Rationally Designed Cu(I) Ligand to Prevent CuAß-Generated ROS Production in the Alzheimer's Disease Context.

In the context of Alzheimer's disease, copper (Cu) can be loosely bound to the amyloid-ß (Aß) peptide, leading to the formation of CuAß, which can catalytically generate reactive oxygen species that contribute to oxidative stress. To fight against this phenomenon, the chelation therapy approach has been developed and consists of using a ligand able to remove Cu from Aß and to redox-silence it, thus stopping the reactive oxygen species (ROS) production. A large number of Cu(II) chelators has been studied, allowing us to define and refine the properties required to design a "good" ligand, but without strong therapeutic outcomes to date. Those chelators targeted the Cu(II) redox state. Herein, we explore a parallel and relevant alternative pathway by designing a chelator able to target the Cu(I) redox state. To that end, we designed LH2 ([1N3S] binding set) and demonstrated that (i) it is perfectly able to extract Cu(I) from Cu(I)Aß even in the presence of an excess of Zn(II) and (ii) it redox-silences the Cu, preventing the formation of ROS. We showed that LH2 that is sensitive to oxidation can efficiently replace the [Zn(II)L] complex without losing its excellent ability to stop the ROS production while increasing its resistance to oxidation.

Biomimetic catalysis of nitrite reductase enzyme using copper complexes in chemical and electrochemical reduction of nitrite.

Copper nitrite reductase mimetics were synthesized using three new tridentate ligands sharing the same N,N,N motif of coordination. The ligands were based on L-proline modifications, attaching a pyridine and a triazole to the pyrrolidine ring, and differ by a pendant group (R = phenyl, n-butyl and n-propan-1-ol). All complexes coordinate nitrite, as evidenced by cyclic voltammetry, UV-Vis, FTIR and electron paramagnetic resonance (EPR) spectroscopies. The coordination mode of nitrite was assigned by FTIR and EPR as κ2O chelate mode. Upon acidification, EPR experiments indicated a shift from chelate to monodentate κO mode, and 15N NMR experiments of a Zn2+ analogue, suggested that the related Cu(II) nitrous acid complex may be reasonably stable in solution, but in equilibrium with free HONO under non catalytic conditions. Reduction of nitrite to NO was performed both chemically and electrocatalytically, observing the highest catalytic activities for the complex with n-propan-1-ol as pendant group. These results support the hypothesis that a hydrogen bond moiety in the secondary coordination sphere may aid the protonation step.

Ability of Azathiacyclen Ligands To Stop Cu(Aß)-Induced Production of Reactive Oxygen Species: [3N1S] Is the Right Donor Set.

Alzheimer's disease (AD) is an incurable neurodegenerative disease that leads to the progressive and irreversible loss of mental functions. The amyloid beta (Aß) peptide involved in the disease is responsible for the production of damaging reactive oxygen species (ROS) when bound to Cu ions. A therapeutic approach that consists of removing Cu ions from Aß to alter this deleterious interaction is currently being developed. In this context, we report the ability of five different 12-membered thiaazacyclen ligands to capture Cu from Aß and to redox silence it. We propose that the presence of a sole sulfur atom in the ligand increases the rate of Cu capture and removal from Aß, while the kinetic aspect of the chelation was an issue encountered with the 4N parent ligand. The best ligand for removing Cu from Aß and inhibiting the associated ROS production is the 1-thia-4,7,10-triazacyclododecane [3N1S]. Indeed the replacement of more N by S atoms makes the corresponding Cu complexes easier to reduce and thus able to produce ROS on their own. In addition, the ligand with three sulfur atoms has a weaker affinity for CuII than Aß, and is thus unable to remove Cu from CuAß.

Sequence-Activity Relationship of ATCUN Peptides in the Context of Alzheimer's Disease.

Amino-terminal CuII and NiII (ATCUN) binding sequences are widespread in the biological world. Here, we report on the study of eight ATCUN peptides aimed at targeting copper ions and stopping the associated formation of reactive oxygen species (ROS). This study was actually more focused on Cu(Aß)-induced ROS production in which the Aß peptide is the "villain" linked to Alzheimer's disease. The full characterization of CuII binding to the ATCUN peptides, the CuII extraction from CuII(Aß), and the ability of the peptides to prevent and/or stop ROS formation are described in the relevant biological conditions. We highlighted in this research that all the ATCUN motifs studied formed the same thermodynamic complex but that the addition of a second histidine in position 1 or 2 allowed for an improvement in the CuII uptake kinetics. This kinetic rate was directly related to the ability of the peptide to stop the CuII(Aß)-induced production of ROS, with the most efficient motifs being HWHG and HGHW.

