Quest for a stable Cu-ligand complex with a high catalytic activity to produce reactive oxygen species.

Metal ion-catalyzed overproduction of reactive oxygen species (ROS) is believed to contribute significantly to oxidative stress and be involved in several biological processes, from immune defense to development of diseases. Among the essential metal ions, copper is one of the most efficient catalysts in ROS production in the presence of O2 and a physiological reducing agent such as ascorbate. To control this chemistry, Cu ions are tightly coordinated to biomolecules. Free or loosely bound Cu ions are generally avoided to prevent their toxicity. In the present report, we aim to find stable Cu-ligand complexes (Cu-L) that can efficiently catalyze the production of ROS in the presence of ascorbate under aerobic conditions. Thermodynamic stability would be needed to avoid dissociation in the biological environment, and high ROS catalysis is of interest for applications as antimicrobial or anticancer agents. A series of Cu complexes with the well-known tripodal and tetradentate ligands containing a central amine linked to three pyridyl-alkyl arms of different lengths were investigated. Two of them with mixed arm length showed a higher catalytic activity in the oxidation of ascorbate and subsequent ROS production than Cu salts in buffer, which is an unprecedented result. Despite these high catalytic activities, no increased antimicrobial activity toward Escherichia coli or cytotoxicity against eukaryotic AGS cells in culture related to Cu-L-based ROS production could be observed. The potential reasons for discrepancy between in vitro and in cell data are discussed.

Restoring cellular copper homeostasis in Alzheimer disease: a novel peptide shuttle is internalized by an ATP-dependent endocytosis pathway involving Rab5- and Rab14-endosomes.

CPPs, or Cell-Penetrating Peptides, offer invaluable utility in disease treatment due to their ability to transport various therapeutic molecules across cellular membranes. Their unique characteristics, such as biocompatibility and low immunogenicity, make them ideal candidates for delivering drugs, genes, or imaging agents directly into cells. This targeted delivery enhances treatment efficacy while minimizing systemic side effects. CPPs exhibit versatility, crossing biological barriers and reaching intracellular targets that conventional drugs struggle to access. This capability holds promise in treating a wide array of diseases, including cancer, neurodegenerative disorders, and infectious diseases, offering a potent avenue for innovative and targeted therapies, yet their precise mechanism of cell entry is far from being fully understood. In order to correct Cu dysregulation found in various pathologies such as Alzheimer disease, we have recently conceived a peptide Cu(II) shuttle, based on the αR5W4 CPP, which, when bound to Cu(II), is able to readily enter a neurosecretory cell model, and release bioavailable Cu in cells. Furthermore, this shuttle has the capacity to protect cells in culture against oxidative stress-induced damage which occurs when Cu binds to the Aß peptide. The aim of this study was therefore to characterize the cell entry route used by this shuttle and determine in which compartment Cu is released. Pharmacological treatments, siRNA silencing and colocalization experiments with GFP-Rab fusion proteins, indicate that the shuttle is internalized by an ATP-dependent endocytosis pathway involving both Rab5 and Rab14 endosomes route and suggest an early release of Cu from the shuttle.

Evaluation of Candidate Theranostics for 227Th/89Zr Paired Radioimmunotherapy of Lymphoma.

