Polymeric Linkers Research Articles

Constructing Carbon Nanotube/Pt Nanoparticle Hybrids Using an Imidazolium‐Salt‐Based Ionic Liquid as a Linker

Carbon nanotube (CNT)/Pt nanoparticle (NP) hybrids are synthesized at room temperature using an imidazolium-salt-based ionic liquid (IS-ILs) as a linker. The new three-component CNT/IS-ILs/metal heterostructure has uniformly distributed Pt NPs, which form a conducting network on the CNTs that leads to high electrocatalytic activity. The hybrid is shown to exhibit higher electrocatalytic activity towards methanol oxidation reactions than a commercial hybrid catalyst and a hybrid containing polymeric linkers.

Probing the Heterogeneity in the Distribution of Binding Properties of Immobilized Surface Sites through Bayesian Analysis

When studying the binding of ligands to macromolecules immobilized at a surface, the question arises whether the surface binding sites are heterogeneous, as one might expect considering many factors such as intrinsic surface roughness, nonuniform density distribution of polymeric linkers and nonuniform chemical attachment producing different protein orientations and conformations. We previously developed a computational tool to determine the distribution of affinity and kinetic rate constants from the analysis of experimental surface binding data.

Polyelectrolyte-linked film assemblies of nanoparticles and nanoshells: Growth, stability, and optical properties

Multi-layer films of nanoparticles and nanoshells featuring various polymeric linkage molecules have been assembled and their optical properties characterized. The growth dynamics, including molecular weight effects, and stability of the various nanoparticle film constructions, using both single polymer as well as combinations of alternating charge polyelectrolytes as linking mechanisms, are presented. The polymeric linkers studied include poly-L-lysine, poly-L-arginine, poly(allylamine hydrochloride), and polyamidoamine dendrimers. Significantly air stable films were achieved with the use of multi-layered polymeric bridges between the nanoparticles and nanoshells. Optical sensitivity normally observed with these nanomaterials in solution was observed for their corresponding film geometries, with the nanoshell films exhibiting a markedly higher ability to report their local dielectric environment.

Bayesian analysis of heterogeneity in the distribution of binding properties of immobilized surface sites.

Once a homogeneous ensemble of a protein ligand is taken from solution and immobilized to a surface, for many reasons the resulting ensemble of surface binding sites to soluble analytes may be heterogeneous. For example, this can be due to the intrinsic surface roughness causing variations in the local microenvironment, nonuniform density distribution of polymeric linkers, or nonuniform chemical attachment producing different protein orientations and conformations. We previously described a computational method for determining the distribution of affinity and rate constants of surface sites from analysis of experimental surface binding data. It fully exploits the high signal/noise ratio and reproducibility provided by optical biosensor technology, such as surface plasmon resonance. Since the computational analysis is ill conditioned, the previous approach used a regularization strategy assuming a priori all binding parameters to be equally likely, resulting in the broadest possible parameter distribution consistent with the experimental data. We now extended this method in a Bayesian approach to incorporate the opposite assumption, i.e., that the surface sites a priori are expected to be uniform (as one would expect in free solution). This results in a distribution of binding parameters as close to monodispersity as possible given the experimental data. Using several model protein systems immobilized on a carboxymethyl dextran surface and probed with surface plasmon resonance, we show microheterogeneity of the surface sites in addition to broad populations of significantly altered affinity. The distributions obtained are highly reproducible. Immobilization conditions and the total surface density of immobilized sites can have a substantial impact on the functional distribution of the binding sites.

Rootletin forms centriole-associated filaments and functions in centrosome cohesion

After duplication of the centriole pair during S phase, the centrosome functions as a single microtubule-organizing center until the onset of mitosis, when the duplicated centrosomes separate for bipolar spindle formation. The mechanisms regulating centrosome cohesion and separation during the cell cycle are not well understood. In this study, we analyze the protein rootletin as a candidate centrosome linker component. As shown by immunoelectron microscopy, endogenous rootletin forms striking fibers emanating from the proximal ends of centrioles. Moreover, rootletin interacts with C-Nap1, a protein previously implicated in centrosome cohesion. Similar to C-Nap1, rootletin is phosphorylated by Nek2 kinase and is displaced from centrosomes at the onset of mitosis. Whereas the overexpression of rootletin results in the formation of extensive fibers, small interfering RNA–mediated depletion of either rootletin or C-Nap1 causes centrosome splitting, suggesting that both proteins contribute to maintaining centrosome cohesion. The ability of rootletin to form centriole-associated fibers suggests a dynamic model for centrosome cohesion based on entangling filaments rather than continuous polymeric linkers.

