Alessandro Pezzella

Chemical and structural diversity in eumelanins : unexplored bio-optoelectronic materials

Eumelanins, the characteristic black, insoluble, and heterogeneous biopolymers of human skin, hair, and eyes, have intrigued and challenged generations of chemists, physicists, and biologists because of their unique structural and optoelectronic properties. Recently, the methods of organic chemistry have been combined with advanced spectroscopic and imaging techniques, theoretical calculations, and methods of condensed-matter physics to gradually force these materials to reveal their secrets. Herein we review the latest advances in the field with a view to showing how the emerging knowledge is not only helping to explain eumelanin functionality, but may also be translated into effective strategies for exploiting their properties to create a new class of biologically inspired high-tech materials.

Melanins and melanogenesis: methods, standards, protocols

Despite considerable advances in the past decade, melanin research still suffers from the lack of universally accepted and shared nomenclature, methodologies, and structural models. This paper stems from the joint efforts of chemists, biochemists, physicists, biologists, and physicians with recognized and consolidated expertise in the field of melanins and melanogenesis, who critically reviewed and experimentally revisited methods, standards, and protocols to provide for the first time a consensus set of recommended procedures to be adopted and shared by researchers involved in pigment cell research. The aim of the paper was to define an unprecedented frame of reference built on cutting‐edge knowledge and state‐of‐the‐art methodology, to enable reliable comparison of results among laboratories and new progress in the field based on standardized methods and shared information.

Melanins and melanogenesis: from pigment cells to human health and technological applications

During the past decade, melanins and melanogenesis have attracted growing interest for a broad range of biomedical and technological applications. The burst of polydopamine‐based multifunctional coatings in materials science is just one example, and the list may be expanded to include melanin thin films for organic electronics and bioelectronics, drug delivery systems, functional nanoparticles and biointerfaces, sunscreens, environmental remediation devices. Despite considerable advances, applied research on melanins and melanogenesis is still far from being mature. A closer intersectoral interaction between research centers is essential to raise the interests and increase the awareness of the biomedical, biomaterials science and hi‐tech sectors of the manifold opportunities offered by pigment cells and related metabolic pathways. Starting from a survey of biological roles and functions, the present review aims at providing an interdisciplinary perspective of melanin pigments and related pathway with a view to showing how it is possible to translate current knowledge about physical and chemical properties and control mechanisms into new bioinspired solutions for biomedical, dermocosmetic, and technological applications.

An integrated approach to the structure of Sepia melanin. Evidence for a high proportion of degraded 5,6-dihydroxyindole-2-carboxylic acid units in the pigment backbone

Abstract The relative proportion of 5,6-dihydroxyindole (DHI) versus 5,6-dihydroxyindole-2-carboxylic acid (DHICA) units in Sepia melanin and their degree of structural integrity were assessed by an integrated approach involving quantitative determination of pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), specific degradation products of DHI- and DHICA-derived units; carboxyl content; rate of ferricyanide consumption; and absorption spectrum. Analyses of both intact and surfactant-solubilised pigment samples, in comparison with synthetic DHI and DHICA melanins, revealed that Sepia melanin consists of a mixture of oligomeric structures incorporating over 75% of DHICA-derived units and only 20% of DHI-derived units, occurring for the most part in an irreversibly degraded form, possibly as pyrrolecarboxylic acids.

Role of Solvent, pH, and Molecular Size in Excited-State Deactivation of Key Eumelanin Building Blocks : Implications for Melanin Pigment Photostability

Ultrafast time-resolved fluorescence spectroscopy has been used to investigate the excited-state dynamics of the basic eumelanin building block 5,6-dihydroxyindole-2-carboxylic acid (DHICA), its acetylated, methylated, and carboxylic ester derivatives, and two oligomers, a dimer and a trimer in the O-acetylated forms. The results show that (1) excited-state decays are faster for the trimer relative to the monomer; (2) for parent DHICA, excited-state lifetimes are much shorter in aqueous acidic medium (380 ps) as compared to organic solvent (acetonitrile, 2.6 ns); and (3) variation of fluorescence spectra and excited-state dynamics can be understood as a result of excited-state intramolecular proton transfer (ESIPT). The dependence on the DHICA oligomer size of the excited-state deactivation and its ESIPT mechanism provides important insight into the photostability and the photoprotective function of eumelanin. Mechanistic analogies with the corresponding processes in DNA and other biomolecules are recognized.

