Trinuclear Ruthenium Clusters as Bivalent Electrochemical Probes for Ligand–Receptor Binding Interactions

Despite their popularity, electrochemical biosensors often suffer from low sensitivity. One possible approach to overcome low sensitivity in protein biosensors is to utilize multivalent ligand–receptor interactions. Controlling the spatial arrangement of ligands on surfaces is another crucial aspect of electrochemical biosensor design. We have synthesized and characterized five biotinylated trinuclear ruthenium clusters as potential new biosensor platforms: [Ru₃O(OAc)₆CO(4-BMP)(py)]⁰ (<b>3</b>), [Ru₃O(OAc)₆CO(4-BMP)₂]⁰ (<b>4</b>), [Ru₃O(OAc)₆L(4-BMP)(py)]⁺ (<b>8</b>), [Ru₃O(OAc)₆L(4-BMP)₂]⁺ (<b>9</b>), and [Ru₃O(OAc)₆L(py)₂]⁺ (<b>10</b>) (OAc = acetate, 4-BMP = biotin aminomethylpyridine, py = pyridine, L = pyC16SH). HABA/avidin assays and isothermal titration calorimetry were used to evaluate the avidin binding properties of <b>3</b> and <b>4</b>. The binding constants were found to range from (6.5–8.0) × 10⁶ M^–1. Intermolecular protein binding of <b>4</b> in solution was determined by native gel electrophoresis. QM, MM, and MD calculations show the capability for the bivalent cluster, <b>4</b>, to intramolecularly bind to avidin. Electrochemical measurements in solution of <b>3a</b> and <b>4a</b> show shifts in <i>E</i>_1/2 of −58 and −53 mV in the presence of avidin, respectively. Self-assembled monolayers formed with <b>8</b>–<b>10</b> were investigated as a model biosensor system. Diluent/cluster ratio and composition were found to have a significant effect on the ability of avidin to adequately bind to the cluster. Complexes <b>8</b> and <b>10</b> showed negligible changes in <i>E</i>_1/2, while complex <b>9</b> showed a shift in <i>E</i>_1/2 of −43 mV upon avidin addition. These results suggest that multivalent interactions can have a positive impact on the sensitivity of electrochemical protein biosensors

Nanodiscs as a Modular Platform for Multimodal MR-Optical Imaging

Nanodiscs are monodisperse, self-assembled discoidal particles that consist of a lipid bilayer encircled by membrane scaffold proteins (MSP). Nanodiscs have been used to solubilize membrane proteins for structural and functional studies and deliver therapeutic phospholipids. Herein, we report on tetramethylrhodamine (TMR) tagged nanodiscs that solubilize lipophilic MR contrast agents for generation of multimodal nanoparticles for cellular imaging. We incorporate both multimeric and monomeric Gd­(III)-based contrast agents into nanodiscs and show that particles containing the monomeric agent (<b>ND2</b>) label cells with high efficiency and generate significant image contrast at 7 T compared to nanodiscs containing the multimeric agent (<b>ND1</b>) and Prohance, a clinically approved contrast agent

Axial Ligand Exchange of <i>N</i>‑heterocyclic Cobalt(III) Schiff Base Complexes: Molecular Structure and NMR Solution Dynamics

The kinetic and thermodynamic ligand exchange dynamics are important considerations in the rational design of metal-based therapeutics and therefore, require detailed investigation. Co­(III) Schiff base complex derivatives of bis­(acetylacetone)­ethylenediimine [acacen] have been found to be potent enzyme and transcription factor inhibitors. These complexes undergo solution exchange of labile axial ligands. Upon dissociation, Co­(III) irreversibly interacts with specific histidine residues of a protein, and consequently alters structure and causes inhibition. To guide the rational design of next generation agents, understanding the mechanism and dynamics of the ligand exchange process is essential. To investigate the lability, pH stability, and axial ligand exchange of these complexes in the absence of proteins, the pD- and temperature-dependent axial ligand substitution dynamics of a series of <i>N</i>-heterocyclic [Co­(acacen)­(X)₂]⁺ complexes [where X = 2-methylimidazole (2MeIm), 4-methylimidazole (4MeIm), ammine (NH₃), <i>N</i>-methylimidazole (NMeIm), and pyridine (Py)] were characterized by NMR spectroscopy. The pD stability was shown to be closely related to the nature of the axial ligand with the following trend toward aquation: 2MeIm > NH₃ ≫ 4MeIm > Py > Im > NMeIm. Reaction of each [Co­(III)­(acacen)­(X)₂]⁺ derivative with 4MeIm showed formation of a mixed ligand Co­(III) intermediate via a dissociative ligand exchange mechanism. The stability of the mixed ligand adduct was directly correlated to the pD-dependent stability of the starting Co­(III) Schiff base with respect to [Co­(acacen)­(4MeIm)₂]⁺. Crystal structure analysis of the [Co­(acacen)­(X)₂]⁺ derivatives confirmed the trends in stability observed by NMR spectroscopy. Bond distances between the Co­(III) and the axial nitrogen atoms were longest in the 2MeIm derivative as a result of distortion in the planar tetradentate ligand, and this was directly correlated to axial ligand lability and propensity toward exchange

