Computational Insights of Selective Intramolecular O-atom Transfer Mediated by Bioinspired Copper Complexes.

The stereoselective copper-mediated hydroxylation of intramolecular C-H bonds from tridentate ligands is reinvestigated using DFT calculations. The computational study aims at deciphering the mechanism of C-H hydroxylation obtained after reaction of Cu(I) precursors with dioxygen, using ligands bearing either activated (L1 ) or non-activated (L2 ) C-H bonds. Configurational analysis allows rationalization of the experimentally observed regio- and stereoselectivity. The computed mechanism involves the formation of a side-on peroxide species (P) in equilibrium with the key intermediate bis-(µ-oxo) isomer (O) responsible for the C-H activation step. The P/O equilibrium yields the same activation barrier for the two complexes. However, the main difference between the two model complexes is observed during the C-H activation step, where the complex bearing the non-activated C-H bonds yields a higher energy barrier, accounting for the experimental lack of reactivity of this complex under those conditions.

Electrochemical Water Oxidation and Stereoselective Oxygen Atom Transfer Mediated by a Copper Complex.

Water oxidation by copper-based complexes to form dioxygen has attracted attention in recent years, with the aim of developing efficient and cheap catalysts for chemical energy storage. In addition, high-valent metal-oxo species produced by the oxidation of metal complexes in the presence of water can be used to achieve substrate oxygenation with the use of H2 O as an oxygen source. To date, this strategy has not been reported for copper complexes. Herein, a copper(II) complex, [(RPY2)Cu(OTf)2 ] (RPY2=N-substituted bis[2-pyridyl(ethylamine)] ligands; R=indane; OTf=triflate), is used. This complex, which contains an oxidizable substrate moiety (indane), is used as a tool to monitor an intramolecular oxygen atom transfer reaction. Electrochemical properties were investigated and, upon electrolysis at 1.30 V versus a normal hydrogen electrode (NHE), both dioxygen production and oxygenation of the indane moiety were observed. The ligand was oxidized in a highly diastereoselective manner, which indicated that the observed reactivity was mediated by metal-centered reactive species. The pH dependence of the reactivity was monitored and correlated with speciation deduced from different techniques, ranging from potentiometric titrations to spectroscopic studies and DFT calculations. Water oxidation for dioxygen production occurs at neutral pH and is probably mediated by the oxidation of a mononuclear copper(II) precursor. It is achieved with a rather low overpotential (280 mV at pH 7), although with limited efficiency. On the other hand, oxygenation is maximum at pH 8-8.5 and is probably mediated by the electrochemical oxidation of an antiferromagnetically coupled dinuclear bis(µ-hydroxo) copper(II) precursor. This constitutes the first example of copper-centered oxidative water activation for a selective oxygenation reaction.

Epoxide hydrolase-catalyzed enantioselective conversion of trans-stilbene oxide: Insights into the reaction mechanism from steady-state and pre-steady-state enzyme kinetics.

A detailed kinetic study based on steady-state and pre-steady-state measurements is described for the highly enantioselective epoxide hydrolase Kau2. The enzyme, which is a member of the α/ß-hydrolase fold family, preferentially reacts with the (S,S)-enantiomer of trans-stilbene oxide (TSO) with an E value of ∼200. The enzyme follows a classical two-step catalytic mechanism with formation of an alkyl-enzyme intermediate in the first step and hydrolysis of this intermediate in a rate-limiting second step. Tryptophan fluorescence quenching during TSO conversion appears to correlate with alkylation of the enzyme. The steady-state data are consistent with (S,S) and (R,R)-TSO being two competing substrates with marked differences in k(cat) and K(M) values. The high enantiopreference of the epoxide hydrolase is best explained by pronounced differences in the second-order alkylation rate constant (k2/K(S)) and the alkyl-enzyme hydrolysis rate k3 between the (S,S) and (R,R)-enantiomers of TSO. Our data suggest that during conversion of (S,S)-TSO the two active site tyrosines, Tyr(157) and Tyr(259), serve mainly as electrophilic catalysts in the alkylation half-reaction, polarizing the oxirane oxygen of the bound epoxide through hydrogen bond formation, however, without fully donating their hydrogens to the forming alkyl-enzyme intermediate.

Visible light-driven O2 reduction by a porphyrin-laccase system.

