aim 1: To elucidate the mode of coupling of 5,6-dihydroxyindoles and their oligomers.
Within this aim, main objectives will be the development of synthetic protocols for the preparation of 5,6-dihydroxyindole derivatives; the synthesis, isolation and structural characterization of higher oligomers of 5,6-dihydroxyindoles, e.g. tetramers from DHI and hexamers from DHICA; the elucidation of the mechanism of coupling of the indole monomers to form the dimers, with special reference to the mechanism of formation of the 2,2’-biindolyl by metal promoted oxidation of DHI; the mechanism of coupling of DHI and DHICA dimers to give higher oligomers; the factors controlling the regiochemistry of the reactions and the effect of different oxidation conditions, e.g. chemical oxidation, enzymatic oxidation, electropolymerization..

Specific aim 2: to determine the mode of assemblage of indole oligomers and the mechanism of growth of the eumelanin particles. Expected outcomes of this specific aim would be the definition of the mode of growth of the melanin particle via oligomer chain extension or p-stacking of short oligomers; the mechanism of interaction of monomer units with growing particles, via physical adsorption or redox exchange with the preformed polymer, and the effect on pigment structure and properties of different oxidation conditions, e.g. chemical, enzymatic or electrochemical; the elucidation of the mechanism of aggregation of the particles e.g. by making recourse to different reaction media such as organic solvents, surfactant, ionic liquids, or polymeric matrices into which to perform 5,6-dihydroxyindole oxidations.

Specific aim 3: to investigate the free radical properties of eumelanins and precursor oligomers. Main objectives will be to determine: whether EPR signals are generated during oxidation of DHI in the presence and in the absence of metal ions; the structural features underlying the EPR signal of the eumelanin polymers; the effects of oxidation conditions, e.g. chemical, enzymatic, or electrochemical, on the EPR properties of the pigments; the effect of aggregation and redox state on the free radical properties of indole polymers; the effects of electron-withdrawing (e.g. a carboxyl group) or electron-donating substituents (e.g. an alkyl residue) on the EPR signals of the indole polymers; the consequence of oxidizing dimers rather than monomers on the free radical properties of the final polymers; the factors affecting the light-induced extrinsic free radical population.

Specific aim 4: to develop and apply DFT and other advanced computational models for predicting chemical mechanisms and physicochemical properties of intermediates/polymers. Main objectives will be the computational analysis of oligomer intermediates produced during oxidation of the various 5,6-dihydroxyindoles; the development of a specific model for investigating main transients during oxidative dimer coupling; the calculation of the absorption spectra of putative intermediates in eumelanin polymer build-up; prediction of the EPR properties of transient intermediates and putative eumelanin building blocks.

Specific aim 5: to explore viable routes for controlling 5,6-dihydroxyindole polymerization or for blending with classical polymers to obtain novel biocompatible materials and composites with suitable properties for UV absorbing systems, sensors, organic conductors, or sunscreen products.

If successful, the project will provide unprecedented insights into the complex structural organization of eumelanin polymers, perhaps one of the last enigmas in the chemistry of organic natural pigments and biopolymers, and will offer an improved background to exploit the peculiar physicochemical properties of suitably tailored eumelanin polymers for innovative applications.
Realization of the above goals appears realistic in the light of the following considerations:
1) The scientific coordinator and his group at Naples have a solid expertise in the field of 5,6-dihydroxyindole chemistry, which is equalled by few other groups in the world; and it is the first time that the crucial issues of 5,6-dihydroxyindole oxidation and eumelanin structure are addressed from different anglepoints by a highly coordinated panel of experts.
2) Preliminary and ongoing studies in the coordinator’s laboratory have already set the basis for promising developments in the synthesis of 5,6-diihydroxyindole oligomers, in the mechanisms of oxidative coupling and in the computational investigation of the mechanisms of pigment formation. Most of the results have been gained through ongoing collaborative programmes with Dr E.J. Land at Daresbury Laboratory where pulse radiolysis facilities are available.
3) The groups involved in the project cover a broad range of expertises, ranging from organic chemistry to computational chemistry, physical chemistry, electron paramagnetic resonance, mass spectrometry and materials science. These expertises are documented by several high quality publications and will be synergistically integrated into solid links of scientific cooperation.
4) The programme has been shaped so that each group closely interacts with partner groups and is involved in virtually all aspects of the research. Results from each groups will be used by all others and it is expected that most of the main objectives will be pursued through exchange of personnel between interacting laboratories. <<<

