Tristhiolato Pseudopeptides Bind Arsenic(III) in an AsS3 Coordination Environment Imitating Metalloid Binding Sites in Proteins.

The AsIII binding of two NTA-based tripodal pseudopeptides, possessing three cysteine (ligand L1) or d-penicillamine residues (ligand L2) as potential coordinating groups for soft semimetals or metal ions, was studied by experimental (UV, CD, NMR, and ESI-MS) and theoretical (DFT) methods. All of the experimental data, obtained with the variation of the AsIII:ligand concentration ratios or pH values in some instances, evidence the exclusive formation of species with an AsS3-type coordination mode. The UV-monitored titration of the ligands with arsenous acid at pH = 7.0 provided an absorbance data set that allowed for the determination of apparent stability constants of the forming species. The obtained stabilities (logK' = 5.26 (AsL1) and logK' = 3.04 (AsL2)) reflect high affinities, especially for the sterically less restricted cysteine derivative. DFT calculated structures correlate well with the spectroscopic results and, in line with the 1H NMR data, indicate a preference for the all-endo conformers resembling the AsIII environment at the semimetal binding sites in various metalloproteins.

A Series of Ni Complexes Based on a Versatile ATCUN-Like Tripeptide Scaffold to Decipher Key Parameters for Superoxide Dismutase Activity.

The cellular level of reactive oxygen species (ROS) has to be controlled to avoid some pathologies, especially those linked to oxidative stress. One strategy for designing antioxidants consists of modeling natural enzymes involved in ROS degradation. Among them, nickel superoxide dismutase (NiSOD) catalyzes the dismutation of the superoxide radical anion, O2•-, into O2 and H2O2. We report here Ni complexes with tripeptides derived from the amino-terminal CuII- and NiII-binding (ATCUN) motif that mimics some structural features found in the active site of the NiSOD. A series of six mononuclear NiII complexes were investigated in water at physiological pH with different first coordination spheres, from compounds with a N3S to N2S2 set, and also complexes that are in equilibrium between the N-coordination (N3S) and S-coordination (N2S2). They were fully characterized by a combination of spectroscopic techniques, including 1H NMR, UV-vis, circular dichroism, and X-ray absorption spectroscopy, together with theoretical calculations and their redox properties studied by cyclic voltammetry. They all display SOD-like activity, with a kcat ranging between 0.5 and 2.0 × 106 M-1 s-1. The complexes in which the two coordination modes are in equilibrium are the most efficient, suggesting a beneficial effect of a nearby proton relay.

Determining the selectivity of a tetra-phosphorylated biomimetic peptide towards uranium in the presence of competing cations through the simultaneous coupling of HILIC to ESI-MS and ICP-MS.

A cyclic tetra-phosphorylated biomimetic peptide (pS1368) has been proposed as a promising starting structure to design a decorporating agent of uranyl (UO22+) due to its affinity being similar to that of osteopontin (OPN), a target UO22+ protein in vivo. The determination of this peptide's selectivity towards UO22+ in the presence of competing endogenous elements is also crucial to validate this hypothesis. In this context, the selectivity of pS1368 towards UO22+ in the presence of Ca2+, Cu2+ and Zn2+ was determined by applying the simultaneous coupling of hydrophilic interaction chromatography (HILIC) to electrospray ionization (ESI-MS) and inductively coupled plasma (ICP-MS) mass spectrometry. Sr2+ was used as Ca2+ simulant, providing less challenging ICP-MS measurements. The separation of the complexes by HILIC was first set up. The selectivity of pS1368 towards UO22+ was determined in the presence of Sr2+, by adding several proportions of the latter to UO2(pS1368). UO22+ was not displaced from UO2(pS1368) even in the presence of a ten-fold excess of Sr2+. The same approach has been undertaken to demonstrate the selectivity of pS1368 towards UO22+ in the presence of Cu2+, Zn2+ and Sr2+ as competing endogenous cations. Hence, we showed that pS1368 was selective towards UO22+ in the presence of Sr2+, but also in the presence of Cu2+ and Zn2+. This study highlights the performance of HILIC-ESI-MS/ICP-MS simultaneous coupling to assess the potential of molecules as decorporating agents of UO22+.

