Cysteine in the R3 Tau Peptide Modulates Hemin Binding and Reactivity.

Tau is a neuronal protein involved in axonal stabilization; however under pathological conditions, it triggers the deposition of insoluble neurofibrillary tangles, which are one of the biomarkers for Alzheimer's disease. The factors that might influence the fibrillation process are i) two cysteine residues in two pseudorepetitive regions, called R2 and R3, which can modulate protein-protein interaction via disulfide cross-linking; ii) an increase of reactive oxygen species affecting the post-translational modification of tau; and iii) cytotoxic levels of metals, especially ferric-heme (hemin), in hemolytic processes. Herein, we investigated how the cysteine-containing R3 peptide (R3C) and its Cys→Ala mutant (R3A) interact with hemin and how their binding affects the oxidative damage of the protein. The calculated binding constants are remarkably higher for the hemin-R3C complex (LogK1 = 5.90; LogK2 = 5.80) with respect to R3A (LogK1 = 4.44; LogK2 < 2), although NMR and CD investigations excluded the direct binding of cysteine as an iron axial ligand. Both peptides increase the peroxidase-like activity of hemin toward catecholamines and phenols, with a double catalytic efficiency detected for hemin-R3C systems. Moreover, the presence of cysteine significantly alters the susceptibility of R3 toward oxidative modifications, easily resulting in peptide dopamination and formation of cross-linked S-S derivatives.

Neuromelanins in brain aging and Parkinson's disease: synthesis, structure, neuroinflammatory, and neurodegenerative role.

Neuromelanins are compounds accumulating in neurons of human and animal brain during aging, with neurons of substantia nigra and locus coeruleus having the highest levels of neuromelanins. These compounds have melanic, lipid, peptide, and inorganic components and are contained inside special autolysosomes. Neuromelanins can participate in neuroprotective or toxic processes occurring in Parkinson's disease according to cellular environment. Their synthesis depends on the concentration of cytosolic catechols and is a protective process since it prevents the toxic accumulation of catechols-derived reactive compounds. Neuromelanins can be neuroprotective also by binding reactive/toxic metals to produce stable and non-toxic complexes. Extraneuronal neuromelanin released by dying dopamine neurons in Parkinson's disease activates microglia which generate reactive oxygen species, reactive nitrogen species, and proinflammatory molecules, thus producing still neuroinflammation and neuronal death. Synthetic neuromelanins have been prepared with melanic, protein structure, and metal content closely mimicking the natural brain pigment, and these models are also able to activate microglia. Neuromelanins have different structure, synthesis, cellular/subcellular distribution, and role than melanins of hair, skin, and other tissues. The main common aspect between brain neuromelanin and peripheral melanin is the presence of eumelanin and/or pheomelanin moieties in their structure.

Asymmetric Sulfoxidation by a Tyrosinase Biomimetic Dicopper Complex with a Benzimidazolyl Derivative of L-Phenylalanine.

A challenge in mimicking tyrosinase activity using model compounds is to reproduce its enantioselectivity. Good enantioselection requires rigidity and a chiral center close to the active site. In this study, the synthesis of a new chiral copper complex, [Cu2(mXPhI)]4+/2+, based on an m-xylyl-bis(imidazole)-bis(benzimidazole) ligand containing a stereocenter with a benzyl residue directly bound on the copper chelating ring, is reported. Binding experiments show that the cooperation between the two metal centers is weak, probably due to steric hindrance given by the benzyl group. The dicopper(II) complex [Cu2(mXPhI)]4+ has catalytic activity in the oxidations of enantiomeric couples of chiral catechols, with an excellent discrimination capability for Dopa-OMe enantiomers and a different substrate dependence, hyperbolic or with substrate inhibition, for the L- or D- enantiomers, respectively. [Cu2(mXPhI)]4+ is active in a tyrosinase-like sulfoxidation of organic sulfides. The monooxygenase reaction requires a reducing co-substrate (NH2OH) and yields sulfoxide with significant enantiomeric excess (e.e.). Experiments with 18O2 and thioanisole yielded sulfoxide with 77% incorporation of 18O, indicating a reaction occurring mostly through direct oxygen transfer from the copper active intermediate to the sulfide. This mechanism and the presence of the chiral center of the ligand in the immediate copper coordination sphere are responsible for the good enantioselectivity observed.

Role of the Cysteine in R3 Tau Peptide in Copper Binding and Reactivity.