Axial Ligand Exchange of <i>N</i>‑heterocyclic Cobalt(III) Schiff Base Complexes: Molecular Structure and NMR Solution Dynamics

The kinetic and thermodynamic ligand exchange dynamics are important considerations in the rational design of metal-based therapeutics and therefore, require detailed investigation. Co­(III) Schiff base complex derivatives of bis­(acetylacetone)­ethylenediimine [acacen] have been found to be potent enzyme and transcription factor inhibitors. These complexes undergo solution exchange of labile axial ligands. Upon dissociation, Co­(III) irreversibly interacts with specific histidine residues of a protein, and consequently alters structure and causes inhibition. To guide the rational design of next generation agents, understanding the mechanism and dynamics of the ligand exchange process is essential. To investigate the lability, pH stability, and axial ligand exchange of these complexes in the absence of proteins, the pD- and temperature-dependent axial ligand substitution dynamics of a series of <i>N</i>-heterocyclic [Co­(acacen)­(X)₂]⁺ complexes [where X = 2-methylimidazole (2MeIm), 4-methylimidazole (4MeIm), ammine (NH₃), <i>N</i>-methylimidazole (NMeIm), and pyridine (Py)] were characterized by NMR spectroscopy. The pD stability was shown to be closely related to the nature of the axial ligand with the following trend toward aquation: 2MeIm > NH₃ ≫ 4MeIm > Py > Im > NMeIm. Reaction of each [Co­(III)­(acacen)­(X)₂]⁺ derivative with 4MeIm showed formation of a mixed ligand Co­(III) intermediate via a dissociative ligand exchange mechanism. The stability of the mixed ligand adduct was directly correlated to the pD-dependent stability of the starting Co­(III) Schiff base with respect to [Co­(acacen)­(4MeIm)₂]⁺. Crystal structure analysis of the [Co­(acacen)­(X)₂]⁺ derivatives confirmed the trends in stability observed by NMR spectroscopy. Bond distances between the Co­(III) and the axial nitrogen atoms were longest in the 2MeIm derivative as a result of distortion in the planar tetradentate ligand, and this was directly correlated to axial ligand lability and propensity toward exchange

Synapse-Binding Subpopulations of Aβ Oligomers Sensitive to Peptide Assembly Blockers and scFv Antibodies

Amyloid β42 self-assembly is complex, with multiple pathways leading to large insoluble fibrils or soluble oligomers. Oligomers are now regarded as most germane to Alzheimer’s pathogenesis. We have investigated the hypothesis that oligomer formation itself occurs through alternative pathways, with some leading to synapse-binding toxins. Immediately after adding synthetic peptide to buffer, solutions of Aβ42 were separated by a 50 kDa filter and fractions assessed by SDS-PAGE silver stain, Western blot, immunoprecipitation, and capacity for synaptic binding. Aβ42 rapidly assembled into aqueous-stable oligomers, with similar protein abundance in small (<50 kDa) and large (>50 kDa) oligomer fractions. Initially, both fractions were SDS-labile and resolved into tetramers, trimers, and monomers by SDS-PAGE. Upon continued incubation, the larger oligomers developed a small population of SDS-stable 10–16mers, and the smaller oligomers generated gel-impermeant complexes. The two fractions associated differently with neurons, with prominent synaptic binding limited to larger oligomers. Even within the family of larger oligomers, synaptic binding was associated with only a subset of these species, as a new scFv antibody (NUsc1) immunoprecipitated only a small portion of the oligomers while eliminating synaptic binding. Interestingly, low doses of the peptide KLVFFA blocked assembly of the 10–16mers, and this result was associated with loss of the smaller clusters of oligomers observed at synaptic sites. What distinguishes these smaller clusters from the unaffected larger clusters is not yet known. Results indicate that distinct species of Aβ oligomers are generated by alternative assembly pathways and that synapse-binding subpopulations of Aβ oligomers could be specifically targeted for Alzheimer’s therapeutics