Andrea Madabeni

Tyrosinase contains a coupled binuclear copper (CBC) active site, which in its bicuprous form (deoxy-Ty) binds O2 to form a side-on peroxide [Cu(II)2O2]2+ intermediate (oxy-Ty) that performs the regioselective monooxygenation of monophenols to catechols and their subsequent 2e− oxidation to quinones. Previously, we used spectroscopic, kinetic, and computational methods to elucidate the mechanism of the initial steps in the monooxygenation reaction. Herein, we investigated the final step in catecholate formation and provide experimental and computational results elucidating the catechol oxidation reaction, formally a proton-coupled 2e− process. We employed single-turnover stopped-flow absorption to observe the elusive catecholate-Cu(II)2OH intermediate via the anaerobic reaction of resting Cu(II)2OH Ty (met-Ty) with the catecholate substrate. This intermediate was cryo-trapped and characterized by electron paramagnetic resonance and X-ray absorption spectroscopies. These experimental results were correlated to quantum mechanics/molecular mechanics (QM/MM) and QM calculations to describe the complete catalytic cycle of tyrosinase, revealing that the 2e− oxidation of catechol to quinone involves two steps: (i) coupled 1H+/1e− transfer from the bound monoanionic catecholate to the bridged hydroxide of the met-Ty active site, followed by (ii) the second 1e− transfer. The latter is calculated to be the rate-limiting step in catechol oxidation, confirmed by experimental solvent kinetic isotope effect studies. Our computational data suggest that quinone release from the protein provides the necessary driving force for this reaction. These results establish a detailed description of this oxidase cycle of the tyrosinase mechanism and more broadly provide molecular-level insights into the diverse reactivity of CBC sites in biology.

A reaction between azide, alkyne and 2H-azirine resulted in C-C bond formation at position five of 1,2,3-triazole, instead of previously misidentified C-N bond connectivity. The reaction mechanism of this C-C bond formation on the triazole ring was fully explained by employing calibrated QM(DFT-D3) calculations. Functionalization of primary products provided substituted pyrimidine, furan or 1,3-oxazepine.

Chalcogenide exchange reactions are an important class of bimolecular nucleophilic substitution reactions (S_N2) involving sulfur and selenium species as nucleophile, central atom, and/or leaving group, which are fundamental throughout redox biology and metabolism. While thiol‐disulfide exchange reactions have been deeply investigated, those involving selenium are less understood, especially with regards to the polarised selenenyl sulfides RSe–SR’. This functional group, which is fundamental in the biochemistry of glutathione peroxidase and thioredoxin reductase enzymes, was recently incorporated in the molecular scaffold of a TrxR1 specific probe, “RX1”. Here, we investigate the S_N2@S and S_N2@Se reactions of selenenyl sulfides in silico to provide the first comprehensive overview of their kinetic and thermodynamic trends, referencing against symmetrical disulfides and diselenides. Then, the role of S_N2@S and S_N2@Se reactions in RX1 chemistry is explored, and a mechanistic picture of its biological chemistry is provided. Additionally, we quantify the role of alternative exchange reactions in the double‐exchange chemistry of RX1. This analysis rationalises the origins of RX1’s TrxR‐specificity even within thiol‐rich cellular environments and can support the design and applications of a range of selenenyl sulfide‐based bioactive probes. Particularly, we observe that the intramolecular S_N2@Se reaction which restores RX1 ground state is an effective protective mechanism against unspecific activation by thiols, explaining its capacity to work in high‐thiol concentration.

The reactivity of Zn²⁺ tetrahedral complexes with H₂O₂ was investigated in silico, as a first step in their disruption process. The substrates were chosen to represent the cores of three different zinc finger protein motifs, i. e., a Zn²⁺ ion coordinated to four cysteines (CCCC), to three cysteines and one histidine (CCCH), and to two cysteines and two histidines (CCHH). The cysteine and histidine ligands were further simplified to methyl thiolate and imidazole, respectively. H₂O₂ was chosen as an oxidizing agent due to its biological role as a metabolic product and species involved in signaling processes. The mechanism of oxidation of a coordinated cysteinate to sulfenate‐κS and the trends for the different substrates were rationalized through activation strain analysis and energy decomposition analysis in the framework of scalar relativistic Density Functional Theory (DFT) calculations at ZORA‐M06/TZ2P ae // ZORA‐BLYP‐D3(BJ)/TZ2P. CCCC is oxidized most easily, an outcome explained considering both electrostatic and orbital interactions. The isomerization to sulfenate‐κO was attempted to assess whether this step may affect the ligand dissociation; however, it was found to introduce a kinetic barrier without improving the energetics of the dissociation. Lastly, ligand exchange with free thiolates and selenolates was investigated as a trigger for ligand dissociation, possibly leading to metal ejection; molecular docking simulations also support this hypothesis.