Timescale

24 months

National and international background

This research plan is focused on the structural characterisation, mechanism of formation and properties of eumelanins, the principal pigments of man and mammals. It will develop through an integrated approach aimed at addressing the oxidative polymerization of 5,6-dihydroxyindoles, the physicochemical properties of the resulting polymers and their potential for practical applications in material sciences. Human pigmentation is the natural phenomenon that provided the initial stimulus for eumelanin research. In man and non-human mammals, almost all normal pigmentation is due to varying amounts, type and distribution of melanins and related pigments throughout the epidermis (Prota, 1992). Melanin pigments are usually classified into two main classes, the black-to-dark brown, insoluble eumelanins, which are widely distributed in the animal kingdom, and the yellow-to-reddish brown, sulphur-containing variants termed pheomelanins. Besides mammals, eumelanin pigments are found also in lower organisms, e.g. the cephalopods, where they are produced in the characteristic ink gland and form the black ink that the animals discharge when frightened. In mammalian skin, eumelanins, like pheomelanins, are produced by highly specialised cells termed the melanocytes. Within these cells, eumelanin biosynthesis involves the tyrosinase-catalyzed conversion of the amino acid tyrosine to dopaquinone, an unstable orthoquinone (Land et al., 2003). This undergoes oxidative cyclisation to give eventually 5,6-dihydroxyindole (DHI) and its 2-carboxylic acid (DHICA) by way of a red-orange 2,3-dihydro-1H-indole-5,6-dione intermediate commonly referred to as dopachrome. Oxidative polymerization of DHI and DHICA eventually leads to eumelanin formation and deposition within subcellular organelles known as melanosomes.
The role of 5,6-dihydroxyindoles as the basic building blocks of eumelanins has been known since the beginnings of the past century (Raper, 1927) and is illustrated by their rapid conversion in vitro to black insoluble pigments visually resembling natural eumelanins when exposed to oxidizing enzymes, chemical oxidants, UV radiation, or even on standing at neutral physiologic pH in the presence of air.
The socio-economical and biomedical importance of eumelanins has been recognized since ancient times and stems from the obvious relevance of these pigments to racial pigmentation, skin photoprotection, sun tanning and a variety of pigmentary disorders, such as albinism, vitiligo and melanoma (Prota, 1992) . In addition, a eumelanin-related pigment, known as neuromelanin, is found in the pigmented dopaminergic neurones of substantia nigra pars compact and has been implicated the etiopathogenesis of Parkinson’s disease, a neurodegenerative disorder of the elderly (Fedorow et al., 2005). Due to their strong absorption of visible light, eumelanins have long been thought to serve an important photoprotective role in the skin and eyes. Support to his view comes from the decreased UV susceptibility and relatively low incidence of skin cancers in dark skinned individuals. Moreover, in the eyes eumelanin is found in the retinal pigment epithelium (RPE), where it has been suggested to serve a photoprotective role by absorbing radiation and by scavenging reactive free radicals (Seagle et al., 2005). Eumelanin photoprotection is of especial relevance because RPE cell death is a major feature of the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the human population older than 60 years of age in the developed world.
More recently, the eumelanins have attracted the attention of biophysicists and the soft matter and functional organic materials communities because of their intriguing and rather unique set of physicochemical properties (Meredith et al., 2006). Eumelanins are amorphous solids with particulate character (Sarna and Swartz, 1998; Nofsinger et al., 2000). An important issue, most relevant to eumelanin structure, concerns the smallest molecular units that may be considered to have the properties of melanin and from which aggregation in solution occurs. Wide-angle x-ray scattering (WAXS) experiments have led to models for a “local structure” for melanin (Cheng et al., 1994) wherein five to seven 5,6-indolequinone units are arranged in planes which are p-stacked with a spacing of 3.4 Å (Simon and Ito, 2004, Clancy and Simon, 2001). Scanning tunneling microscopy (STM) was used to image the same melanin precipitated from highly dispersed, dilute solutions onto highly oriented pyrolytic graphite substrates (Zajac et al., 1994). The compact three-dimensional structure observed in the STM image is compatible with the model of stacked layers of 5,6-indolequinone. Atomic force microscopy (AFM) images of DHI melanin all show a particulate character; the colloidal samples have average particle heights of 13.8 Å, while the average height of electrochemically-deposited particles is 27.9 Å which corresponds to 6-7 