227Th is a promising radioisotope for targeted α-particle therapy. It produces 5 α-particles through its decay, with the clinically approved 223Ra as its first daughter. There is an ample supply of 227Th, allowing for clinical use; however, the chemical challenges of chelating this large tetravalent f-block cation are considerable. Using the CD20-targeting antibody ofatumumab, we evaluated chelation of 227Th4+ for α-particle-emitting and radiotheranostic applications. Methods: We compared 4 bifunctional chelators for thorium radiopharmaceutical preparation: S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), 2-(4-isothicyanatobenzyl)-1,2,7,10,13-hexaazacyclooctadecane-1,4,7,10,13,16-hexaacetic acid (p-SCN-Bn-HEHA), p-isothiacyanatophenyl-1-hydroxy-2-oxopiperidine-desferrioxamine (DFOcyclo*-p-Phe-NCS), and macrocyclic 1,2-HOPO N-hydroxysuccinimide (L804-NHS). Immunoconstructs were evaluated for yield, purity, and stability in vitro and in vivo. Tumor targeting of the lead 227Th-labeled compound in vivo was performed in CD20-expressing models and compared with a companion 89Zr-labeled PET agent. Results: 227Th-labeled ofatumumab-chelator constructs were synthesized to a radiochemical purity of more than 95%, excepting HEHA. 227Th-HEHA-ofatumumab showed moderate in vitro stability. 227Th-DFOcyclo*-ofatumumab presented excellent 227Th labeling efficiency; however, high liver and spleen uptake was revealed in vivo, indicative of aggregation. 227Th-DOTA-ofatumumab labeled poorly, yielding no more than 5%, with low specific activity (0.08 GBq/g) and modest long-term in vitro stability (<80%). 227Th-L804-ofatumumab coordinated 227Th rapidly and efficiently at high yields, purity, and specific activity (8 GBq/g) and demonstrated extended stability. In vivo tumor targeting confirmed the utility of this chelator, and the diagnostic analog, 89Zr-L804-ofatumumab, showed organ distribution matching that of 227Th to delineate SU-DHL-6 tumors. Conclusion: Commercially available and novel chelators for 227Th showed a range of performances. The L804 chelator can be used with potent radiotheranostic capabilities for 89Zr/227Th quantitative imaging and α-particle therapy.

A luminescent ATCUN peptide variant with enhanced properties for copper(II) sensing in biological media.

The measurement of labile CuII in biological samples is fundamental for understanding Cu metabolism and has been emerging as a promising diagnostic marker for Cu-related pathologies such as Wilson's and Alzheimer's diseases. The use of fluorescent chelators may be useful to circumvent separation steps employed by current methods. For this purpose, we recently designed a selective and suited-affinity turn-off luminescent probe based on a peptide bearing the CuII-binding Xxx-Zzz-His (Amino-Terminal CuII- and NiII-binding, ATCUN) motif and a TbIII-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) complex. Here, we present an analogue probe bearing the ATCUN motif variant Xxx-His-His. This probe showed much faster response in biologically-relevant media and higher stability than the previous motif at low pH. These features could be beneficial to the measurement of dynamic CuII fluctuations and the application in slightly acidic media, such as urine.

A novel DOTA-like building block with a picolinate arm for the synthesis of lanthanide complex-peptide conjugates with improved luminescence properties.

Combination of complexes of lanthanide cations (Ln3+) for their luminescent properties and peptides for their recognition properties is interesting in view of designing responsive luminescent probes. The octadentate DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelate is the most popular chelate to design Ln3+ complex-peptide conjugates. We describe a novel building block, DO3Apic-tris(allyl)ester, which provides access to peptides with a conjugated nonadentate chelate, namely DO3Apic, featuring a picolinate arm in place of one of the acetate arms compared to DOTA, for improved luminescence properties. This building block, with allyl protecting groups, is readily obtained by solid phase synthesis. We show that it is superior to its analogue with tBu protecting groups for the preparation of peptide conjugates because of the difficult removal of the tBu protecting groups for the latter. Then, we compare two luminescent zinc fingers (LZF) comprising (i) a zinc finger peptide for selective Zn2+ binding, (ii) a Eu3+ complex and (iii) an acridone antenna (ACD) for long-wavelength sensitization of Eu3+ luminescence. The first one, LZF3ACD|Eu, incorporates a DOTA chelate for Eu3+ whereas the other, LZF4ACD|Eu, incorporates a DO3Apic chelate. Both act as Zn2+-responsive luminescent probes but we show that changing DOTA for DO3Apic results in a higher Eu3+ luminescence lifetime and in a doubling of the quantum yield, confirming the interest of the DO3Apic chelate and the DO3Apic(tris(allyl)ester building block for the preparation of Ln3+ complex-peptide conjugates. Additionally, the DO3Apic chelate provides self-calibration for LZF4ACD|Eu luminescence upon excitation of its picolinamide chromophore, making LZF4ACD|Eu a ratiometric sensor for Zn2+.

