Screening, separation and identification of metal-chelating peptides for nutritional, cosmetics and pharmaceutical applications.

Metal-chelating peptides, which form metal-peptide coordination complexes with various metal ions, can be used as biofunctional ingredients notably to enhance human health and prevent diseases. This review aims to discuss recent insights into food-derived metal-chelating peptides, the strategies set up for their discovery, their study, and identification. After understanding the overall properties of metal-chelating peptides, their production from food-derived protein sources and their potential applications will be discussed, particularly in nutritional, cosmetics and pharmaceutical fields. In addition, the review provides an overview of the last decades of progress in discovering food-derived metal-chelating peptides, addressing several screening, separation and identification methodologies. Furthermore, it emphasizes the methods used to assess peptide-metal interaction, allowing for better understanding of chemical and thermodynamic parameters associated with the formation of peptide-metal coordination complexes, as well as the specific amino acid residues that play important roles in the metal ion coordination.

Clickable C-Glycosyl Scaffold for the Development of a Dual Fluorescent and [18F]fluorinated Cyanine-Containing Probe and Preliminary In Vitro/Vivo Evaluation by Fluorescence Imaging.

Considering the individual characteristics of positron emission tomography (PET) and optical imaging (OI) in terms of sensitivity, spatial resolution, and tissue penetration, the development of dual imaging agents for bimodal PET/OI imaging is a growing field. A current major breakthrough in this field is the design of monomolecular agent displaying both a radioisotope for PET and a fluorescent dye for OI. We took advantage of the multifunctionalities allowed by a clickable C-glycosyl scaffold to gather the different elements. We describe, for the first time, the synthesis of a cyanine-based dual PET/OI imaging probe based on a versatile synthetic strategy and its direct radiofluorination via [18F]F-C bond formation. The non-radioactive dual imaging probe coupled with two c(RGDfK) peptides was evaluated in vitro and in vivo in fluorescence imaging. The binding on αvß3 integrin (IC50 = 16 nM) demonstrated the efficiency of the dimeric structure and PEG linkers in maintaining the affinity. In vivo fluorescence imaging of U-87 MG engrafted nude mice showed a high tumor uptake (40- and 100-fold increase for orthotopic and ectopic brain tumors, respectively, compared to healthy brain). In vitro and in vivo evaluations and resection of the ectopic tumor demonstrated the potential of the conjugate in glioblastoma cancer diagnosis and image-guided surgery.

Bio-Inspired Casein-Derived Antioxidant Peptides Exhibiting a Dual Direct/Indirect Mode of Action.

Antioxidant compounds are chemicals of primary importance, especially for their applications in nutrition and healthcare, thanks to their abilities to prevent oxidation processes and to limit and/or rebalance the oxidative stress, well-known for its impact on a wide variety of diseases. While several biomolecules are well-known for their antioxidant properties (e.g., ascorbic acid, carotenoids, phenolic derivatives), bio-sourced antioxidants have drawn considerable attention in the last decades, especially bioactive peptides, mainly obtained by the hydrolysis process. Antioxidant peptide sequences are mainly identified a posteriori, thanks to fastidious and time-consuming approaches and techniques, limiting the discovery of new efficient peptides. In this context and taking inspiration from nature, we report herein on a new series of three bio-inspired antioxidant peptides derived from the milk protein casein. These phosphopeptides, designed to chelate the redox-active iron(III) and forming highly soluble complexes up to pH 9, act both as indirect (i.e., inhibition of the metal redox activity) and direct (i.e., radical scavenging) antioxidants.

Quantitative Tissue Pharmacokinetics and EPR Effect of AGuIX Nanoparticles: A Multimodal Imaging Study in an Orthotopic Glioblastoma Rat Model and Healthy Macaque.

AGuIX are emerging radiosensitizing nanoparticles (NPs) for precision radiotherapy (RT) under clinical evaluation (Phase 2). Despite being accompanied by MRI thanks to the presence of gadolinium (Gd) at its surface, more sensitive and quantifiable imaging technique should further leverage the full potential of this technology. In this study, it is shown that 89 Zr can be labeled on such NPs directly for positron emission tomography (PET) imaging with a simple and scalable method. The stability of such complexes is remarkable in vitro and in vivo. Using a glioblastoma orthotopic rat model, it is shown that injected 89 Zr-AGuIX is detectable inside the tumor for at least 1 week. Interestingly, the particles seem to efficiently infiltrate the tumor even in necrotic areas, which places great hope for the treatment of radioresistant tumor. Lastly, the first PET/MR whole-body imaging is performed in non-human primate (NHP), which further demonstrates the translational potential of these bimodal NP.