Tau is a widespread neuroprotein that regulates the cytoskeleton assembly. In some neurological disorders, known as tauopathies, tau is dissociated from the microtubule and forms insoluble neurofibrillary tangles. Tau comprises four pseudorepeats (R1-R4), containing one (R1, R2, R4) or two (R3) histidines, that potentially act as metal binding sites. Moreover, Cys291 and Cys322 in R2 and R3, respectively, might have an important role in protein aggregation, through possible disulfide bond formation, and/or affecting the binding and reactivity of redox-active metal ions, as copper. We, therefore, compare the interaction of copper with octadeca-R3-peptide (R3C) and with the mutant containing an alanine residue (R3A) to assess the role of thiol group. Spectrophotometric titrations allow to calculate the formation constant of the copper(I) complexes, showing a remarkable stronger interaction in the case of R3C (log Kf = 13.4 and 10.5 for copper(I)-R3C and copper(I)-R3A, respectively). We also evaluate the oxidative reactivity associated to these copper complexes in the presence of dopamine and ascorbate. Both R3A and R3C peptides increase the capability of copper to oxidize catechols, but copper-R3C displays a peculiar mechanism due to the presence of cysteine. HPLC-MS analysis shows that cysteine can form disulfide bonds and dopamine-Cys covalent adducts, with potential implication in tau aggregation process.

Water-Soluble Melanin-Protein-Fe/Cu Conjugates Derived from Norepinephrine as Reliable Models for Neuromelanin of Human Brain Locus Coeruleus.

Water-soluble melanin-protein-Fe/Cu conjugates derived from norepinephrine and fibrillar ß-lactoglobulin are reliable models for neuromelanin (NM) of human brain locus coeruleus. Both iron and copper promote catecholamine oxidation and exhibit strong tendency to remain coupled in oligonuclear aggregates. The Fe-Cu clusters are EPR silent and affect the 1 H NMR spectra of the conjugates through a specific sequence of signals. Derivatives containing only Fe or Cu exhibit different NMR patterns. The EPR spectra show weak signals of paramagnetic FeIII in conjugates containing Fe or mixed Fe-Cu sites due to small amounts of mononuclear centers. The latter derivatives exhibit EPR signals for isolated CuII centers. These features parallel the EPR behavior of NM from locus coeruleus. The spectral data indicate that FeIII is bound to the melanic fraction, whereas CuII is bound on the protein fibrils, suggesting that the Fe-Cu clusters occur at the interface between the two components of the synthetic NMs.

A Cu-bis(imidazole) Substrate Intermediate Is the Catalytically Competent Center for Catechol Oxidase Activity of Copper Amyloid-ß.

Interaction of copper ions with Aß peptides alters the redox activity of the metal ion and can be associated with neurodegeneration. Many studies deal with the characterization of the copper binding mode responsible for the reactivity. Oxidation experiments of dopamine and related catechols by copper(II) complexes with the N-terminal amyloid-ß peptides Aß16 and Aß9, and the Aß16[H6A] and Aß16[H13A] mutant forms, both in their free amine and N-acetylated forms show that efficient reactivity requires the oxygenation of a CuI-bis(imidazole) complex with a bound substrate. Therefore, the active intermediate for catechol oxidation differs from the proposed "in-between state" for the catalytic oxidation of ascorbate. During the catechol oxidation process, hydrogen peroxide and superoxide anion are formed but give only a minor contribution to the reaction.

Oxidase Reactivity of CuII Bound to N-Truncated Aß Peptides Promoted by Dopamine.

The redox chemistry of copper(II) is strongly modulated by the coordination to amyloid-ß peptides and by the stability of the resulting complexes. Amino-terminal copper and nickel binding motifs (ATCUN) identified in truncated Aß sequences starting with Phe4 show very high affinity for copper(II) ions. Herein, we study the oxidase activity of [Cu-Aß4-x] and [Cu-Aß1-x] complexes toward dopamine and other catechols. The results show that the CuII-ATCUN site is not redox-inert; the reduction of the metal is induced by coordination of catechol to the metal and occurs through an inner sphere reaction. The generation of a ternary [CuII-Aß-catechol] species determines the efficiency of the oxidation, although the reaction rate is ruled by reoxidation of the CuI complex. In addition to the N-terminal coordination site, the two vicinal histidines, His13 and His14, provide a second Cu-binding motif. Catechol oxidation studies together with structural insight from the mixed dinuclear complexes Ni/Cu-Aß4-x reveal that the His-tandem is able to bind CuII ions independently of the ATCUN site, but the N-terminal metal complexation reduces the conformational mobility of the peptide chain, preventing the binding and oxidative reactivity toward catechol of CuII bound to the secondary site.