Optimized Coordination of Uranyl in Engineered Calmodulin Site 1 Provides a Subnanomolar Affinity for Uranyl and a Strong Uranyl versus Calcium Selectivity.

As an alpha emitter and chemical toxicant, uranium toxicity in living organisms is driven by its molecular interactions. It is therefore essential to identify main determinants of uranium affinity for proteins. Others and we showed that introducing a phosphoryl group in the coordination sphere of uranyl confers a strong affinity of proteins for uranyl. In this work, using calmodulin site 1 as a template, we modulate the structural organization of a metal-binding loop comprising carboxylate and/or carbonyl ligands and reach affinities for uranyl comparable to that provided by introducing a strong phosphoryl ligand. Shortening the metal binding loop of calmodulin site 1 from 12 to 10 amino acids in CaMΔ increases the uranyl-binding affinity by about 2 orders of magnitude to log KpH7 = 9.55 ± 0.11 (KdpH7 = 280 ± 60 pM). Structural analysis by FTIR, XAS, and molecular dynamics simulations suggests an optimized coordination of the CaMΔ-uranyl complex involving bidentate and monodentate carboxylate groups in the uranyl equatorial plane. The main role of this coordination sphere in reaching subnanomolar dissociation constants for uranyl is supported by similar uranyl affinities obtained in a cyclic peptide reproducing CaMΔ binding loop. In addition, CaMΔ presents a uranyl/calcium selectivity of 107 that is even higher in the cyclic peptide.

A Simple Fluorescence Affinity Assay to Decipher Uranyl-Binding to Native Proteins.

Determining the affinity of proteins for uranyl is key to understand the toxicity of this cation and to further develop decorporation strategies. However, usual techniques to achieve that goal often require specific equipment and expertise. Here, we propose a simple, efficient, fluorescence-based method to assess the affinity of proteins and peptides for uranyl, at equilibrium and in buffered solution. We first designed and characterized an original uranyl-binding fluorescent probe. We then built a reference scale for uranyl affinity in solution, relying on signal quenching of our fluorescent probe in presence of high-affinity uranyl-binding peptides. We finally validated our approach by re-evaluating the uranyl-binding affinity of four native proteins. We envision that this tool will facilitate the reliable and reproducible assessment of affinities of peptides and proteins for uranyl.

A Bioinspired NiII Superoxide Dismutase Catalyst Designed on an ATCUN-like Binding Motif.

Nickel superoxide dismutase (NiSOD) is an enzyme that protects cells against O2·-. While the structure of its active site is known, the mechanism of the catalytic cycle is still not elucidated. Its active site displays a square planar NiII center with two thiolates, the terminal amine and an amidate. We report here a bioinspired NiII complex built on an ATCUN-like binding motif modulated with one cysteine, which demonstrates catalytic SOD activity in water (kcat = 8.4(2) × 105 M-1 s-1 at pH = 8.1). Its reactivity with O2·- was also studied in acetonitrile allowing trapping two different short-lived species that were characterized by electron paramagnetic resonance or spectroelectrochemistry and a combination of density functional theory (DFT) and time-dependent DFT calculations. Based on these observations, we propose that O2·- interacts first with the complex outer sphere through a H-bond with the peptide scaffold in a [NiIIO2·-] species. This first species could then evolve into a NiIII hydroperoxo inner sphere species through a reaction driven by protonation that is thermodynamically highly favored according to DFT calculations.

Phosphate-Rich Biomimetic Peptides Shed Light on High-Affinity Hyperphosphorylated Uranyl Binding Sites in Phosphoproteins.