Several recent studies have shown that the combination of photosensitizers with metalloenzymes can support a light-driven multielectron reduction of molecules such as CO(2) or HCN. Here we show that the association of the zinc tetramethylpyridinium porphyrin (ZnTMPyP(4+)) photosensitizer with the multicopper oxidase (MCO) laccase allows to link the oxidation of an organic molecule to the four electrons reduction of dioxygen into water. The enzyme is photoreduced within minutes with porphyrin/enzyme ratio as low as 1:40. With a 1:1 ratio, the dioxygen consumption rate is 1.7 µmol L(-1) s(-1). Flash photolysis experiments support the formation of the triplet excited state of ZnTMPyP(4+) which reduces the enzyme to form a radical cation of the porphyrin with a k(ET) ≈ 10(7) s(-1) M(-1). The long-lived triplet excited state of the ZnTMPyP(4+) (τ(0) = 0.72 ms) accounts for a substantial electron-transfer quantum yield, φ(ET) = 0.35. Consequently, the enzyme-dependent photo-oxidation of the electron donor occurs with a turnover of 8 min(-1) for the one-electron oxidation process, thereby supporting the suitability of such enzyme/sensitizer hybrid systems for aerobic photodriven transformations on substrates. This study is the first example of a phorphyrin-sensitized four-electron reduction of an enzyme of the MCO family, leading to photoreduction of dioxygen into water.

Controlled Immobilization of a Palladium Complex/Laccase Hybrid into a Macrocellular Siliceous Host.

This study investigates the site-directed immobilization of a hybrid catalyst bearing a biquinoline-based-Pd(II) complex (1) and a robust laccase within cavities of a silica foam to favor veratryl alcohol oxidation. We performed the grafting of 1 at a unique surface located lysine of two laccase variants, either at closed (1⊂UNIK157 ) or opposite position (1⊂UNIK71 ) of the enzyme oxidation site. After immobilization into the cavities of silica monoliths bearing hierarchical porosity, we show that catalytic activity is dependent on the orientation and loading of each hybrid, 1⊂UNIK157 being twice as active than 1⊂UNIK71 (203 TON vs 100 TON) when operating under continuous flow. These systems can be reused 5 times, with an operational activity remaining as high as 40 %. We show that the synergy between 1 and laccase can be tuned within the foam. This work is a proof of concept for controlling the organization of a heterogeneous hybrid catalyst using a Pd/laccase/silica foam.

Modulation of laccase catalysed oxidations at the surface of magnetic nanoparticles.

We explored the coupling of laccases to magnetic nanoparticles (MNPs) with different surface chemical coating. Two laccase variants offering two opposite and precise orientations of the substrate oxidation site were immobilised onto core-shell MNPs presenting either aliphatic aldehyde, aromatic aldehyde or azide functional groups at the particles surface. Oxidation capabilities of the six-resulting laccase-MNP hybrids were compared on ABTS and coniferyl alcohol. Herein, we show that the original interfaces created differ substantially in their reactivities with an amplitude from 1 to > 4 folds depending on the nature of the substrate. Taking enzyme orientation into account in the design of surface modification represents a way to introduce selectivity in laccase catalysed reactions.

Hydrogen evolution reaction mediated by an all-sulfur trinuclear nickel complex.

We report the synthesis and the characterization of a trinuclear nickel complex. Solid state and solution studies using X-ray diffraction, NMR and UV-vis spectroscopy highlight the square planar geometries around the metal centers and an all-sulfur coordination sphere. It exhibits significant electrocatalytic activity for hydrogen evolution in DMF using Et3NHCl as the proton source. DFT studies suggest that sulfur atoms act as proton relay, as proposed in [NiFe] hydrogenases.

Binding of 2-hydroxypyridine-N-oxide on dicopper(II) centers: insights into tyrosinase inhibition mechanism by transition-state analogs.

2-Hydroxypyridine-N-oxide (HOPNO) is described as a new and efficient transition-state analog (TS-analog) inhibitor for the mushroom tyrosinase with an IC(50) = 1.16 microM and a K(I) = 1.8 microM. Using the binuclear copper(II) complex [Cu(2)(BPMP)(mu-OH)](ClO(4))(2) (2) known as a functional model for the tyrosinase catecholase activity, we isolated and fully characterized a 1:1 (2)/OPNO adduct in which the HOPNO is deprotonated and chelates only one Cu-atom of the binuclear site in a bidentate mode. On the basis of these results, a structural model for the tyrosinase inhibition by HOPNO is proposed.

The role of methylation in the copper(ii) coordination properties of a His-containing decapeptide.

