Olivier Sénèque

Coordination Properties of Zinc Finger Peptides Revisited: Ligand Competition Studies Reveal Higher Affinities for Zinc and Cobalt

Zinc fingers are ubiquitous small protein domains which have a Zn(Cys)(4-x)(His)(x) site. They possess great diversity in their structure and amino acid composition. Using a family of six peptides, it was possible to assess the influence of hydrophobic amino acids on the metal-peptide affinities and on the rates of metal association and dissociation. A model of a treble-clef zinc finger, a model of the zinc finger site of a redox-switch protein, and four variants of the classical ββα zinc finger were used. They differ in their coordination set, their sequence length, and their hydrophobic amino acid content. The speciation, metal binding constants, and structure of these peptides have been investigated as a function of pH. The zinc binding constants of peptides, which adopt a well-defined structure, were found to be around 10(15) at pH 7.0. The rates of zinc exchange between EDTA and the peptides were also assessed. We evidenced that the packing of hydrophobic amino acids into a well-defined hydrophobic core can have a drastic influence on both the binding constant and the kinetics of metal exchange. Notably, well-packed hydrophobic amino acids can increase the stability constant by 4 orders of magnitude. The half-life of zinc exchange was also seen to vary significantly depending on the sequence of the zinc finger. The possible causes for this behavior are discussed. This work will help in understanding the dynamics of zinc exchange in zinc-containing proteins.

Calix[6]arene-BasedN3-Donors − A Versatile Supramolecular System with Tunable Electronic and Steric Properties − Study on the Formation of Tetrahedral Dicationic Zinc Complexes in a Biomimetic Environment

Novel tridentate N-ligands containing tertiary amines, pyrazoles, or benzimidazole groups were synthesized from tBu-calix[6]arene. Together with the previously described pyridine and imidazole-based ligands, they form a large family of biomimetic ligands (X6Me3N3) with different electronic and steric properties. Their capacity at stabilizing a tetrahedral Zn dicationic center in acetonitrile was investigated. Tertiary amines were too basic and sterically hindered, leading to precipitation of Zn(OH)2. The resulting protonated ligand was, in one case, structurally characterized by X-ray analysis. Ligands with pyrazole, benzimidazole and imidazole donors, all formed a stable Zn complex under stoichiometric conditions in acetonitrile. An 1H NMR spectroscopic study together with X-ray crystallography showed that the metal ion is coordinated to the three nitrogen arms with MeCN as a fourth ligand included in the calixarene conic pocket. These complexes provide new but rare examples of stable dicationic tetrahedral Zn species. The calixarene functionalized by three pyridine groups, on the other hand, did not appear to be a good ligand, which stands in contrast with its remarkable ability at stabilizing copper(I). Finally, these funnel complexes are chiral due to their helical shape. In solution, both enantiomers are in equilibrium. However, sterically hindered N-donors increased the enantiomerization barrier above 16 kcal/mol.

Supramolecular Assembly with Calix[6]arene and Copper Ions − Formation of a Novel Tetranuclear Core Exhibiting Unusual Redox Properties and Catecholase Activity

The supramolecular biomimetic chemistry based on calix[6]arene N-ligands has been further explored. A tris(imidazole)CuI complex was treated with 1 mol-equiv. of cuprous ion under dioxygen to produce a tetranuclear cupric species. X-ray structural determination of this novel Cu4 complex revealed that the self-inclusion of an imidazolyl coordinating arm into the hydrophobic calixarene cavity provides the base of coordination for a {ClImCu(OH)2CuIm2}2 assembly. The Cu4 core is maintained in solution and is stable even in a coordinating solvent such as acetonitrile. Magnetic susceptibility measurements evidenced a strong antiferromagnetic coupling in each Cu(OH)2Cu subunit with J = −408 cm−1. The complex displayed catecholase activity in the presence of 3,5-di-tert-butylcatechol behaving as a four-electron hole with, however, a sluggish CuI4 ⇄ CuII4 regeneration through O2 autooxidation. Finally, electrochemical studies revealed two oxidative reversible processes that successively gave rise to a {CuIICuIII}{CuII2} and a {CuIICuIII}2 mixed-valence species that could be characterized by UV/Vis and EPR spectroscopy. The overall structure and behavior of this tetranuclear complex is reminiscent of multicopper enzymes. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

Cooperative Metal Binding and Helical Folding in Model Peptides of Treble‐Clef Zinc Fingers

Two peptides, L(TC) and L(TC)(T) have been synthesised to model the treble-clef zinc fingers encountered in many Zn(Cys)(4)-site-containing proteins. Both are cyclic peptides with a linear tail grafted on a glutamate side chain of the cycle. They differ by the length of this tail, which lacks five amino acids in L(TC)(T) compared to L(TC). Both peptides bind Zn(2+) and Co(2+) in 1:1 metal/peptide ratio and the structure of these complexes have been characterised by NMR, UV/Vis and CD spectroscopy. Both peptides fold the same way around the metal ion and they fully reproduce the classical fold of treble-clef zinc fingers and display an extended hydrogen-bond network around the coordinating sulfur atoms. The structures of the ML(TC) complexes reveal that the linear tail forms a short two-turn alpha-helix, present in the metallated form only. The formation of this helix constitutes a rare example of metal-induced folding. The second turn of this helix is composed of the five amino acids that are absent in L(TC)(T). The study of the pH-dependence of the Zn(2+) binding constants shows that the metal ion is bound by four cysteinates above pH 5.2 and the binding constants are the highest reported so far. Interestingly, the binding constant of Zn x L(TC) is about tenfold higher than that of Zn x L(TC)(T). This difference clearly indicates that the helix, present in Zn x L(TC) only, stabilises the Zn(2+) complex by about 1.2 kcal mol(-1). The origin of this stabilisation is ascribed to an electrostatic interaction between the [ZnS(4)](2-) centre and the helix. This reveals a cooperative effect: zinc binding allows the folding of the tail into a helix which, in turn, strengthens the zinc complex.