Hybrid Bis-Histidine Phenanthroline-Based Ligands to Lessen Aß-Bound Cu ROS Production: An Illustration of Cu(I) Significance.

We here report the synthesis of three new hybrid ligands built around the phenanthroline scaffold and encompassing two histidine-like moieties: phenHH, phenHGH and H'phenH', where H correspond to histidine and H' to histamine. These ligands were designed to capture Cu(I/II) from the amyloid-ß peptide and to prevent the formation of reactive oxygen species produced by amyloid-ß bound copper in presence of physiological reductant (e.g., ascorbate) and dioxygen. The amyloid-ß peptide is a well-known key player in Alzheimer's disease, a debilitating and devasting neurological disorder the mankind has to fight against. The Cu-Aß complex does participate in the oxidative stress observed in the disease, due to the redox ability of the Cu(I/II) ions. The complete characterization of the copper complexes made with phenHH, phenHGH and H'phenH' is reported, along with the ability of ligands to remove Cu from Aß, and to prevent the formation of reactive oxygen species catalyzed by Cu and Cu-Aß, including in presence of zinc, the second metal ions important in the etiology of Alzheimer's disease. The importance of the reduced state of copper, Cu(I), in the prevention and arrest of ROS is mechanistically described with the help of cyclic voltammetry experiments.

Solid-state and solution characterizations of [(TMPA)Cu(II)(SO3)] and [(TMPA)Cu(II)(S2O3)] complexes: Application to sulfite and thiosulfate fast detection.

Sulfite (SO32-) and thiosulfate (S2O32-) ions are used as food preservative and antichlor agent respectively. To detect low levels of such anions we used Cu(II) complex of the Tris-Methyl Pyridine Amine (TMPA) ligand, denoted L. Formation of [LCu(SO3)] (1) and [LCu(S2O3)] (2) in solution were monitored using UV-Vis, EPR and cyclic voltammetry, while the solid-state X-ray structures of both complexes were solved. In addition, we also evaluated the pH range in which the complexes are stable, and the anions binding affinity values for the [LCu(solvent)]2+ (3) parent complex. As a matter of illustration, we determined the sulfite content in a commercial crystal sugar.

Measurement of Interpeptidic CuII Exchange Rate Constants of CuII-Amyloid-ß Complexes to Small Peptide Motifs by Tryptophan Fluorescence Quenching.

The interpeptidic CuII exchange rate constants were measured for two Cu amyloid-ß complexes, Cu(Aß1-16) and Cu(Aß1-28), to fluorescent peptides GHW and DAHW using a quantitative tryptophan fluorescence quenching methodology. The second-order rate constants were determined at three pH values (6.8, 7.4, and 8.7) important to the two Cu(Aß) coordination complexes, components Cu(Aß)I and Cu(Aß)II. The interpeptidic CuII exchange rate constant is approximately 104 M-1 s-1 but varies in magnitude depending on many variables. These include pH, length of the Aß peptide, location of the anchoring histidine ligand in the fluorescent peptide, number of amide deprotonations required in the tryptophan peptide to coordinate CuII, and interconversion between Cu(Aß)I and Cu(Aß)II. We also present EPR data probing the CuII exchange between peptides and the formation of ternary species between Cu(Aß) and GHW. As the nonfluorescent GHK and DAHK peptides are important motifs found in the blood and serum, their ability to sequester CuII ions from Cu(Aß) complexes may be relevant for the metal homeostasis and its implication in Alzheimer's disease. Thus, their kinetic CuII interpeptidic exchange rate constants are important chemical rate constants that can help elucidate the complex CuII trafficking puzzle in the synaptic cleft.

Unexpected Trends in Copper Removal from Aß Peptide: When Less Ligand Is Better and Zn Helps.