OXIDATIVE DEGRADATION OF MELANINS TO PYRROLE ACIDS : A MODEL STUDY

Abstract The origin of pyrrole-2,3,5-tricarboxylic acid (PTCA), the most characteristic degradation product of melanins, was investigated by use of synthetic pigments prepared from the major biosynthetic precursors, 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Under the reported conditions, i.e. acidic permanganate, oxidative degradation of DHI- and DHICA-melanins afforded PTCA in 0.15 and 3.0% w/w yield, respectively. A significant improvement of PTCA yields up to 7% was obtained using alkaline hydrogen peroxide as the oxidising agent. Under these conditions pyrrole-2,3-dicarboxylic acid (PDCA) was also obtained in significant yields. Investigation of the oxidative degradation of some model indole oligomers (1–4) provided unambiguous evidence that PTCA may originate from 2-linked DHI-units in the pigment polymer as well as from DHICA-derived units, whereas PDCA arises from DHI-units not substituted at 2-position. Monitoring of the oxidation of dimer 1 to PTCA showed the intermediate formation of DHICA. Accordingly, a mechanistic route is proposed for the degradation of 2-substituted DHI-units in melanin polymer to PTCA, which also accounts for the observed yields of formation of the pyrrole acid from the model oligomers.

Identification of Partially Degraded Oligomers of 5,6-Dihydroxyindole-2-carboxylic Acid inSepia Melanin by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Despite extensive efforts over more than half a century, the direct structural investigation of natural melanins has, so far, been largely unsuccessful, because of the adverse physical and chemical properties of these pigments. In the present study, we applied matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to the direct analysis of fresh melanin samples from the cuttlefish Sepia officinalis, and succeeded in identifying clearly distinct patterns of low molecular weight ionic species, ranging from 450 to 1200 Da. Detailed analysis of the molecular weights and mass differences between the major species, aided by comparison with MALDI-MS spectra of synthetic melanins, allowed formulation of the major components as oligomers of 5,6-dihydroxyindole-2-carboxylic acid partially degraded by peroxidative fission of the o-diphenol moieties with concomitant decarboxylation and oxygenation reactions. These results provide the first direct insight into the structure of Sepia melanin and confirm the unique value of MALDI-MS techniques for the investigation of highly complex and heterogeneous polymers such as melanins.

Functionality of epidermal melanin pigments: current knowledge on UV-dissipative mechanisms and research perspectives

So far it is not known whether epidermal melanins are solely photoprotective or also phototoxic. Also largely unknown are underlying UV-induced mechanisms and impact of the chemical structure. This perspective will focus on the current insights into the UV-dissipative mechanisms in melanin model systems and the implications for the in vivo pigments. We will also identify future research perspectives towards a full understanding of the functionality of epidermal pigments.

Oxidative polymerisation of 5,6-dihydroxyindole-2-carboxylic acid to melanin: A new insight

Abstract Previous studies on the oxidative polymerisation of 5,6-dihydroxyindole-2-carboxylic acid, a key intermediate in the biosynthesis of eumelanins, had delineated a reaction pathway involving mainly repeated coupling of the indole units through the 4- and 7- positions. Using an improved HPLC methodology for the direct analysis of oligomer intermediates, we have now obtained evidence for a more complex mode of polymerisation, involving formation, besides the 4,4′ and 4,7′ coupled dimers 5 and 6, of three new dimers, which have been isolated and identified as the 3,4′-, 3,7′- and 7,7′-biindolyls 7–9. The observed implication of the 3-position is unprecedented in the oxidative polymerisation of 5,6-dihydroxyindoles and yields important clues for future studies aimed at elucidating the chemical constitution of natural and synthetic eumelanins.

Structural Analysis of Synthetic Melanins from 5,6-Dihydroxyindole by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Despite extensive investigation, the structure of melanins, the major determinants of skin colour differences in man and other mammals, is still poorly defined, mainly because of the unfavourable properties of the materials. In this study, analysis of model pigments prepared by enzymically- or chemically-induced oxidative polymerization of 5,6-dihydroxyindole (DHI), the ultimate biosynthetic precursor, was investigated by means of matrix-assisted laser desorption/ionization mass spectrometry. All DHI melanins exhibited quite distinct pattern of ionic species of low m/z ranging from 500 to 1500, none of which corresponded to intact DHI oligomers. Analysis of the molecular weights and mass differences between the oligomer species provided evidence for a significant breakdown of the pigment backbone by peroxidative fission of the indole units with concomitant decarboxylation and oxygenation reactions. These processes, which take place to a different extent depending on the preparation conditions, are possibly initiated by hydrogen peroxide, either added as the oxidant or slowly generated in the reaction medium. In support of this, melanin samples prepared by tyrosinase oxidation in the presence of catalase comprised, as major components, intact DHI oligomers up to hexamers. Overall, these results offer a new picture of the structure of DHI melanins which may significantly contribute to the understanding of the high degree of molecular heterogeneity of the natural pigments.