Membrane Binding Strongly Affecting the Dopamine Reactivity Induced by Copper Prion and Copper/Amyloid-ß (Aß) Peptides. A Ternary Copper/Aß/Prion Peptide Complex Stabilized and Solubilized in Sodium Dodecyl Sulfate Micelles.

The combination between dyshomeostatic levels of catecholamine neurotransmitters and redox-active metals such as copper and iron exacerbates the oxidative stress condition that typically affects neurodegenerative diseases. We report a comparative study of the oxidative reactivity of copper complexes with amyloid-ß (Aß40) and the prion peptide fragment 76-114 (PrP76-114), containing the high-affinity binding site, toward dopamine and 4-methylcatechol, in aqueous buffer and in sodium dodecyl sulfate micelles, as a model membrane environment. The competitive oxidative and covalent modifications undergone by the peptides were also evaluated. The high binding affinity of Cu/peptide to micelles and lipid membranes leads to a strong reduction (Aß40) and quenching (PrP76-114) of the oxidative efficiency of the binary complexes and to a stabilization and redox silencing of the ternary complex CuII/Aß40/PrP76-114, which is highly reactive in solution. The results improve our understanding of the pathological and protective effects associated with these complexes, depending on the physiological environment.

Binding and Reactivity of Copper to R1 and R3 Fragments of tau Protein.

Tau protein is present in significant amounts in neurons, where it contributes to the stabilization of microtubules. Insoluble neurofibrillary tangles of tau are associated with several neurological disorders known as tauopathies, among which is Alzheimer's disease. In neurons, tau binds tubulin through its microtubule binding domain which comprises four imperfect repeats (R1-R4). The histidine residues contained in these fragments are potential binding sites for metal ions and are located close to the regions that drive the formation of amyloid aggregates of tau. In this study, we present a detailed characterization through potentiometric and spectroscopic methods of the binding of copper in both oxidation states to R1 and R3 peptides, which contain one and two histidine residues, respectively. We also evaluate how the redox cycling of copper bound to tau peptides can mediate oxidation that can potentially target exogenous substrates such as neuronal catecholamines. The resulting quinone oxidation products undergo oligomerization and can competitively give post-translational peptide modifications yielding catechol adducts at amino acid residues. The presence of His-His tandem in the R3 peptide strongly influences both the binding of copper and the reactivity of the resulting copper complex. In particular, the presence of the two adjacent histidines makes the copper(I) binding to R3 much stronger than in R1. The copper-R3 complex is also much more active than the copper-R1 complex in promoting oxidative reactions, indicating that the two neighboring histidines activate copper as a catalyst in molecular oxygen activation reactions.

Interaction between Hemin and Prion Peptides: Binding, Oxidative Reactivity and Aggregation.

We investigate the interaction of hemin with four fragments of prion protein (PrP) containing from one to four histidines (PrP106-114, PrP95-114, PrP84-114, PrP76-114) for its potential relevance to prion diseases and possibly traumatic brain injury. The binding properties of hemin-PrP complexes have been evaluated by UV-visible spectrophotometric titration. PrP peptides form a 1:1 adduct with hemin with affinity that increases with the number of histidines and length of the peptide; the following log K1 binding constants have been calculated: 6.48 for PrP76-114, 6.1 for PrP84-114, 4.80 for PrP95-114, whereas for PrP106-114, the interaction is too weak to allow a reliable binding constant calculation. These constants are similar to that of amyloid-ß (Aß) for hemin, and similarly to hemin-Aß, PrP peptides tend to form a six-coordinated low-spin complex. However, the concomitant aggregation of PrP induced by hemin prevents calculation of the K2 binding constant. The turbidimetry analysis of [hemin-PrP76-114] shows that, once aggregated, this complex is scarcely soluble and undergoes precipitation. Finally, a detailed study of the peroxidase-like activity of [hemin-(PrP)] shows a moderate increase of the reactivity with respect to free hemin, but considering the activity over long time, as for neurodegenerative pathologies, it might contribute to neuronal oxidative stress.

Condition-Dependent Coordination and Peroxidase Activity of Hemin-Aß Complexes.