Cu, Zn, and amyloid-ß (Aß) peptides play an important role in the etiology of Alzheimer's disease (AD). Their interaction indeed modifies the self-assembly propensity of the peptide that is at the origin of the deposition of insoluble peptide aggregates in the amyloid plaque, a hallmark found in AD brains. Another even more important fallout of the Cu binding to Aß peptide is the formation of reactive oxygen species (ROS) that contributes to the overall oxidative stress detected in the disease and is due to the redox ability of the Cu ions. Many therapeutic approaches are currently developed to aid fighting against AD, one of them targeting the redox-active Cu ions. Along this research line, we report in the present article the use of a phenanthroline-based peptide-like ligand (L), which is able to withdraw Cu from Aß and redox-silence it in a very stable 4N Cu(II) binding site even in the presence of Zn(II). In addition and in contrast to what is usually observed, the presence of excess of L lessens the searched effect of ROS production prevention, but it is counterbalanced by the co-presence of Zn(II). To explain such unprecedented trends, we proposed a mechanism that involves the redox reaction between Cu(II)L and Cu(I)L2. We thus illustrated (i) how speciation and redox chemistry can weaken the effect of a ligand that would have appeared perfectly suitable if only tested in a 1:1 ratio and on CuAß and (ii) how Zn overcomes the undesired lessening of ROS arrest due to excess of ligand. In brief, we have shown how working in biologically relevant conditions is important for the understanding of all of the reactions at play and this must be taken into consideration for the further rational design of ligands aiming to become drug candidates.

Impact of N-Truncated Aß Peptides on Cu- and Cu(Aß)-Generated ROS: CuI Matters!

In vitro Cu(Aß1-x )-induced ROS production has been extensively studied. Conversely, the ability of N-truncated isoforms of Aß to alter the Cu-induced ROS production has been overlooked, even though they are main constituents of amyloid plaques found in the human brain. N-Truncated peptides at the positions 4 and 11 (Aß4-x and Aß11-x ) contain an amino-terminal copper and nickel (ATCUN) binding motif (H2 N-Xxx-Zzz-His) that confer them different coordination sites and higher affinities for CuII compared to the Aß1-x peptide. It has further been proposed that the role of Aß4-x peptide is to quench CuII toxicity in the brain. However, the role of CuI coordination has not been investigated to date. In contrast to CuII , CuI coordination is expected to be the same for N-truncated and N-intact peptides. Herein, we report in-depth characterizations and ROS production studies of Cu (CuI and CuII ) complexes of the Aß4-16 and Aß11-16 N-truncated peptides. Our findings show that the N-truncated peptides do produce ROS when CuI is present in the medium, albeit to a lesser extent than the unmodified counterpart. In addition, when used as competitor ligands (i.e., in the presence of Aß1-16 ), the N-truncated peptides are not able to fully preclude Cu(Aß1-16 )-induced ROS production.

The reactivity of molecular oxygen and reactive oxygen species with [FeFe] hydrogenase biomimetics: reversibility and the role of the second coordination sphere.

The development of oxygen-tolerant H2-evolving catalysts plays a vital role for a future H2 economy. For example, the [FeFe] hydrogenase enzymes are excellent catalyst for H2 evolution but rapidly become inactivated in the presence of O2. The mechanistic details of the enzyme's inactivation by molecular oxygen still remain unclear. Here, two H2-evolving diiron complexes [Fe2(µ-SCH2NHCH2S)(CO)6] (1adt) and [Fe2(µ-SCH2CH2CH2S)(CO)6] (2pdt), inspired by the active site of [FeFe] hydrogenase, were investigated for their reactivity with molecular oxygen and reactive oxygen species. A one-electron reduced and oxygenated 1adt species was identified and characterized spectroscopically, which can be directly generated by reacting with molecular oxygen and chemical reductants at room temperature but it is unstable and gradually decomposes. Interestingly, the whole process is reversible and the addition of protons can facilitate the deoxygenation process and prevent further degradation at room temperature. This new identification of intermediate species serves as a model for studying the reversible inactivation and degradation of oxygen-sensitive [FeFe] hydrogenases by O2, and provides chemical precedence for such processes. In comparison, the complex lacking the nitrogen bridgehead, 2pdt, exhibits reduced reactivity towards O2 in the presence of reductants, highlighting that the importance of the second coordination sphere on modulating the oxygenation processes. These results provide new directions to design molecular electrocatalysts for proton reduction operated at ambient conditions and the re-engineering of [FeFe] hydrogenases for improving oxygen tolerance.

Influence of Copper Coordination Spheres on Nitrous Oxide Reductase (N2Or) Activity of a Mixed-Valent Copper Complex Containing a {Cu2S} Core.