Some phosphoproteins such as osteopontin (OPN) have been identified as high-affinity uranyl targets. However, the binding sites required for interaction with uranyl and therefore involved in its toxicity have not been identified in the whole protein. The biomimetic approach proposed here aimed to decipher the nature of these sites and should help to understand the role of the multiple phosphorylations in UO2 2+ binding. Two hyperphosphorylated cyclic peptides, pS168 and pS1368 containing up to four phosphoserine (pSer) residues over the ten amino acids present in the sequences, were synthesized with all reactions performed in the solid phase, including post-phosphorylation. These ß-sheet-structured peptides present four coordinating residues from four amino acid side chains pointing to the metal ion, either three pSer and one glutamate in pS168 or four pSer in pS1368 . Significantly, increasing the number of pSer residues up to four in the cyclodecapeptide scaffolds produced molecules with an affinity constant for UO2 2+ that is as large as that reported for osteopontin at physiological pH. The phosphate-rich pS1368 can thus be considered a relevant model of UO2 2+ coordination in this intrinsically disordered protein, which wraps around the metal ion to gather four phosphate groups in the UO2 2+ coordination sphere. These model hyperphosphorylated peptides are highly selective for UO2 2+ with respect to endogenous Ca2+ , which makes them good starting structures for selective UO2 2+ complexation.

Mononuclear Ni(II) Complexes with a S3O Coordination Sphere Based on a Tripodal Cysteine-Rich Ligand: pH Tuning of the Superoxide Dismutase Activity.

The superoxide dismutase (SOD) activity of mononuclear NiII complexes, whose structures are inspired by the NiSOD, has been investigated. They have been designed with a sulfur-rich pseudopeptide ligand, derived from nitrilotriacetic acid (NTA), where the three acid functions are grafted with cysteines (L3S). Two mononuclear complexes, which exist in pH-dependent proportions, have been fully characterized by a combination of spectroscopic techniques including 1H NMR, UV-vis, circular dichroism, and X-ray absorption spectroscopy, together with theoretical calculations. They display similar square-planar S3O coordination, with the three thiolates of the three cysteine moieties from L3S coordinated to the NiII ion, together with either a water molecule at physiological pH, as [NiL3S(OH2)]-, or a hydroxo ion in more basic conditions, as [NiL3S(OH)]2-. The 1H NMR study has revealed that contrary to the hydroxo ligand, the bound water molecule is labile. The cyclic voltammogram of both complexes displays an irreversible one-electron oxidation process assigned to the NiII/NiIII redox system with Epa = 0.48 and 0.31 V versus SCE for NiL3S(OH2) and NiL3S(OH), respectively. The SOD activity of both complexes has been tested. On the basis of the xanthine oxidase assay, an IC50 of about 1 µM has been measured at pH 7.4, where NiL3S(OH2) is mainly present (93% of the NiII species), while the IC50 is larger than 100 µM at pH 9.6, where NiL3S(OH) is the major species (92% of the NiII species). Interestingly, only NiL3S(OH2) displays SOD activity, suggesting that the presence of a labile ligand is required. The SOD activity has been also evaluated under catalytic conditions at pH 7.75, where the ratio between NiL3S(OH2)/ NiL3S(OH) is about (86:14), and a rate constant, kcat = 1.8 × 105 M-1 s-1, has been measured. NiL3S(OH2) is thus the first low-molecular weight, synthetic, bioinspired Ni complex that displays catalytic SOD activity in water at physiological pH, although it does not contain any N-donor ligand in its first coordination sphere, as in the NiSOD. Overall, the data show that a key structural feature is the presence of a labile ligand in the coordination sphere of the NiII ion.

Quantification of Surface GalNAc Ligands Decorating Nanostructured Lipid Carriers by UPLC-ELSD.