The peroxidase activity of hemin-peptide complexes remains a potential factor in oxidative damage relevant to neurodegeneration. Here, we present the effect of temperature, ionic strength, and pH relevant to pathophysiological conditions on the dynamic equilibrium between high-spin and low-spin hemin-Aß40 constructs. This influence on peroxidase activity was also demonstrated using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and dopamine (DA) oxidation rate analyses with increasing ratios of Aß16 and Aß40 (up to 100 equivalents). Interaction and reactivity studies of aggregated Aß40-hemin revealed enhanced peroxidase activity versus hemin alone. Comparison of the results obtained using Aß16 and Aß40 amyloid beta peptides revealed marked differences and provide insight into the potential effects of hemin-Aß on neurological disease progression.

A Stereoselective Tyrosinase Model Compound Derived from an m-Xylyl-l-histidine Ligand.

The aim of mimicking enzyme activity represents an important motivation for the development of new catalysts. A challenging objective is the development of chiral complexes for bioinspired enantioselective oxidation reactions. Herein, we report a new chiral dinuclear copper(II) complex based on a m-xylyl-bis(histidine) ligand (mXHI) as a biomimetic catalyst for tyrosinase and catechol oxidase. The new ligand improves a previous system also containing two tridentate N3 units derived from l-histidine that were connected by a short, rigid ethanediamine bridge. In mXHI the bridge is provided by the more extended m-xylyl moiety. The dicopper(II) complex [Cu2(mXHI)]4+ was studied as a catalyst for stereoselective oxidations of enantiomeric couples of chiral catechols of biological interest (L/D-dopa, L/D-dopa methyl ester, and ( R/ S)-norepinephrine), showing excellent discrimination capability, particularly for the methyl esters of dopa enantiomers. The catechol oxidation was studied in acetate buffer as slightly acidic medium, and a role of acetate as bridging ligand between the two coppers, preorganizing the dinuclear center in a more catalytic efficient structure, could be established. The oxidation of ß-naphthol and 3,5-ditertbutylphenol was studied as a model monophenolase reaction. The oxidation proceeds stoichiometrically, and the partial incorporation of 18O into ß-naphthol when the reaction was performed using 18O2 suggests the existence of two competitive reaction pathways, a genuine monooxygenase mechanism and a radical pathway. However, the more challenging reaction on derivatives of l-/d-tyrosine did not lead to the desired monooxygenase product but only to products of radical oxidation. Complex [Cu2(mXHI)]4+ was also used for the catalytic sulfoxidation of thioanisole in the presence of hydroxylamine as cosubstrate, in a preliminary attempt to model the reaction of external monooxygenases. The reaction proceeds with 25 turnovers, but the enantiomeric excess of sulfoxide was modest.

Dopamine, Oxidative Stress and Protein-Quinone Modifications in Parkinson's and Other Neurodegenerative Diseases.

Dopamine (DA) is the most important catecholamine in the brain, as it is the most abundant and the precursor of other neurotransmitters. Degeneration of nigrostriatal neurons of substantia nigra pars compacta in Parkinson's disease represents the best-studied link between DA neurotransmission and neuropathology. Catecholamines are reactive molecules that are handled through complex control and transport systems. Under normal conditions, small amounts of cytosolic DA are converted to neuromelanin in a stepwise process involving melanization of peptides and proteins. However, excessive cytosolic or extraneuronal DA can give rise to nonselective protein modifications. These reactions involve DA oxidation to quinone species and depend on the presence of redox-active transition metal ions such as iron and copper. Other oxidized DA metabolites likely participate in post-translational protein modification. Thus, protein-quinone modification is a heterogeneous process involving multiple DA-derived residues that produce structural and conformational changes of proteins and can lead to aggregation and inactivation of the modified proteins.

Prion Peptides Are Extremely Sensitive to Copper Induced Oxidative Stress.

Copper(II) binding to prion peptides does not prevent Cu redox cycling and formation of reactive oxygen species (ROS) in the presence of reducing agents. The toxic effects of these species are exacerbated in the presence of catecholamines, indicating that dysfunction of catecholamine vesicular sequestration or recovery after synaptic release is a dangerous amplifier of Cu induced oxidative stress. Cu bound to prion peptides including the high affinity site involving histidines adjacent to the octarepeats exhibits marked catalytic activity toward dopamine and 4-methylcatechol. The resulting quinone oxidation products undergo parallel oligomerization and endogenous peptide modification yielding catechol adducts at the histidine binding ligands. These modifications add to the more common oxidation of Met and His residues produced by ROS. Derivatization of Cu-prion peptides is much faster than that undergone by Cu-ß-amyloid and Cu-α-synuclein complexes in the same conditions.