A new mixed-valent dicopper complex [5] was generated from ligand exchange by dissolving a bis(CH3CN) precursor [3] in acetone. Introduction of a water molecule in place of an acetonitrile ligand was evidenced by base titration and the presence of a remaining coordinated CH3CN by IR, 19F NMR, and theoretical methods. The proposed structure (CH3CN-Cu-(SR)-Cu-OH2) was successfully DFT-optimized and the calculated parameters are in agreement with the experimental data. [5] has a unique temperature-dependence EPR behavior, with a localized valence from 10 to 120 K that undergoes delocalized at room temperature. The electrochemical signatures are in the line of the other aquo parent [2] and sensibly different from the rest of the series. Similar to the case of [2], [5] was finally capable of single turnover N2O reduction at room temperature. N2 was detected by GC-MS, and the redox character was confirmed by EPR and ESI-MS. Kinetic data indicate a reaction rate order close to 1 and a rate 10 times faster compared to [2]. [5] is thus the second example of that kind and highlights not only the main role of the Cu-OH2 motif, but also that the adjacent Cu-X partner (X = OTf- in [2] and CH3CN in [5]) is a new actor in the casting to establish structure/activity correlations.

Metal vs. ligand protonation and the alleged proton-shuttling role of the azadithiolate ligand in catalytic H2 formation with FeFe hydrogenase model complexes.

Electron and proton transfer reactions of diiron complexes [Fe2adt(CO)6] (1) and [Fe2adt(CO)4(PMe3)2] (4), with the biomimetic azadithiolate (adt) bridging ligand, have been investigated by real-time IR- and UV-vis-spectroscopic observation to elucidate the role of the adt-N as a potential proton shuttle in catalytic H2 formation. Protonation of the one-electron reduced complex, 1- , occurs on the adt-N yielding 1H and the same species is obtained by one-electron reduction of 1H+ . The preference for ligand vs. metal protonation in the Fe2(i,0) state is presumably kinetic but no evidence for tautomerization of 1H to the hydride 1Hy was observed. This shows that the adt ligand does not work as a proton relay in the formation of hydride intermediates in the reduced catalyst. A hydride intermediate 1HHy+ is formed only by protonation of 1H with stronger acid. Adt protonation results in reduction of the catalyst at much less negative potential, but subsequent protonation of the metal centers is not slowed down, as would be expected according to the decrease in basicity. Thus, the adtH+ complex retains a high turnover frequency at the lowered overpotential. Instead of proton shuttling, we propose that this gain in catalytic performance compared to the propyldithiolate analogue might be rationalized in terms of lower reorganization energy for hydride formation with bulk acid upon adt protonation.

Copper-Targeting Approaches in Alzheimer's Disease: How To Improve the Fallouts Obtained from in Vitro Studies.

According to the amyloid cascade hypothesis, metal ions, mainly Cu and Zn ions, bound to the amyloid-ß (Aß) peptides are implicated in Alzheimer's disease (AD), a widespread neurodegenerative disease. They indeed impact the aggregation pathways of Aß and are involved in the catalytic generation of reactive oxygen species (ROS) that participate in oxidative stress, while Aß aggregation and oxidative stress are regarded as two key events in AD etiology. Cu ions due to their redox ability have been considered to be the main potential therapeutic targets in AD. A considerable number of ligands have been developed in order to modulate the toxicity associated with Cu in this context, via disruption of the Aß-Cu interaction. Among them, small synthetic ligands and small peptide scaffolds have been designed and studied for their ability to remove Cu from Aß. Some of those ligands are able to prevent Cu(Aß)-induced ROS production and can modify the aggregation pathways of Aß in vitro and in cellulo. Examples of such ligands are gathered in this Viewpoint, as a function of their structures and discussed with respect to their properties against Cu(Aß) deleterious fallouts. Nevertheless, the beneficial activities of the most promising ligands detected in vitro and in cellulo have not been transposed to human yet. Some parameters that might explain this apparent contradiction and key concepts to consider for the design of "more" efficient ligands are thus reported and discussed. En passant, this Viewpoint sheds light on the difficulties in comparing the results from one study to another that hamper significant advances in the field.

Monitoring H-cluster assembly using a semi-synthetic HydF protein.

The [FeFe] hydrogenase enzyme interconverts protons and molecular hydrogen with remarkable efficiency. The reaction is catalysed by a unique metallo-cofactor denoted as the H-cluster containing an organometallic dinuclear Fe component, the [2Fe] subsite. The HydF protein delivers a precursor of the [2Fe] subsite to the apo-[FeFe] hydrogenase, thus completing the H-cluster and activating the enzyme. Herein we generate a semi-synthetic form of HydF by loading it with a synthetic low valent dinuclear Fe complex. We show that this semi-synthetic protein is practically indistinguishable from the native protein, and utilize this form of HydF to explore the mechanism of H-cluster assembly. More specifically, we show that transfer of the precatalyst from HydF to the hydrogenase enzyme results in the release of CO, underscoring that the pre-catalyst is a four CO species when bound to HydF. Moreover, we propose that an electron transfer reaction occurs during H-cluster assembly, resulting in an oxidation of the [2Fe] subsite with concomitant reduction of the [4Fe4S] cluster present on the HydF protein.