Using Native Chemical Ligation for Site-Specific Synthesis of Hetero-bis-lanthanide Peptide Conjugates: Application to Ratiometric Visible or Near-Infrared Detection of Zn2.

The interest in ratiometric luminescent probes that detect and quantify a specific analyte is growing. Owing to their special luminescence properties, lanthanide(III) cations offer attractive opportunities for the design of dual-color ratiometric probes. Here, the design principle of hetero-bis-lanthanide peptide conjugates by using native chemical ligation is described for perfect control of the localization of each lanthanide cation within the molecule. Two zinc-responsive probes, r-LZF1Tb|Cs124|Eu and r-LZF1Eu|Cs124|Tb are described on the basis of a zinc finger peptide and two DOTA (DOTA=1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid) complexes of terbium and europium. Both display dual-color ratiometric emission in response to the presence of Zn2+ . By using a screening approach, anthracene was identified for the sensitization of the luminescence of two near-infrared-emitting lanthanides, Yb3+ and Nd3+ . Thus, two novel zinc-responsive hetero-bis-lanthanide probes, r-LZF3Yb|Anthra|Nd and r-LZF3Nd|Anthra|Yb were assembled, the former offering a neat ratiometric response to Zn2+ with emission in the near-infrared around 1000 nm, which is unprecedented.

A terbium(iii) luminescent ATCUN-based peptide sensor for selective and reversible detection of copper(ii) in biological media.

The measurement of exchangeable Cu2+ levels in biological samples is gaining interest in the context of copper-related pathologies. Here, we report a Tb3+ luminescent turn-off sensor for Cu2+ based on the specific and suitable-affinity Xxx-Zzz-His (ATCUN) peptide motif, enabling Cu2+ detection in the presence of a biological fluorescent background.

N-Terminal Cu-Binding Motifs (Xxx-Zzz-His, Xxx-His) and Their Derivatives: Chemistry, Biology and Medicinal Applications.

Peptides and proteins with N-terminal amino acid sequences NH2 -Xxx-His (XH) and NH2 -Xxx-Zzz-His (XZH) form well-established high-affinity CuII -complexes. Key examples are Asp-Ala-His (in serum albumin) and Gly-His-Lys, the wound healing factor. This opens a straightforward way to add a high-affinity CuII -binding site to almost any peptide or protein, by chemical or recombinant approaches. Thus, these motifs, NH2 -Xxx-Zzz-His in particular, have been used to equip peptides and proteins with a multitude of functions based on the redox activity of Cu, including nuclease, protease, glycosidase, or oxygen activation properties, useful in anticancer or antimicrobial drugs. More recent research suggests novel biological functions, mainly based on the redox inertness of CuII in XZH, like PET imaging (with 64 Cu), chelation therapies (for instance in Alzheimer's disease and other types of neurodegeneration), antioxidant units, Cu transporters and activation of biological functions by strong CuII binding. This Review gives an overview of the chemical properties of Cu-XH and -XZH motifs and discusses the pros and cons of the vastly different biological applications, and how they could be improved depending on the application.

Selectively Activatable Latent Thiol and Selenolesters Simplify the Access to Cyclic or Branched Peptide Scaffolds.

The cyclic dichalcogenides based on the bis(2-chalcogenoethyl)amide structure are latent N,S (SEA, chalcogen = S) or N,Se (SeEA, chalcogen = Se) acyl shift systems. The large difference in the reducing potential between SEA and SeEA dichalcogenides allows their sequential and selective activation by reduction. Based on these concepts, one-pot three or four peptide segment assembly processes were designed, facilitating access to branched or cyclic peptide scaffolds.

Synthesis of Unprotected Linear or Cyclic O-Acyl Isopeptides in Water Using Bis(2-sulfanylethyl)amido Peptide Ligation.