Copper-Aß Peptides and Oxidation of Catecholic Substrates: Reactivity and Endogenous Peptide Damage.

The oxidative reactivity of copper complexes with Aß peptides 1-16 and 1-28 (Aß16 and Aß28) against dopamine and related catechols under physiological conditions has been investigated in parallel with the competitive oxidative modification undergone by the peptides. It was found that both Aß16 and Aß28 markedly increase the oxidative reactivity of copper(II) towards the catechol compounds, up to a molar ratio of about 4:1 of peptide/copper(II). Copper redox cycling during the catalytic activity induces the competitive modification of the peptide at selected amino acid residues. The main modifications consist of oxidation of His13/14 to 2-oxohistidine and Phe19/20 to ortho-tyrosine, and the formation of a covalent His6-catechol adduct. Competition by the endogenous peptide is rather efficient, as approximately one peptide molecule is oxidized every 10 molecules of 4-methylcatechol.

Anti-hypertensive property of a nickel-piperazine/NO donor in spontaneously hypertensive rats.

The nickel-piperazine/NO donor compound, Ni(PipNONO)Cl, belonging to the family of compounds labelled as "metal-nonoates", due to its promising vasodilating activity, has been considered as a potential drug candidate in anti-hypertensive therapy. Drug efficacy has been evaluated in spontaneously hypertensive rats (SHR) in comparison with normotensive animals (C57BL/6 mice and WKY rats). In normotensive animals the metal-nonoate maintained blood pressure at basal level both following acute administration and after 30 days of treatment. In SHR, Ni(PipNONO)Cl reduced blood pressure in the dose range of 3-10mg/kg. When compared with a commercial NONOate, DETA/NO, used at the same doses, Ni(PipNONO)Cl was more active in reducing blood pressure in SHR than DETA/NO in the first two weeks, while the effect of the two molecules was similar in the third and fourth week. The degradation and control compound Ni(Pip)Cl2 had no effect on blood pressure and heart rate in same animal models. Remarkably, the blood pressure reduction induced by the new NO-donor Ni(PipNONO)Cl does not evoke changes in the heart rate and tolerance. Considering the mechanisms of vascular protection, 30 days of administration of Ni(PipNONO)Cl improved endothelial function in SHR by upregulating endothelial NO synthase (eNOS) through increased eNOS protein levels and downregulated Caveolin-1 (Cav-1), and by increasing superoxide dismutase 1 (SOD1) protein level in aortae. In cultured endothelial cells Ni(PipNONO)Cl restored the cell functions (cytoskeletal protein expression, migration and proliferation) altered by the inflammatory mediator interleukin-1ß (IL-1ß), impairing the endothelial to mesenchimal transition. In conclusion, Ni(PipNONO)Cl maintained unaltered blood pressure in normotensive mice and rats, and it exerted anti-hypertensive effect in SHR through the restoration of vascular endothelial protective functions.

Copper(I) Forms a Redox-Stable 1:2 Complex with α-Synuclein N-Terminal Peptide in a Membrane-Like Environment.

α-Synuclein (αS) is the main protein component of Lewy bodies, characterizing the pathogenesis of Parkinson's disease. αS is unstructured in solution but adopts a helical structure in its extended N-terminal segment upon association with membranes. In vitro the protein binds avidly Cu(II), but in vivo the protein is N-acetylated, and Cu(II) binding is lost. We have now clarified the binding characteristics of the Cu(I) complex with the truncated αS peptide 1-15, both in N-acetylated and free amine forms, in a membrane mimetic environment and found that complexation occurs with a 1:2 Cu(I)-αS stoichiometry, where Cu(I) is bound to Met1 and Met5 residues of two helical peptide chains. The resulting tetrahedral Cu(I) center is redox-stable, does not form reactive oxygen species, and is unreactive against dopamine in the presence of O2. This suggests that, unlike cytosolic Cu(I)-αS, which retains the capacity to activate O2 and promote oxidative reactions, membrane-bound Cu(I)-αS may serve as a sink for unreactive copper.

A Mn(II)-Mn(II) center in human prolidase.