Metabolomics approach based on LC-HRMS for the fast screening of iron(II)-chelating peptides in protein hydrolysates.

Production of iron-chelating peptides from protein hydrolysates requires robust and adequate screening methods to optimize their purification and subsequently valorize their potential antioxidant properties. An original methodology was developed for direct and sensitive screening of iron(II)-chelating peptides based on ion-pair reverse phase liquid chromatography (IP-RPLC) coupled to high-resolution mass spectrometry (HRMS). Peptide mixture was first added to iron(II) solution to form iron(II)-peptide complexes. Then IP-RPLC-HRMS analysis was conducted on this iron-peptide mixture and on the iron-free peptide solution for comparative mass spectra analysis. This protocol, initially applied to a range of low molecular weight standard peptides, allowed detection of [(Peptide-H)+56FeII]+ complex ion for iron(II)-chelating peptides (GGH, EAH, DAH, ßAH, DMH, DTH, DSH). GGH was added in complex peptide mixtures and targeted analysis of [(GGH-H)+56FeII]+ complex showed a limit of detection (LOD) below 0.77 mg L-1 of GGH. This protocol was finally tested in combination with metabolomics software and additional digital processing for non-targeted search for iron(II)-chelating peptides. Applicability of this new screening methodology has been validated by detection of GGH as iron(II)-chelating peptide when added at 0.77 mg L-1 in casein hydrolysate. Graphical abstract.

Synthesis of a DOTA-C-glyco bifunctional chelating agent and preliminary in vitro and in vivo study of [68Ga]Ga-DOTA-C-glyco-RGD.

The design of bifunctional chelating agents (BFCA) allowing straightforward radiometal labelling of biomolecules is a current challenge. We report herein the development of a bifunctional chelating agent based on a DOTA chelator linked to a C-glycosyl compound, taking advantage of the robustness and hydrophilicity of this type of carbohydrate derivative. This new BFCA was coupled with success by CuAAC with c(RGDfK) for αvß3 integrin targeting. As attested by in vitro evaluation, the conjugate DOTA-C-glyco-c(RGDfC) demonstrated high affinity for αvß3 integrins (IC50 of 42 nM). [68Ga]Ga-DOTA-C-glyco-c(RGDfK) was radiosynthesized straightforwardly and showed high hydrophilic property (log D 7.4 = -3.71) and in vitro stability (>120 min). Preliminary in vivo PET study of U87MG engrafted mice gave evidence of an interesting tumor-to-non-target area ratio. All these data indicate that [68Ga]Ga-DOTA-C-glyco-c(RGDfK) allows monitoring of αvß3 expression and could thus be used for cancer diagnosis. The DOTA-C-glycoside BFCA reported here could also be used with various ligands and chelating other (radio)metals opening a broad scope of applications in imaging modalities and therapy.

Both metal-chelating and free radical-scavenging synthetic pentapeptides as efficient inhibitors of reactive oxygen species generation.

Reactive oxygen species (ROS) are major sources of oxidative stress playing prominent roles in the development of several pathologies including cardiovascular and neurodegenerative diseases or cancers. The presence of transition biometal ions, specifically copper and iron, induces ROS formation by catalyzing the reduction of molecular oxygen to superoxide anion (O2˙-), hydrogen peroxide (H2O2) and hydroxyl (HO˙) radical. To limit ROS production and their detrimental effects, we report on the synthesis, physicochemical studies and antioxidant assays of an innovative series of synthetic pentapeptides exhibiting a dual direct/indirect mode of action, both as iron(iii)-chelators and as radical scavengers. These combined effects lead to a drastic reduction of in vitro reactive oxygen species production up to 95% for the more reactive hydroxyl radical.

Chromatographic separation simulation of metal-chelating peptides from surface plasmon resonance binding parameters.

Some metal-chelating peptides have antioxidant properties, with potential nutrition, health, and cosmetics applications. This study aimed to simulate their separation on immobilized metal ion affinity chromatography from their affinity constant for immobilized metal ion determined in surface plasmon resonance, both technics are based on peptide-metal ion interactions. In our approach, first, the affinity constant of synthetic peptides was determined by surface plasmon resonance and used as input data to numerically simulate the chromatographic separation with a transport-dispersive model based on Langmuir adsorption isotherm. Then, chromatographic separation was applied on the same peptides to determine their retention time and compare this experimental tR with the simulated tR obtained from simulation from surface plasmon resonance data. For the investigated peptides, the relative values of tR were comparable. Hence, our study demonstrated the pertinence of such numerical simulation correlating immobilized metal ion affinity chromatography and surface plasmon resonance.