Bio-inspired Calix[6]Arene–Zinc Funnel Complexes

A bio-inspired supramolecular system is presented. A calix[6]arene possessing three imidazolyl arms on alternate phenolic positions binds a zinc ion. The resulting complex contains a hydrophobic pocket, which has a flattened conic shape. The system behaves as a selective molecular funnel for neutral guests that bind the metal centre. The exceptional stability of these tetrahedral dicationic complexes is exemplified by the acetaldehyde ternary adduct that was analysed by X-ray crystallography. The ligand is deeply buried in the heart of the calixarene cavity, pointing its methyl group selectively towards the centre of one of the aromatic walls, thereby establishing a stabilizing CH/π interaction. Protic guests undergo hydrogen bonding with the phenolic oxygens of the calixarene structure. The selectivity of the binding in the cavity is based on both the affinity of the donor atom of the guest ligand for the zinc ion and the relative host–guest geometries. The helical shape of the tris-imidazolyl groups binding the metal centre is the base of the chirality of the system. The twisted calix[6]arene structure of the zinc funnel complexes is shown to provide a new example of a cavity suitable for host–guest chiral induction. Formation of helical zinc complexes.

Is the cytoplasmic loop of MerT, the mercuric ion transport protein, involved in mercury transfer to the mercuric reductase?

In MerT, the mercury transporter, a first cysteine pair, located in the first trans‐membrane helix, receives mercury from the periplasm. Then, a second cysteine pair, housed in a cytoplasmic loop connecting the second and the third trans‐membrane helices, is thought to transfer the metal to another cysteine pair located in the N‐terminal extension of the mercuric reductase. We found that a 23‐amino acid synthetic peptide corresponding to the cytoplasmic loop can bind one mercury atom per molecule and that this mercury atom can be transferred specifically to MerAa. The solution structure of Hg‐bound ppMerT has been solved by 1H NMR spectroscopy.

Supramolecular control of an organic radical coupled to a metal ion embedded at the entrance of a hydrophobic cavity

A novel N3ArO-calix[6]arene-based system is presented. It allows the formation of an aryloxy radical bound to a metal ion (CuII or ZnII) that presents a free coordination site in a concave cavity. Its oxidative activity appears highly controlled by the supramolecular system hence providing a good model for radical enzymes such as Galactose oxidase.

Cyclic Peptides Bearing a Side-Chain Tail: A Tool to Model the Structure and Reactivity of Protein Zinc Sites

A recent bioinformatic study has evaluated to about 1000 (or ca 3 % of the total protein number) the number of human proteins possessing a tetracysteinate zinc site. These sites were initially supposed to have a structural role since they were associated to zinc finger proteins where they fold the protein chain in a conformation suitable to its binding to DNA. They were later found in several proteins and enzymes involved in demethylation processes such as the DNA repair protein Ada and various transferases. More recently, such sites were discovered in the heat shock protein Hsp33, and the disulfide reductase Trx2 where their interaction with reactive oxygen species (ROS) contributes to the oxidative stress response. This is of special interest since tetracysteinate zinc sites, especially in zinc finger proteins, have been considered to be likely targets of ROS. Free cysteines are commonly involved in peroxide sensing and response and their reactivity has been thoroughly studied over the past twenty years. A reasonable reactivity picture has emerged that points to the importance of hydrogen-bonding to increase the nucleophilic character of the cysteine sulfur. No such rationale is available for metal-bound cysteinates.

A Diiron(III,IV) Imido Species Very Active in Nitrene‐Transfer Reactions

Metal-catalyzed nitrene transfer reactions arouse intense interest as clean and efficient procedures for amine synthesis. Efficient Rh- and Ru-based catalysts exist but Fe alternatives are actively pursued. However, reactive iron imido species can be very short-lived and getting evidence of their occurrence in efficient nitrene-transfer reactions is an important challenge. We recently reported that a diiron(III,II) complex is a very efficient nitrene-transfer catalyst to various substrates. We describe herein how, by combining desorption electrospray ionization mass spectrometry, quantitative chemical quench experiments, and DFT calculations, we obtained conclusive evidence for the occurrence of an {Fe(III) Fe(IV) NTosyl} intermediate that is very active in H-abstraction and nitrene-transfer reactions. DFT calculations revealed a strong radical character of the tosyl nitrogen atom in very low-lying electronic configurations of the Fe(IV) ion which are likely to confer its high reactivity.

Selective functionalization at the small rim of calix[6]arene. Synthesis of novel non-symmetrical N3, N4 and N3ArO biomimetic ligands

Abstract Eight novel calix[6]arene-based biomimetic ligands for transition metal ions have been synthesized. They display a non-symmetrical N 3 , N 4 or N 3 Ar O binding core that mimics enzyme active sites presenting histidine and tyrosine residues. The key step for their synthesis is the mono-alkylation at the small rim of the C 3v symmetrical trimethyl ether derivative of t Bu-calix[6]arene with N -Boc-2-chloroethylamine to yield a novel calix[6]arene synthon. Its combined O -alkylation with a chloromethyl aromatic amine and N -deprotection or alkylation or reductive alkylation with a salicylaldehyde derivative yielded the calix[6]arene-based ligands with mixed N / O donors.