Nanoparticles have been extensively studied for drug delivery and targeting to specific organs. The functionalization of the nanoparticle surface by site-specific ligands (antibodies, peptides, saccharides) can ensure efficient recognition and binding with relevant biological targets. One of the main challenges in the development of these decorated nanocarriers is the accurate quantification of the amount of ligands on the nanoparticle surface. In this study, nanostructured lipid carriers (NLC) were functionalized with N-acetyl-D-galactosamine (GalNAc) units, known to target the asialoglycoprotein receptor (ASGPR). Different molar percentages of GalNAc-functionalized surfactant (0%, 2%, 5%, and 14%) were used in the formulation. Based on ultra-high-performance liquid chromatography separation and evaporative light-scattering detection (UPLC-ELSD), an analytical method was developed to specifically quantify the amount of GalNAc units present at the NLC surface. This method allowed the accurate quantification of GalNAc surfactant and therefore gave some insights into the structural parameters of these multivalent ligand systems. Our data show that the GalNAc decorated NLC possess large numbers of ligands at their surface and suitable distances between them for efficient multivalent interaction with the ASGPR, and therefore promising liver-targeting efficiency.

A Constrained Tetrapeptide as a Model of Cu(I) Binding Sites Involving Cu4S6 Clusters in Proteins.

Peptide design is an efficient strategy to create relevant models of natural metal binding sites found in proteins. The two short tetrapeptides Ac-Cys-dPro-Pro-Cys-NH2 (CdPPC) and Ac-Cys-Pro-Gly-Cys-NH2 (CPGC) were synthesized and studied as mimics of Cu(I) binding sites involved in Cu homeostasis. Both sequences contain ß turn inducing motifs to rigidify the peptide backbone structure and thereby preorganize the metal-binding side chains. The more constrained structure of the peptide CdPPC with respect to CPGC was evidenced by the measurements of the temperature coefficients of the amide protons by 1H NMR, which suggest a solvent-shielded intramolecular hydrogen bond in CdPPC, and no H-bond in CPGC. The Cu(I) complexes were studied by UV, circular dichroism (CD), and NMR spectroscopies as well as electrospray ionization mass spectrometry (ESI-MS) experiments in aqueous solution at physiological pH. The complexes formed with CPGC showed a complicated speciation with the possible formation of many polymetallic species. By contrast, the better preorganization in CdPPC leads to the formation of a unique Cu4L3 complex involving a Cu4S6 core. The formation of this specific cluster was confirmed by ESI-MS and by diffusion-ordered NMR spectroscopy in solution. The affinity of CdPPC for Cu(I) (ß11pH7.4 = 1017.5 calculated for a CuL complex) is more than 1 order of magnitude larger than the affinity measured for the less constrained peptide CPGC. Besides, this stability constant value is very similar to those reported with proteins. Therefore, the Cu(I) complex formed with the simple tetrapeptide CdPPC in water at physiological pH represents a very good model of Cu(I)-thiolate clusters found in proteins. The extremely large selectivity (1011) in favor of Cu(I) with respect to Zn(II), an abundant competitor in cells, makes it a promising candidate to be targeted to the liver cells for the localized treatment of Cu overload in Wilson's disease.

Mercury Trithiolate Binding (HgS3) to a de Novo Designed Cyclic Decapeptide with Three Preoriented Cysteine Side Chains.

Mercury(II) is an unphysiological soft ion with high binding affinity for thiolate ligands. Its toxicity lies in the interactions with low molecular weight thiols including glutathione and cysteine-containing proteins that disrupt the thiol balance and alter vital functions. However, mercury can also be detoxified via interactions with Hg(II)-responsive regulatory proteins such as MerR, which coordinates Hg(II) with three cysteine residues in a trigonal planar fashion (HgS3 coordination). The model cyclodecapeptide P3C, c(GCTCSGCSRP) was designed to promote Hg(II) chelation in a HgS3 coordination environment through the parallel orientation of three cysteine side chains. The binding motif is derived from the dicysteine P2C cyclodecapeptide validated previously as a model for d10 metal transporters containing the binding sequence CxxC. The formation of the mononuclear HgP3C complex with a HgS3 coordination is demonstrated using electrospray ionization mass spectrometry, UV absorption, and 199Hg NMR. Hg LIII-edge extended X-ray absorption fine structure (EXAFS) spectroscopy indicates that the Hg(II) coordination environment is T-shaped with two short Hg-S distances at 2.45 Å and one longer distance at 2.60 Å. The solution structure of the HgP3C complex was refined based on 1H-1H NMR constraints and EXAFS results. The cyclic peptide scaffold has a rectangular shape with the three binding cysteine side chains pointing toward Hg(II). The HgP3CH complex has a p Ka of 4.3, indicating that the HgS3 coordination mode is stable over a large range of pH. This low p Ka value suggests that the preorientation of the three cysteine groups is particularly well-achieved for Hg(II) trithiolate coordination in P3C.