An easy-to-implement combinatorial approach involving an activity-based assay for the discovery of a peptidyl copper complex mimicking superoxide dismutase.

A combinatorial approach using a one-bead-one-compound method and a screening based on a SOD-activity assay was set up for the discovery of an efficient peptidyl copper complex. The complex exhibited good stability constants, suitable redox potentials and excellent intrinsic activity. This complex was further assayed in cells for its antioxidant properties and showed beneficial effects when cells were subjected to oxidative stress.

SPR screening of metal chelating peptides in a hydrolysate for their antioxidant properties.

There is a growing need in the industrial sector (health, nutrition and cosmetic) to discover new biomolecules with various physico-chemical and bioactive properties. Various beneficial effects of peptides - notably those produced from protein hydrolysis - are reported in the literature. The antioxidant activity involves various mechanisms, among them metal chelation, studied by UV-visible spectrophotometry. In this paper, we set up an original method of screening metal chelating peptides in a hydrolysate using Surface Plasmon Resonance (SPR) for their antioxidant properties. To date, the empirical approach used several cycles of hydrolysate fractionation and bioactivity evaluation until the isolation of the pure bioactive molecule and its identification. Besides, the detection of metal-chelating peptide is not sensitive enough by spectrophotometry. For the first time, metal chelating peptides were screened in hydrolysates using SPR and a correlation was established between affinity constant determined in SPR and metal chelation capacity determined from UV-visible spectrophotometry.

l-Alanylglycylhistamine dihydro-chloride.

In the title compound {systematic name: 4-[2-({N-[(2S)-2-ammonio-propano-yl]glyc-yl}amino)-eth-yl]-1H-imidazol-3-ium dichloride}, C(10)H(19)N(5)O(2) (2+)·2Cl(-), the pseudo-tripeptide l-alanyl-glycyl-histamine is protonated at both the terminal amino group and the histidine N2 atom. The resulting positive charges are neutralized by two chloride anions. In the crystal, the organic cation adopts a twisted conformation about the CH(2)-CH(2) bond of histamine and about the C-N bond in the main chain, stabilized by a short intra-molecular C-H⋯O contact. In the crystal, N(+)-H⋯O and N(+)-H⋯Cl(-) hydrogen bonds link the mol-ecules into infinite sheets parallel to the (100) plane. The stacking of these sheets along the a axis is supported by N(amide)-H⋯Cl(-) hydrogen bonds.

The role of terminal amino group and histidine at the fourth position in the metal ion binding of oligopeptides revisited: Copper(II) and nickel(II) complexes of glycyl-glycyl-glycyl-histamine and its N-Boc protected derivative.

Copper(II) and nickel(II) binding properties of two pseudo tetrapeptides, N-Boc-Gly-Gly-Gly-Histamine (BGGGHa) and Gly-Gly-Gly-Histamine (GGGHa) have been investigated by pH-potentiometric titrations, UV-visible-, EPR-, NMR- and ESI-HRMS (electrospray ionization high resolution MS) spectroscopies, in order to compare the role of N-terminal amino group and imidazole moiety at the fourth position in the complex formation processes. Substantially higher stabilities were determined for the ML complexes of GGGHa, compared to those of BGGGHa, supporting the coordination of the terminal amino group and the histamine imidazole of the non-protected ligand. A dimeric Cu(2)H(-2)L(2) species, formed through the deprotonation of peptide groups of the ligands, was found in the GGGHa-copper(II) system. Deprotonation and coordination of further amide nitrogens led to CuH(-2)L and, above pH~10, CuH(-3)L. Experimental data supports a {NH(2), 2 × N(amide),N(im)} macrochelate structure in CuH(-2)L whereas a {NH(2), 3 × N(amide)} coordination environment in CuH(-3)L. The first two amide deprotonation processes were found to be strongly cooperative with nickel(II) and spectroscopic studies proved the transformation of the octahedral parent complexes to square planar, yellow, diamagnetic species, NiH(-2)L and above pH~9, NiH(-3)L. In the basic pH-range deprotonation and coordination of the amide groups also took place in the BGGGHa containing systems, leading to complexes with a {3 × N(amide),N(im)} donor set, and in parallel the re-dissolving of precipitate. Above pH~11, a further proton release from the pyrrolic NH group of the imidazole ring of BGGGHa occurred providing an additional proof for the different binding modes of the two ligands.