Human prolidase, the enzyme responsible for the hydrolysis of the Xaa-Pro/Hyp peptide bonds, is a key player in the recycling of imino acids during the final stage of protein catabolism and extracellular matrix remodeling. Its metal active site composition corresponding to the maximal catalytic activity is still unknown, although prolidase function is of increasing interest due to the link with carcinogenesis and mutations in prolidase gene cause a severe connective tissue disorder. Here, using EPR and ICP-MS on human recombinant prolidase produced in Escherichia coli (hRecProl), the Mn(II) ion organized in a dinuclear Mn(II)-Mn(II) center was identified as the protein cofactor. Furthermore, thermal denaturation, CD/fluorescence spectroscopy and limited proteolysis revealed that the Mn(II) is required for the proper protein folding and that a protein conformational modification is needed in the transition from apo- to Mn(II)loaded-enzyme. The collected data provided a better knowledge of the human holo-prolidase and, although limited to the recombinant enzyme, the exact identity and organization of the metal cofactor as well as the conformational change required for activity were proven.

Protective effects of novel metal-nonoates on the cellular components of the vascular system.

At the cardiovascular level, nitric oxide (NO) controls smooth muscle functions, maintains vascular integrity, and exerts an antihypertensive effect. Metal-nonoates are a recently discovered class of NO donors, with NO release modulated through the complexation of the N-aminoethylpiperazine N-diazeniumdiolate ligand to metal ions, and thus representing a significant innovation with respect to the drugs traditionally used. In this study, we characterized the vascular protective effects of the most effective compound of this class, Ni(PipNONO)Cl, compared with the commercial N-diazeniumdiolate group derivate, diethylenetriamine/nitric oxide (DETA/NO). Ni(PipNONO)Cl induced a concentration-dependent relaxation of precontracted rat aortic rings. The ED50 was 0.67 µM, compared with 4.3 µM obtained with DETA/NO. When tested on cultured microvascular endothelial cells, Ni(PipNONO)Cl exerted a protective effect on the endothelium, promoting cell proliferation and survival in the picomolar range. The administration of Ni(PipNONO)Cl to vascular smooth muscle cells reduced the cell number, promoting their apoptosis at a high concentration (10 µM). Inhibition of smooth muscle cell migration, a hallmark of atherosclerosis, was accompanied by cytoskeletal rearrangement and loss of lamellipodia. When added to isolated platelets, Ni(PipNONO)Cl significantly reduced ADP-induced aggregation. Since atherosclerosis is accompanied by an inflammatory environment, cultured endothelial cells were exposed to interleukin (IL)-1ß. In the presence of IL-1ß, Ni(PipNONO)Cl inhibited cyclooxygenase-2 and inducible nitric oxide synthase upregulation, and reduced endothelial permeability and the platelet and monocyte adhesion markers CD31 and CD40 at the plasma membrane. Overall, these data indicate that Ni(PipNONO)Cl exerts vascular protective effects relevant for vascular dysfunction and prevention of atherosclerosis and thrombosis.

Exploiting Principal Component Analysis (PCA) to reveal temperature, buffer and metal ions' role in neuromelanin (NM) synthesis by dopamine (DA) oxidative polymerization.

Neuromelanin (NM) plays a well-established role in neurological disorders pathogenesis; the mechanism of action is still discussed and the investigations in this field are limited by NM's complex and heterogeneous composition, insolubility, and low availability from human brains. An alternative can be offered by synthetic NM obtained from dopamine (DA) oxidative polymerization; however, a deep knowledge of the influence of both physicochemical parameters (T, pH, ionic strength) and other compounds in the reaction media (buffer, metal ions, other catecholamines) on DA oxidation process and, consequently, on synthetic NM features is mandatory to develop reliable NM preparation methodologies. To partially fulfill this aim, the present work focuses on defining the role of temperature, buffer and metal ions on both DA oxidation rate and DA oligomer size. DA oxidation in the specific conditions is monitored by UV-Vis spectroscopy and Principal Component Analysis (PCA) is run either on the raw spectra to model the background absorption increase, related to small DA oligomers formation, or on their first derivative to rationalize DA consumption. After having studied three case studies, 3-Way PCA is applied to directly evaluate the effect of temperature and buffer type on DA oxidation in the presence of different metal ions. Despite the proof-of-concept nature of the work and the number of compounds still to be included in the investigation, the preliminary results and the possibility to further expand the chemometric approach represent an interesting contribution to the field of in vitro simulation of NM synthesis.