A Trishistidine Pseudopeptide with Ability to Remove Both CuΙ and CuΙΙ from the Amyloid-ß Peptide and to Stop the Associated ROS Formation.

The pseudopeptide L, derived from a nitrilotriacetic acid scaffold and functionalized with three histidine moieties, is reminiscent of the amino acid side chains encountered in the Alzheimer's peptide (Aß). Its synthesis and coordination properties for CuΙ and CuΙΙ are described. L efficiently complex CuΙΙ in a square-planar geometry involving three imidazole nitrogen atoms and an amidate-Cu bond. By contrast, CuΙ is coordinated in a tetrahedral environment. The redox behavior is irreversible and follows an ECEC mechanism in accordance with the very different environments of the two redox states of the Cu center. This is in line with the observed resistance of the CuΙ complex to oxidation by oxygen and the CuΙΙ complex reduction by ascorbate. The affinities of L for CuΙΙ and CuΙ at physiological pH are larger than that reported for the Aß peptide. Therefore, due to its peculiar Cu coordination properties, the ligand L is able to target both redox states of Cu, redox silence them and prevent reactive oxygen species production by the CuAß complex. Because reactive oxygen species contribute to the oxidative stress, a key issue in Alzheimer's disease, this ligand thus represents a new strategy in the long route of finding molecular concepts for fighting Alzheimer's disease.

Cyclic Phosphopeptides to Rationalize the Role of Phosphoamino Acids in Uranyl Binding to Biological Targets.

The specific molecular interactions responsible for uranium toxicity are not yet understood. The uranyl binding sites in high-affinity target proteins have not been identified yet and the involvement of phosphoamino acids is still an important question. Short cyclic peptide sequences, with three glutamic acids and one phosphoamino acid, are used as simple models to mimic metal binding sites in phosphoproteins and to help understand the mechanisms involved in uranium toxicity. A combination of peptide design and synthesis, analytical chemistry, extended X-ray absorption fine structure (EXAFS) spectroscopy, and DFT calculations demonstrates the involvement of the phosphate group in the uranyl coordination sphere together with the three carboxylates of the glutamate moieties. The affinity constants measured with a reliable analytical competitive approach at physiological pH are significantly enhanced owing to the presence of the phosphorous moiety. These findings corroborate the importance of phosphoamino acids in uranyl binding in proteins and the relevance of considering phosphoproteins as potential uranyl targets in vivo.

ASGPR-Mediated Uptake of Multivalent Glycoconjugates for Drug Delivery in Hepatocytes.

Liver cells are an essential target for drug delivery in many diseases. The hepatocytes express the asialoglycoprotein receptor (ASGPR), which promotes specific uptake by means of N-acetylgalactosamine (GalNAc) recognition. In this work, we designed two different chemical architectures to treat Wilson's disease by intracellular copper chelation. Two glycoconjugates functionalized with three or four GalNAc units each were shown to enter hepatic cells and chelate copper. Here, we studied two series of compounds derived from these glycoconjugates to find key parameters for the targeting of human hepatocytes. Efficient cellular uptake was demonstrated by flow cytometry using HepG2 human heptic cells that express the human oligomeric ASGPR. Dissociation constants in the nanomolar range showed efficient multivalent interactions with the receptor. Both architectures were therefore concluded to be able to compete with endogeneous asialoglycoproteins and serve as good vehicles for drug delivery in hepatocytes.