Nickel(II)-dipeptidoamine-based tetrameric complex: structural study in solution and in solid state.

The coordination structure of M(4)L(4)H(-8) macromolecules (M = Ni(II), Cu(II), Pd(II)) containing small peptidic ligands (L = Xaa-His or Xaa-His-Yaa) has been predicted primarily on the basis of spectroscopic and potentiometric data in the literature. In this work, the neutral tetranuclear nickel(II) complex 1 formed with four double-deprotonated ligands (L = α-methyl-alanyl-histamine) was prepared, and its crystal structure was determined (C(36)H(56)N(16)Ni(4)O(4)·4.5CH(3)OH·1.5H(2)O: a = 11.2645(4) A, b = 23.5003(8) A, c = 20.9007(7) A, ß = 102.321(1)°, monoclinic, P2(1)/c, Z = 4). In complex 1, the metal ions have a square planar geometry with 4N donor set consisting of the N-terminal amino nitrogen, the deprotonated amide nitrogen, the imidazole N(3) atom, and the deprotonated imidazole N(1) atom of the adjacent ligand. The latter nitrogen atom provides the connection of the four NiLH(-2) units forming a C(1) symmetrical saddle-like shape. The complexation of L with Ni(II) ion has been studied by a potentiometric method combined with UV-visible spectrophotometric titration. At pH 8.0, the predominant species is M(4)L(4)H(-8) with pK(4)(oligomerization) = 5.73. The tetranuclear structure of complex 1 was also studied in solution by (1)H and (13)C NMR spectroscopy suggesting a structure of symmetry S(4). DFT calculations on optimized structure in symmetry C(1) and S(4) have been performed to explain the observed differences in solution and in solid state. The nuclearity was also confirmed in solution by ESI-HRMS analysis.

4-{2-[3-(2-Ammonioacetamido)propanamido]ethyl}-1H-imidazol-3-ium dichloride.

Mol-ecules of the title compound, Gly-ß-Ala-Histamine dihydro-chloride, C(10)H(19)N(5)O(2) (2+)·2Cl(-), are linked by N-H⋯O and N-H⋯Cl hydrogen bonds into two-dimensional polymeric sheets parallel to the (011) plane, forming a stacked structure along the a axis. The parallel layers are also inter-linked alternately by different N-H⋯Cl hydrogen bonds, forming a three-dimensional framework.

Structural, kinetic, and theoretical studies on models of the zinc-containing phosphodiesterase active center: medium-dependent reaction mechanisms.

Dinuclear zinc(II) complexes [Zn(2)(bpmp)(mu-OH)](ClO(4))(2) (1) and [Zn(2)(bpmp)(H(2)O)(2)](ClO(4))(3) (2) (H-BPMP=2,6-bis[bis(2-pyridylmethyl)aminomethyl]-4-methylphenol) have been synthesized, structurally characterized, and pH-driven changes in metal coordination observed. The transesterification reaction of 2-hydroxypropyl p-nitrophenyl phosphate (HPNP) in the presence of the two complexes was studied both in a water/DMSO (70:30) mixture and in DMSO. Complex 2 was not reactive whereas for 1 considerable rate enhancement of the spontaneous hydrolysis reaction was observed. A detailed mechanistic investigation by kinetic studies, spectroscopic measurements ((1)H, (31)P NMR spectroscopy), and ESI-MS analysis in conjunction with ab initio calculations was performed on 1. Based on these results, two medium-dependent mechanisms are presented and an unusual bridging phosphate intermediate is proposed for the process in DMSO.

Synthetic models of the active site of catechol oxidase: mechanistic studies.

The ability of copper proteins to process dioxygen at ambient conditions has inspired numerous research groups to study their structural, spectroscopic and catalytic properties. Catechol oxidase is a type-3 copper enzyme usually encountered in plant tissues and in some insects and crustaceans. It catalyzes the conversion of a large number of catechols into the respective o-benzoquinones, which subsequently auto-polymerize, resulting in the formation of melanin, a dark pigment thought to protect a damaged tissue from pathogens. After the report of the X-ray crystal structure of catechol oxidase a few years earlier, a large number of publications devoted to the biomimetic modeling of its active site appeared in the literature. This critical review (citing 114 references) extensively discusses the synthetic models of this enzyme, with a particular emphasis on the different approaches used in the literature to study the mechanism of the catalytic oxidation of the substrate (catechol) by these compounds. These are the studies on the substrate binding to the model complexes, the structure-activity relationship, the kinetic studies of the catalytic oxidation of the substrate and finally the substrate interaction with (per)oxo-dicopper adducts. The general overview of the recognized types of copper proteins and the detailed description of the crystal structure of catechol oxidase, as well as the proposed mechanisms of the enzymatic cycle are also presented.