stacked sheets, in rough agreement with 2 nanometer sized molecular structures (Gallas et al., 1999, 2000). Eumelanins are characterized by broadband monotonic absorption in the UV-visible range, which has not yet been explained satisfactorily (Sarna and Swartz, 1998). The observable optical properties appear to be a complex function of the ability of the pigment to absorb, reflect and scatter light at different wavelengths. All eumelanins, be they natural or synthetic, have persistent (intrinsic) and inducible free radical centers. In the presence of visible or UV light, the CW EPR signal grows to a steady state, accompanied by the photoproduction of extrinsic free radicals, and then reversibly decays back to the original dark intrinsic signal (Seagle et al., 2005). Eumelanins efficiently bind metal ions and drugs (Sarna and Swartz, 1998). They are susceptible to redox changes, and show non-radiative conversion of photo-excited electronic states (Meredith and Riesz, 2004), anti-oxidant and free radical scavenging ability; and some electrical conductivity in the condensed phase. In relation to the last property, it has been speculated that these systems may be bio-organic amorphous semiconductors (McGinness et al., 1974; Tran et al., 2006).
This combination of properties has led to the proposition that that they may be useful from a technological perspective as functional “electronic soft-solid” (Meredith et al, 2006) with good biocompatibility and bioavailability. Accordingly, interest in 5,6-dihydroxyindoles and eumelanin chemistry has grown exponentially during the past decade far exceeding the original biomedical relevance (d’Ischia et al., 2005).
Despite intense research efforts spanning over more than half a century, however, the structural properties of eumelanins and the origin of their physical properties remain an enigma, and even their actual functional role is obscure. Several obstacles account for the slow progress that mark eumelanin research, including the intractable character, chemical heterogeneity and the lack of well defined spectral properties (Prota, 1992; Prota et al., 1998). It is unknown what is the degree of polymerization of 5,6-dihydroxyindoles in the eumelanin pigment, what is the basic three-dimensional structural unit (if any) and what are the mechanisms of oligomer assemblage. Some interesting insight into the structural properties of eumelanins has been gained through use of MALDI mass spectrometry (Napolitano et al., 1996; Pezzella et al., 1997a). The results of a series of experiments revealed the presence in both natural and synthetic pigments of species that could be compatible with partially degraded oligomers of DHI and DHICA.
Because of the many difficulties in the direct investigation of eumelanins, approaches toward definition of the mode of formation of these pigments as well as for developing a consistent structural model have relied to a considerable degree on the investigation of the oxidative polymerization of 5,6-dihydroxyindoles. Interest in this process is also warranted by the prospects of mimicking nature to produce new materials, and of tailoring melanins to improve functionality and prepare synthetic pigments with controlled structural properties and functionalities for technology applications. (Meredith et al., 2006).
Chemical efforts to elucidate the early species formed in the oxidation of 5,6-dihydroxyindole led to the isolation of a collection of dimers and trimers (d’Ischia et al., 2005). The structural features of the isolated oligomers revealed an unanticipated propensity to couple through 2,2'-, 2,4'- and 2,7'-linkages, as highlighted by the structures of the main dimers isolated. In particular, 2,2’-biindolyls are obtained exclusively in the presence of metal cations such as copper, zinc and nickel, whereas 2,4- and 2,7’-biindolyls are obtained in the absence of transition metal ions. Such modes of coupling stem from the particular dioxygenation pattern pertaining to the 5,6-dihydroxyindole system, which resembles the 2,3-dihydroxynaphthalene system, and directs significant electron density toward the 2-position of the indole ring. The course of the oxidation depends on the pH of the medium. Thus, in acidic medium oxidation of DHI leads to the remarkable formation of isomeric hexahydroxydiindolocarbazoles as dark blue pigments which separate from the reaction mixture (Manini et al., 1998). Oxidation of DHICA leads to coupling products mainly at the 4- and 7-positions, with only minor involvement of the 3-position (Pezzella et al., 1997b). Linear trimers of DHICA have been isolated in different atropoisomeric forms, giving the first evidence for the chiral nature of these oligomeric species. Investigations of chirality in such systems allowed the isolation of a regiosymmetric tetramer by means of a model approach involving oxidation of the 4,4'-biindolyl. The resolution of the enantiomers of the tetramer was achieved and their absolute stereochemistry was deduced by the exciton chirality method(Pezzella et al., 2002, 2003)..