Pseudo-peptides based on methyl cysteine or methionine inspired from Mets motifs found in the copper transporter Ctr1.

Most proteins involved in Cu homeostasis bind to intracellular Cu(I) in stable Cu(S-Cys)x environments, thanks to well-conserved cysteine-rich sequences. Similarly, the Cu(I) transport protein Ctr1, responsible for copper acquisition, binds Cu(I) in Cu(S-Met)3 environments in conserved methionine-rich MXMXXM sequences, referred as Mets motifs. Pseudo-peptides based on a nitrilotriacetic acid scaffold and functionalized with three amino acids bearing thioether side chains, either methyl cysteine in T(1) or methionine in T(2), were synthesized as mimics of the Mets sequences found in Ctr1. These two ligands were obtained with good overall yields from commercial amino acids and demonstrate efficient chelating ability for Cu(I). Only one species, the mononuclear [CuT(1,2)](+) complex, was evidenced by electrospray ionization-mass spectroscopy (ESI-MS) and the circular dichroism signature obtained for the most constrained CuT(1) complex having the shortest side chains showed reorganization of the pseudo-peptide scaffold upon Cu(I) complexation. Considering that thioether functions are neutral sulfur donors, the stability constants measured by competition with ferrozine are quite large: log K ≈ 10.2-10.3. The CuT(1,2) complexes are significantly more stable that those formed with linear peptides, mimicking isolated Mets motifs MXMXXM of the Cu transport protein Ctr1 (log K ≈ 5-6). This may be attributed to the preorganized pseudo-peptide scaffold, which arranges the three neutral sulfur donors toward the metal center. Such moderate affinity Cu(I) chelators are interesting for applications in chelation therapy, for instance, to induce minimum disturbance of Cu homeostasis in Wilson's disease treatments.

Preorganized Peptide Scaffolds as Mimics of Phosphorylated Proteins Binding Sites with a High Affinity for Uranyl.

Cyclic peptides with two phosphoserines and two glutamic acids were developed to mimic high-affinity binding sites for uranyl found in proteins such as osteopontin, which is believed to be a privileged target of this ion in vivo. These peptides adopt a ß-sheet structure that allows the coordination of the latter amino acid side chains in the equatorial plane of the dioxo uranyl cation. Complementary spectroscopic and analytical methods revealed that these cyclic peptides are efficient uranyl chelating peptides with a large contribution from the phosphorylated residues. The conditional affinity constants were measured by following fluorescence tryptophan quenching and are larger than 10(10) at physiological pH. These compounds are therefore promising models for understanding uranyl chelation by proteins, which is relevant to this actinide ion toxicity.

XAS Investigation of Silver(I) Coordination in Copper(I) Biological Binding Sites.

Silver(I) is an unphysiological ion that, as the physiological copper(I) ion, shows high binding affinity for thiolate ligands; its toxicity has been proposed to be due to its capability to replace Cu(I) in the thiolate binding sites of proteins involved in copper homeostasis. Nevertheless, the nature of the Ag(I)-thiolate complexes formed within cells is poorly understood, and the details of Ag(I) coordination in such complexes in physiologically relevant conditions are mostly unknown. By making use of X-ray absorption spectroscopy (XAS), we characterized the Ag(I) binding sites in proteins related to copper homeostasis, such as the chaperone Atox1 and metallothioneins (MTs), as well as in bioinspired thiolate Cu(I) chelators mimicking these proteins, in solution and at physiological pH. Different Ag(I) coordination environments were revealed: the Ag-S bond length was found to correlate to the Ag(I) coordination number, with characteristic values of 2.40 and 2.49 Å in AgS2 and AgS3 sites, respectively, comparable to the values reported for crystalline Ag(I)-thiolate compounds. The bioinspired Cu(I) chelator L(1) is proven to promote the unusual trigonal AgS3 coordination and, therefore, can serve as a reference compound for this environment. In the Cu(I)-chaperone Atox1, Ag(I) binds in digonal coordination to the two Cys residues of the Cu(I) binding loop, with the AgS2 characteristic bond length of 2.40 ± 0.01 Å. In the multinuclear Ag(I) clusters of rabbit and yeast metallothionein, the average Ag-S bond lengths are 2.48 ± 0.01 Å and 2.47 ± 0.01 Å, respectively, both indicative of the predominance of trigonal AgS3 sites. This work lends insight into the coordination chemistry of silver in its most probable intracellular targets and might help in elucidating the mechanistic aspects of Ag(I) toxicity.