Catecholase activity of a copper(II) complex with a macrocyclic ligand: unraveling catalytic mechanisms.

We report the structure, properties and a mechanism for the catecholase activity of a tetranuclear carbonato-bridged copper(II) cluster with the macrocyclic ligand [22]pr4pz (9,22-dipropyl-1,4,9,14,17,22,27,28,29, 30-decaazapentacyclo[22.2.1.1(4,7).1(11,14). 1(17,20)]triacontane-5,7(28),11(29),12,18, 20(30),24(27),25-octaene). In this complex, two copper ions within a macrocyclic unit are bridged by a carbonate anion, which further connects two macrocyclic units together. Magnetic susceptibility studies have shown the existence of a ferromagnetic interaction between the two copper ions within one macrocyclic ring, and a weak antiferromagnetic interaction between the two neighboring copper ions of two different macrocyclic units. The tetranuclear complex was found to be the major compound present in solution at high concentration levels, but its dissociation into two dinuclear units occurs upon dilution. The dinuclear complex catalyzes the oxidation of 3,5-di-tert-butylcatechol to the respective quinone in methanol by two different pathways, one proceeding via the formation of semiquinone species with the subsequent production of dihydrogen peroxide as a byproduct, and another proceeding via the two-electron reduction of the dicopper(II) center by the substrate, with two molecules of quinone and one molecule of water generated per one catalytic cycle. The occurrence of the first pathway was, however, found to cease shortly after the beginning of the catalytic reaction. The influence of hydrogen peroxide and di-tert-butyl-o-benzoquinone on the catalytic mechanism has been investigated. The crystal structures of the free ligand and the reduced dicopper(I) complex, as well as the electrochemical properties of both the Cu(II) and the Cu(I) complexes are also reported.

Catecholase activity of a mu-hydroxodicopper(II) macrocyclic complex: structures, intermediates and reaction mechanism.

The monohydroxo-bridged dicopper(II) complex (1), its reduced dicopper(I) analogue (2) and the trans-mu-1,2-peroxo-dicopper(II) adduct (3) with the macrocyclic N-donor ligand [22]py4pz (9,22-bis(pyridin-2'-ylmethyl)-1,4,9,14,17,22,27,28,29,30- decaazapentacyclo -[22.2.1(14,7).1(11,14).1(17,20)]triacontane-5,7(28),11(29),12,18,20(30), 24(27),25-octaene), have been prepared and characterized, including a 3D structure of 1 and 2. These compounds represent models of the three states of the catechol oxidase active site: met, deoxy (reduced) and oxy. The dicopper(II) complex 1 catalyzes the oxidation of catechol model substrates in aerobic conditions, while in the absence of dioxygen a stoichiometric oxidation takes place, leading to the formation of quinone and the respective dicopper(I) complex. The catalytic reaction follows a Michaelis-Menten behavior. The dicopper(I) complex binds molecular dioxygen at low temperature, forming a trans-mu-1,2-peroxo-dicopper adduct, which was characterized by UV-Vis and resonance Raman spectroscopy and electrochemically. This peroxo complex stoichiometrically oxidizes a second molecule of catechol in the absence of dioxygen. A catalytic mechanism of catechol oxidation by 1 has been proposed, and its relevance to the mechanisms earlier proposed for the natural enzyme and other copper complexes is discussed.

Alkylation of human hemoglobin A0 by the antimalarial drug artemisinin.

In vitro, the heme cofactor of human iron(II) hemoglobin was efficiently and quickly alkylated at meso positions by the peroxide-based antimalarial drug artemisinin, leading to heme-artemisinin-derived covalent adducts. This reaction occurred in the absence of any added protease or in the presence of an excess of an extra non-heme protein, or even when artemisinin was added to hemolysed human blood. This activation of artemisinin by the heme moiety of non-digested hemoglobin clearly indicates the high affinity of this drug for heme, and its efficient alkylating ability under very mild conditions.