Se( iv ) seleninic acid is the active catalyst in aniline oxidation by H ₂ O ₂ due to its favorable peroxide activation kinetics. The slow formation of Se( vi ) selenonic acid and its poorer performance in H ₂ O ₂ activation hinder its catalytic role.

PhSeZnCl, which is also known as Santi's reagent, can catalyze the reduction of hydrogen peroxide by thiols with a GPx-like mechanism. In this work, the first step of this catalytic cycle, i.e., the reduction of H₂O₂ by PhSeZnCl, is investigated in silico using state-of-the-art density functional theory calculations. Then, the role of the metal is evaluated by replacing Zn with its group 12 siblings (Cd and Hg). The thermodynamic and kinetic factors favoring Zn are elucidated. Furthermore, the role of the halogen is considered by replacing Cl with Br in all three metal compounds, and this turns out to be negligible. Finally, the overall GPx-like mechanism of PhSeZnCl and PhSeZnBr is discussed by evaluating the energetics of the mechanistic path leading to the disulfide product.

Sulfoxides and selenoxides oxidize thiols to disulfides while being reduced back to sulfides and selenides. While the reduction mechanism of sulfoxides to sulfides has been thoroughly explored experimentally as well as computationally, less attention has been devoted to the heavier selenoxides. In this work, we explore the reductive mechanism of dimethyl selenoxide, as an archetypal selenoxide and, for the sake of comparison, the reductive mechanism of dimethyl sulfoxide to gain insight into the role of the chalcogen on the reaction substrate. Particular attention is devoted to the key role of sulfurane and selenurane intermediates. Moreover, the capacity of these system to oxidize selenols rather than thiols, leading to the formation of selenyl sulfide bridges, is explored in silico. Notably, this analysis provides molecular insight into the role of selenocysteine in methionine sulfoxide reductase selenoenzyme. The activation strain model of chemical reactivity is employed in the studied reactions as an intuitive tool to bridge the computationally predicted effect of the chalcogen on the chalcogenoxide as well as on the chalcogenol.

The SARS-CoV-2 pandemic has prompted global efforts to develop therapeutics. The main protease of SARS-CoV-2 (Mpro) and the papain-like protease (PLpro) are essential for viral replication and are key targets for therapeutic development. In this work, we investigate the mechanisms of SARS-CoV-2 inhibition by diphenyl diselenide (PhSe)2 which is an archetypal model of diselenides and a renowned potential therapeutic agent. The in vitro inhibitory concentration of (PhSe)2 against SARS-CoV-2 in Vero E6 cells falls in the low micromolar range. Molecular dynamics (MD) simulations and density functional theory (DFT) calculations [level of theory: SMD-B3LYP-D3­(BJ)/6-311G­(d,p), cc-pVTZ] are used to inspect non-covalent inhibition modes of both proteases via π-stacking and the mechanism of covalent (PhSe)2 + Mpro product formation involving the catalytic residue C145, respectively. The in vitro CC50 (24.61 μM) and EC50 (2.39 μM) data indicate that (PhSe)2 is a good inhibitor of the SARS-CoV-2 virus replication in a cell culture model. The in silico findings indicate potential mechanisms of proteases’ inhibition by (PhSe)2; in particular, the results of the covalent inhibition here discussed for Mpro, whose thermodynamics is approximatively isoergonic, prompt further investigation in the design of antiviral organodiselenides.

The reactive oxygen species (ROS) scavenging capacities of ginkgolides and bilobalide, which are the peculiar constituents of the extract of Ginkgo biloba, are investigated in silico (level of theory: (SMD)-M06-2X/6-311+G(d,p)//M06-2X/6-31G(d)). Unlike other popular antioxidant natural substances, the carbon backbones of these compounds are entirely aliphatic and exclusively single C–C bonds are present. The selectivity for alkoxyl radicals via hydrogen-atom transfer (HAT) is assessed; importantly, the scavenging of peroxyl radicals is also possible from a peculiar site, here labeled C10 both for ginkgolides and bilobalide. The energetics are described in detail, and the analysis discloses that the studied compounds are powerful scavengers, with thermodynamic and kinetic properties similar to those of Trolox and melatonin, and that, in addition, they display selectivity for peroxyl radicals. These are all chemical-reactivity features contributing to the therapeutic action of the extract of G. biloba.