In Parkinson’s disease (PD), dopamine neurons containing neuromelanin selectively degenerate. Neuromelanin binds iron and accumulates in aging. Iron accumulates in reactive form during aging, PD, and is involved in neurodegeneration. It is not clear how the interaction of neuromelanin and iron can be protective or toxic by modulating redox processes. Here, we investigated the interaction of neuromelanin from human substantia nigra with iron in the presence of ascorbic acid, dopamine, and hydrogen peroxide. We observed that neuromelanin blocks hydroxyl radical production by Fenton’s reaction, in a dose‐dependent manner. Neuromelanin also inhibited the iron‐mediated oxidation of ascorbic acid, thus sparing this major antioxidant molecule in brain. The protective effect of neuromelanin on ascorbate oxidation occurs even in conditions of iron overload into neuromelanin. The blockade of iron into a stable iron–neuromelanin complex prevents dopamine oxidation, inhibiting the formation of neurotoxic dopamine quinones. The above processes occur intraneuronally in aging and PD, thus showing that neuromelanin is neuroprotective. The iron–neuromelanin complex is completely decomposed by hydrogen peroxide and its degradation rate increases with the amount of iron bound to neuromelanin. This occurs in PD when extraneuronal iron–neuromelanin is phagocytosed by microglia and iron–neuromelanin degradation releases reactive/toxic iron.

Neuromelanins (NMs) are neuronal pigments of melanic-lipidic type which accumulate during aging. They are involved in protective and degenerative mechanisms depending on the cellular context, however their structures are still poorly understood. NMs from nine human brain areas were analyzed in detail. Elemental analysis led to identification of three types of NM, while infrared spectroscopy showed that NMs from neurons of substantia nigra and locus coeruleus, which selectively degenerate in Parkinson’s disease, have similar structure but different from NMs from brain regions not targeted by the disease. Synthetic melanins containing Fe and bovine serum albumin were prepared to model the natural product and help clarifying the structure of NMs. Extensive nuclear magnetic resonance spectroscopy studies showed the presence of dolichols both in the soluble and insoluble parts of NM. Diffusion measurements demonstrated that the dimethyl sulfoxide soluble components consist of oligomeric precursors with MWs in the range 1.4–52 kDa, while the insoluble part contains polymers of larger size but with a similar composition. These data suggest that the selective vulnerability of neurons of substantia nigra and locus coeruleus in Parkinson’s disease might depend on the structure of the pigment. Moreover, they allow to propose a pathway for NM biosynthesis in human brain.

Elucidating the structure and biosynthesis of neuromelanin (NM) would be an important step towards understanding its putative role in the pathogenesis of Parkinson's disease. A useful complement to studies aimed at unraveling the origin and properties of this essentially insoluble natural substance is the preparation of synthetic derivatives that resemble NM. With this aim in mind, water-soluble conjugates between dopamine-derived melanin and bovine serum albumin (BSA) were synthesized. Melanin-BSA adducts were prepared with both eumelanic oligomers obtained through the oxidative polymerization of dopamine and pheomelanic oligomers obtained under the same conditions from dopamine and cysteine. Iron ions were added during the synthesis to understand the interaction between the pigment and this metal ion, as the NM in neurons in several human brain regions contains significant amounts of iron. The structures of the conjugates were analyzed by (1)H NMR spectroscopy and controlled proteolysis/MS experiments. The binding of iron(III) ions was evaluated by ICP analysis and EPR spectroscopy. The EPR signal from bound iron(III) indicated high-spin octahedral sites and, as also seen for NM, the signal is coupled to a signal from a radical associated with the melanic components of the conjugates. However, the intensity of the EPR signal from iron suggested a reduced fraction of the total iron, indicating that most of the iron is strongly coupled in clusters within the matrix. The amount of paramagnetic, mononuclear iron(III) was greater in the pheomelanin-BSA conjugates, suggesting that iron clustering is reduced in the sulfur-containing pigment. Thus, the melanin-BSA conjugates appear to be good models for the natural pigment.

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Four new macrocyclic ligands of varying ring size, [22] (D), show the pyrazole groups and the amine nitrogens all to be involved in the coordination of the copper(II) ions. In the mononuclear compound A the copper ion is in a distorted octahedral geometry, with the equatorial plane formed by four nitrogen donor atoms from the ligand and the axial positions occupied by two oxygen atoms from mono-coordinated perchlorate anions. In compound B each copper ion is in a distorted square pyramidal environment, with the three ligand nitrogens and a chloride atom in the equatorial plane and another chloride atom in the axial position. The cation of compound C contains four copper centers. Two bridging Cl atoms connect the two central copper atoms to form a centrosymmetric four membered ring. Two macrocyclic units are present in the cation, each containing two copper atoms bridged by a chloride atom. One copper is fivefold coordinated and the second copper is distorted octahedral. In compound D both copper(II) ions are in a distorted octahedral N 5 O environment, with the equatorial plane formed by the three ligand nitrogens and a nitrogen from an acetonitrile molecule. A second acetonitrile molecule and a mono-coordinated perchlorate anion are weakly bound in the axial positions. The copper nitrate compounds of these new ligands are to some degree active catalysts in the oxidative coupling of 2,6-dimethylphenol with molecular dioxygen to poly(2,6-dimethyl-1,4-phenylene ether). #

Bioconjugation is a rapidly expanding field because of the numerous potential applications of bioconjugate materials. We explored the usefulness of branched porphyrins as rigid scaffolds, bearing multiple sites for bioconjugation. To this end, we first selected the tetrakis(p-[aminomethyl] phenyl) porphyrin (TAMPP) macrocycle and developed a straightforward synthetic protocol, able to provide the desired tetraphenylporphyrin, carrying four functional amino groups. The partially protection of the amino groups by tert-butoxy-carbonyl allowed the selective and specific decoration of the porphyrin with different peptide sequences. To explore the utility of the macrocycle as molecular scaffold for bioconjugation, we selected peptide sequences able to function as thrombin inhibitors. In particular, two peptide sequences, named CS3 and ES7, able to interact, respectively, with the thrombin catalytic site and the fibrinogen recognition exosite, were joined onto the porphyrin macrocycle, providing the multisite-directed inhibitor CS3-TAMPP-ES7. This multisite inhibitor and its Mn(III) complex are able to inhibit α-thrombin-catalyzed hydrolysis of Tos-Gly-Pro-Arg-nitroanilide with inhibition constants in the micromolar range, as well as the hydrolysis of the natural substrate fibrinogen. The inhibitor is resistant against enzymatic degradation by thrombin and is highly selective. The Mn(III) complex is capable of interacting with clot-bound thrombin and partially inhibits clot growth in the presence of fibrinogen. The results herein reported are very promising, suggesting the potential of the newly developed conjugate as new imaging agents for clot detection.

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