Although the precise mechanism of oxidative coupling of 5,6-dihydroxyindoles remains elusive, product analysis suggests that the process involves nucleophilic attack of the indole system onto the 5,6-indolequinone. Valuable insights have also derived from the application of pulsed radiation techniques. One-electron oxidation of DHI in aqueous solution at pH 7.4 leads to an initial transient species with peaks at 330 nm and 490 nm (Lambert et al., 1989), ascribed to an oxygen-centered radical (semiquinone) (Felix and Sealy, 1981). Although there is little doubt about the nature of the semiquinones resulting from one-electron oxidation of 5,6-dihydroxyindoles, there is much less certainty regarding the products of decay of these indolesemiquinones. The most likely possibility is disproportionation, resulting in the formation of indolequinone, which may rapidly isomerise into quinomethane and/or quinone-imine tautomers.
Virtually nothing is known on the oxidation chemistry of 5,6-dihydroxyindole oligomers: a main difficulty arises from the lack of convenient procedures for the preparation of dimers in sufficient yields for chemical studies. Moreover, all attempts to investigate the species formed far beyond the dimer level were unsuccessful due to the complex mixtures of products formed and their instability and insolubility. This is yet a most important goal since identification of the first formed species derived from oxidation of the oligomer intermediates, to determine their spectrophotometric properties and kinetics of decay, and to map their patterns of reactivity is likely to provide new important information about the mode of formation and key structural features of the eumelanin polymer.
The antioxidant and photoprotective properties of eumelanins have prompted a number of computational studies of 5,6-dihydroxyindoles, their oxidation products and dimers, and oligomeric models of eumelanin (Galvao and Caldas, 1988, 1990a,b). Using B3LYP and PBE0 calculations the relative stabilities and the excitation energies of the tautomers of 5,6-dihydroxyindole and 5,6-indolequinone have been investigated (Il’ichev and Simon, 2003). An ab initio and semi-empirical study has shown that these molecules can all behave as electron acceptors (Bolivar-Marinez et al., 1999). The authors conclude that the electronic properties are consistent with the semi-conductor model proposed for melanins (Longuet-Higgins, 1960). DFT (density functional theory) calculations on the monomers and several dimers (Stark et al., 2003a) gave calculated properties in good agreement with those of the ab initio study (Bolivar-Martinez et al., 1999) and with experimental data. These DFT studies were then extended to calculations of higher oligomers (Stark et al., 2003b) and the calculated oligomeric spectra provide support for the structural model of eumelanin (Cheng et al. 1994; Zajac et al.,1994). More recently, however, experimental and computational results have been provided suggesting that the traditional model of eumelanin as an amorphous organic semiconductor is not required to explain its optical properties, and that a model based on chemical disorder should be considered as more feasible (Tran et al., 2006). A first-principles DFT study of 5,6-dihydroxyindole and its quinone has calculated electronic and vibrational properties and has focused attention on the HOMO-LUMO gap which is twice as large in the dihydroxyindole as in the quinones (Powell et al., 2004). This may be significant in understanding the broadband optical absorption of eumelanins. A semi-empirical INDO study has examined the electronic properties of stacked eumelanin monomers and the results suggest localisation of charge on the monomer subunits and an electron hopping mechanism for electron transfer (Bochenek and Gudowska-Nowak, 2003). However, the majority of computational studies suffers from considerable weakness due to the fact that most calculations were referred to hypothetical monomer and oligomer species completely devoid of experimental support.
The above survey has summarized the scientific background to the proposed research project which will be aimed at addressing a number of critical issues for a deeper understanding of 5,6-dihydroxyindole oxidative chemistry and eumelanin structure and (bio)functionality:
1) what is the mode of coupling of 5,6-dihydroxyindoles and their oligomers;
2) what is the mode of assemblage of indole oligomers and the mechanism of growth of the eumelanin particles, and what is the basic oligomer unit forming the eumelanin particle;
3) what is the origin of the optical and free radical properties of eumelanins, what are the underlying structural features and the effects of electron-withdrawing or electron-donating substituents of the indole momers on the properties of the polymers;
4) what is the potential of 5,6-dihydroxyindole polymers as biocompatible and bioavailable materials for novel technological applications. <<<