Engineering short peptide sequences for uranyl binding.

Peptides are interesting tools to rationalize uranyl-protein interactions, which are relevant to uranium toxicity in vivo. Structured cyclic peptide scaffolds were chosen as promising candidates to coordinate uranyl thanks to four amino acid side chains pre-oriented towards the dioxo cation equatorial plane. The binding of uranyl by a series of decapeptides has been investigated with complementary analytical and spectroscopic methods to determine the key parameters for the formation of stable uranyl-peptide complexes. The molar ellipticity of the uranyl complex at 195 nm is directly correlated to its stability, which demonstrates that the ß-sheet structure is optimal for high stability in the peptide series. Cyclodecapeptides with four glutamate residues exhibit the highest affinities for uranyl with log KC =8.0-8.4 and, therefore, appear as good starting points for the design of high-affinity uranyl-chelating peptides.

D-Penicillamine tripodal derivatives as efficient copper(I) chelators.

New tripodal metal-chelating agents derived from nitrilotriacetic acid (NTA) and extended by three unnatural amino acids D-penicillamine (D-Pen) are presented. D-Pen is actually the drug most extensively used to treat copper (Cu) overload in Wilson's disease and as such is a very attractive building block for the design of chelating agents. D-Pen is also a bulkier analogue of cysteine, with the ß-methylene hydrogen atoms replaced by larger methyl groups. The hindrance of the gem-dimethyl group close to the thiol functions is demonstrated to influence the speciation and stability of the metal complexes. The ligands L(4) (ester) and L(5) (amide) were obtained from NTA and commercial D-Pen synthons in four and five steps with overall yields of 14 and 24%, respectively. Their ability to bind Cu(I), thanks to their three thiolate functions, has been investigated using both spectroscopic and analytical methods. UV, CD, and NMR spectroscopy and mass spectrometry evidence the formation of two Cu(I) complexes with L(5): the mononuclear complex CuL(5) and one cluster (Cu2L(5))2. In contrast, the bulkier ethyl ester derivative L(4) cannot accommodate the mononuclear complex in solution and thus forms exclusively the cluster (Cu2L(4))2. Cu K-edge X-ray absorption spectroscopy (XAS and EXAFS) confirms that Cu(I) is bound in trigonal-planar sulfur-only environments in all of these complexes with Cu- - -S distances ranging from 2.22 to 2.23 Å. Such C3-symmetric CuS3 cores are coordination modes frequently adopted in Cu(I) proteins such as metallothioneins. These two ligands bind Cu(I) tightly and selectively, which makes them promising chelators for intracellular copper detoxification in vivo.

DNA sensing by a Eu-binding peptide containing a proflavine unit.

Synthesis of a lanthanide-binding peptide (LBP) for the detection of double-stranded DNA is presented. A proflavine moiety was introduced into a high affinity LBP involving two unnatural chelating amino acids in the Ln ion coordination. The Eu(3+)-LBP complex is demonstrated to bind to ct-DNA and to sensitize Eu luminescence. The DNA binding process is effectively detected via the Eu-centered luminescence thanks to the intimate coupling between the LBP scaffold and DNA intercalating unit.