Synthesis of a miniaturized [FeFe] hydrogenase model system.

The reaction occurring during artificial maturation of [FeFe] hydrogenase has been recreated using molecular systems. The formation of a miniaturized [FeFe] hydrogenase model system, generated through the combination of a [4Fe4S] cluster binding oligopeptide and an organometallic Fe complex, has been monitored by a range of spectroscopic techniques. A structure of the final assembly is suggested based on EPR and FTIR spectroscopy in combination with DFT calculations. The capacity of this novel H-cluster model to catalyze H2 production in aqueous media at mild potentials is verified in chemical assays.

Generation of a functional, semisynthetic [FeFe]-hydrogenase in a photosynthetic microorganism.

[FeFe]-Hydrogenases are hydrogen producing metalloenzymes with excellent catalytic capacities, highly relevant in the context of a future hydrogen economy. Here we demonstrate the synthetic activation of a heterologously expressed [FeFe]-hydrogenase in living cells of Synechocystis PCC 6803, a photoautotrophic microbial chassis with high potential for biotechnological energy applications. H2-Evolution assays clearly show that the non-native, semi-synthetic enzyme links to the native metabolism in living cells.

In†Vivo EPR Characterization of Semi-Synthetic [FeFe] Hydrogenases.

EPR spectroscopy reveals the formation of two different semi-synthetic hydrogenases in vivo. [FeFe] hydrogenases are metalloenzymes that catalyze the interconversion of molecular hydrogen and protons. The reaction is catalyzed by the H-cluster, consisting of a canonical iron-sulfur cluster and an organometallic [2Fe] subsite. It was recently shown that the enzyme can be reconstituted with synthetic cofactors mimicking the composition of the [2Fe] subsite, resulting in semi-synthetic hydrogenases. Herein, we employ EPR spectroscopy to monitor the formation of two such semi-synthetic enzymes in whole cells. The study provides the first spectroscopic characterization of semi-synthetic hydrogenases in vivo, and the observation of two different oxidized states of the H-cluster under intracellular conditions. Moreover, these findings underscore how synthetic chemistry can be a powerful tool for manipulation and examination of the hydrogenase enzyme under in vivo conditions.

Valence Localization at a Bio-inspired Mixed-Valent {Cu2 S}2+ Motif upon Solvation in Acetonitrile: Effect on Nitrous Oxide Reductase (N2 Or) Activity.

We demonstrate, based on experimental and theoretical evidence, that the isolated [2(CH3 CN)2 ]2+ complex prepared in CH3 CN and containing a mixed-valent {Cu2II,I S} core evolves towards a new [2(CH3 CN)3 ]2+ species upon solvation in CH3 CN. Unlike its type III structural analogue [2(H2 O)(OTf)]+ active toward N2 O reduction, this new type I compound is inactive. This outcome opens new perspectives for a rational for N2 O activation using bio-inspired Cu/S complexes, especially on the role of the valence localization/delocalization and the Cu-Cu bond on the reactivity.

Redox-Innocent Metal-Assisted Cleavage of S-S Bond in a Disulfide-Containing Ligand.

Due to their redox capabilities, thiols have an important role in biological oxidative/reductive processes through the formation of disulfides or their oxidation to into sulfenic, sulfinic, or sulfonic derivatives being also relevant for specific enzyme activities. The mechanisms of these biological pathways often involve metal ion(s). In this case, deciphering metal-assisted transformation of the S-S bond is of primary interest. This report details the reactivity of the disulfide-containing 2,6-bis[(bis(pyridylmethyl)amino)methyl]-4-methylmercaptophenyldisulfide (L(Me(BPA)S-S)) ligand with Cu(II) using different experimental conditions (anaerobic, H2O-only, H2O/O2, or O2-only). Crystallographic snapshots show the formation of tetranuclear disulfide, dinuclear sulfinate, and sulfonate complexes. Mechanistic investigations using Zn(II) as control indicate a non-metal-redox-assisted process in all cases. When present, water acts as nucleophile and attacks at the S-S bond. Under anhydrous conditions, a different pathway involving a direct O2 attack at the disulfide is proposed.