N-Methylation of the peptide amide bond has proven to be a powerful strategy to fine-tune the conformation and properties of peptides. In this context and for the first time, we show that N-methylation can also be used to control the copper(ii) coordination properties of peptides and stabilize at high pH values the copper(ii) species lacking amidate coordination. Namely, we have prepared a derivative of the O-Asp peptide where the copper(ii) coordinating amino acids, i.e. Asp and His residues, were N-methylated (ONMe-Asp). A combined study using potentiometric and spectroscopic (UV-Vis, CD, EPR and NMR) techniques indicates the formation of the wanted major species, [CuH(ONMe-Asp)]2+, where copper(ii) is bound to His4(Nε), His7(Nε), His9(Nε) and Asp2(COO-). With respect to the parent non-methylated O-Asp peptide, [CuH(ONMe-Asp)]2+ is stable at higher pH values but has lower affinity for copper(ii). Additionally, electrochemical studies reveal a Cu(ii) ⇌ Cu(i) redox process with a larger cathodic and anodic peak separation. Species containing copper(ii) coordinating amidates were not observed for this ONMe-Asp peptide.

Characterization of a Dinuclear Copper(II) Complex and Its Fleeting Mixed-Valent Copper(II)/Copper(III) Counterpart.

The synthesis of a dinuclear copper(II) complex, supported by a 1,3-diamino-2-propanol-based tetraamide ligand, is reported. Structural properties in the solid state and in solution, by means of XRD analysis and NMR spectroscopy, respectively, provide evidence of a highly flexible complex that can display several conformations, leading to the image of the wings of a butterfly. The complex was fully characterized and the redox properties were investigated. Room-temperature spectro-electrochemistry was used to monitor the formation of a metastable mono-oxidized product that displayed an absorption band centered at λ=463 nm. EPR investigation of the low-temperature, chemically generated, mono-oxidized product reveals the presence of an intermediate described as a mixed-valent CuII CuIII species, which is a model of the possible highly oxidizing intermediate in particulate methane monooxygenase.

Laccases as palladium oxidases.

The first example of a coupled catalytic system involving an enzyme and a palladium(ii) catalyst competent for the aerobic oxidation of alcohol in mild conditions is described. In the absence of dioxygen, the fungal laccase LAC3 is reduced by a palladium(0) species as evidenced by the UV/VIS and ESR spectra of the enzyme. During the oxidation of veratryl alcohol performed in water, at room temperature and atmospheric pressure, LAC3 regenerates the palladium catalyst, is reduced and catalyzes the four-electron reduction of dioxygen into water with no loss of enzyme activity. The association of a laccase with a water-soluble palladium complex results in a 7-fold increase in the catalytic efficiency of the complex. This is the first step in the design of a family of renewable palladium catalysts for aerobic oxidation.

Reactivity of dinuclear copper(II) complexes towards melanoma cells: Correlation with its stability, tyrosinase mimicking and nuclease activity.

In this work, the influence of two new dinuclear copper(II) complexes in the viability of melanoma cells (B16F10 and TM1MNG3) was investigated, with the aim of verifying possible correlations between their cytotoxicity and their structure. One of the complexes had a polydentate dinucleating amine-imine ligand (complex 2), and the other a tridentate imine and a diamine-bridging ligand (complex 4). The analogous mononuclear copper(II) species (complexes 1 and 3, respectively) were also prepared for comparative studies. Crystal structure determination of complex 2 indicated a square-based pyramidal geometry around each copper, coordinated to three N atoms from the ligand and the remaining sites being occupied by either solvent molecules or counter-ions. Complex 4 has a tetragonal geometry. Interactions of these complexes with human albumin protein (HSA) allowed an estimation of their relative stabilities. Complementary studies of their reactivity towards DNA indicated that all of them are able of causing significant oxidative damage, with single and double strand cleavages, in the presence of hydrogen peroxide. However, nuclease activity of the dinuclear species was very similar and much higher than that of the corresponding mononuclear compounds. Although complex 2, with a more flexible structure, exhibits a much higher tyrosinase activity than complex 4, having a more rigid environment around the metal ion, both complexes showed comparable cytotoxicity towards melanoma cells. Corresponding mononuclear complexes showed to be remarkably less reactive as tyrosinase mimics as well as cytotoxic agents. Moreover, the dinuclear complexes showed higher cytotoxicity towards more melanogenic cells. The obtained results indicated that the structure of these species is decisive for its activity towards the malignant tumor cells tested.