SEA ligation proceeds chemoselectively at pH 3, i.e., at a pH where the O-acyl isopeptides are protected by protonation. This property was used for synthesizing unprotected O-acyl isopeptides in water, starting from peptide segments which are easily accessible by the Fmoc SPPS.

Solid phase protein chemical synthesis.

The chemical synthesis of peptides or small proteins is often an important step in many research projects and has stimulated the development of numerous chemical methodologies. The aim of this review is to give a substantial overview of the solid phase methods developed for the production or purification of polypeptides. The solid phase peptide synthesis (SPPS) technique has facilitated considerably the access to short peptides (<50 amino acids). However, its limitations for producing large homogeneous peptides have stimulated the development of solid phase covalent or non-covalent capture purification methods. The power of the native chemical ligation (NCL) reaction for protein synthesis in aqueous solution has also been adapted to the solid phase by the combination of novel linker technologies, cysteine protection strategies and thioester or N,S-acyl shift thioester surrogate chemistries. This review details pioneering studies and the most recent publications related to the solid phase chemical synthesis of large peptides and proteins.

One-pot chemical synthesis of small ubiquitin-like modifier protein-peptide conjugates using bis(2-sulfanylethyl)amido peptide latent thioester surrogates.

Small ubiquitin-like modifier (SUMO) post-translational modification (PTM) of proteins has a crucial role in the regulation of important cellular processes. This protocol describes the chemical synthesis of functional SUMO-peptide conjugates. The two crucial stages of this protocol are the solid-phase synthesis of peptide segments derivatized by thioester or bis(2-sulfanylethyl)amido (SEA) latent thioester functionalities and the one-pot assembly of the SUMO-peptide conjugate by a sequential native chemical ligation (NCL)/SEA native peptide ligation reaction sequence. This protocol also enables the isolation of a SUMO SEA latent thioester, which can be attached to a target peptide or protein in a subsequent step. It is compatible with 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, and it gives access to homogeneous, reversible and functional SUMO conjugates that are not easily produced using living systems. The synthesis of SUMO-peptide conjugates on a milligram scale takes 20 working days.

Access to large cyclic peptides by a one-pot two-peptide segment ligation/cyclization process.

The use of the N-acetoacetyl protecting group for N-terminal cysteine residue enabled creation of an efficient and mild one-pot native chemical ligation/SEA ligation sequence giving access to large cyclic peptides.

Selenopeptide transamidation and metathesis.

Selenopeptides can be transamidated by cysteinyl peptides in water using mild conditions (pH 5.5, 37 °C) in the presence of an arylthiol catalyst. Similar conditions also catalyze the metathesis of selenopeptides. The usefulness of the selenophosphine derived from TCEP (TCEP═Se) for inhibiting the TCEP-induced deselenization of selenocysteine residue is also reported.

Tidbits for the synthesis of bis(2-sulfanylethyl)amido (SEA) polystyrene resin, SEA peptides and peptide thioesters.

Protein total chemical synthesis enables the atom-by-atom control of the protein structure and therefore has a great potential for studying protein function. Native chemical ligation of C-terminal peptide thioesters with N-terminal cysteinyl peptides and related methodologies are central to the field of protein total synthesis. Consequently, methods enabling the facile synthesis of peptide thioesters using Fmoc-SPPS are of great value. Herein, we provide a detailed protocol for the preparation of bis(2-sulfanylethyl)amino polystyrene resin as a starting point for the synthesis of C-terminal bis(2-sulfanylethyl)amido peptides and of peptide thioesters derived from 3-mercaptopropionic acid.

Bis(2-sulfanylethyl)amido peptides enable native chemical ligation at proline and minimize deletion side-product formation.

Native chemical ligation of C-terminal peptidyl prolyl alkylthioesters with N-terminal cysteinyl peptides usually exhibits poor kinetic rates compared to other C-terminal amino acid residues. It is shown here that the reaction is accompanied by the formation of a deletion side product which is minimized by using a bis(2-sulfanylethyl)amido (SEA) thioester surrogate at a mildly acidic pH.