Determination of fluoxetine and norfluoxetine in rat brain microdialysis samples following intraperitoneal fluoxetine administration

Fluoxetine (FLX) and the N-desmethyl metabolite, norfluoxetine (NFLX) in rat brain microdialysis samples were determined by high-performance liquid chromatography (HPLC) with fluorescence detection using pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). In vitro experiment showed that the relative recovery of FLX across microdialysis membrane was enhanced by adding β-cyclodextrin (β-CD) or β-CD polymer to microdialysis perfusion fluid. The perfusion fluid containing β-CD polymer, which has polymeric glyceryl linkers attached to the hydroxyl group, gave the better recovery with satisfactory precisions. Using 1% β-CD polymer in Ringer’s solution as the perfusate, in vivo rat brain microdialysis experiment on intraperitoneal administration of FLX (10 mg/kg) to rats was carried out. The fluorescence peaks of FLX and NFLX appeared later than 30 min after the administration of FLX, and then, gradually increased with time. Two hours later, FLX reached a plateau level, but NFLX slowly increased, and at 24–48 h, NFLX levels were higher than FLX levels. These data suggest that long distributions of FLX and the potent metabolite, NFLX, in brain contributed to the long-term drug actions in vivo.

Mechanics of semiflexible chains formed by poly(ethylene glycol)-linked paramagnetic particles.

Magnetorheological particles, permanently linked into chains, provide a magnetically actuated means to manipulate microscopic fluid flow. Paramagnetic colloidal particles form reversible chains by acquiring dipole moments in the presence of an external magnetic field. By chemically connecting paramagnetic colloidal particles, flexible magnetoresponsive chains can be created. We link the paramagnetic microspheres using streptavidin-biotin binding. Streptavidin coated microspheres are placed in a flow cell and a magnetic field is applied, causing the particles to form chains. Then a solution of polymeric linkers of bis-biotin-poly(ethylene glycol) molecules is added in the presence of the field. These linked chains remain responsive to a magnetic field; however, in the absence of an external magnetic field these chains bend and flex due to thermal motion. The chain flexibility is determined by the length of the spacer molecule between particles and is quantified by the flexural rigidity or bending stiffness. To understand the mechanical properties of the chains, we use a variety of optical trapping experiments to measure the flexural rigidity. Increasing the length of the poly(ethylene glycol) chain in the linker increases the flexibility of the chains.

Inactivation of BK channels by the NH2 terminus of the beta2 auxiliary subunit: an essential role of a terminal peptide segment of three hydrophobic residues.

An auxiliary β2 subunit, when coexpressed with Slo α subunits, produces inactivation of the resulting large-conductance, Ca2+ and voltage-dependent K+ (BK-type) channels. Inactivation is mediated by the cytosolic NH2 terminus of the β2 subunit. To understand the structural requirements for inactivation, we have done a mutational analysis of the role of the NH2 terminus in the inactivation process. The β2 NH2 terminus contains 46 residues thought to be cytosolic to the first transmembrane segment (TM1). Here, we address two issues. First, we define the key segment of residues that mediates inactivation. Second, we examine the role of the linker between the inactivation segment and TM1. The results show that the critical determinant for inactivation is an initial segment of three amino acids (residues 2–4: FIW) after the initiation methionine. Deletions that scan positions from residue 5 through residue 36 alter inactivation, but do not abolish it. In contrast, deletion of FIW or combinations of point mutations within the FIW triplet abolish inactivation. Mutational analysis of the three initial residues argues that inactivation does not result from a well-defined structure formed by this epitope. Inactivation may be better explained by linear entry of the NH2-terminal peptide segment into the permeation pathway with residue hydrophobicity and size influencing the onset and recovery from inactivation. Examination of the ability of artificial, polymeric linkers to support inactivation suggests that a variety of amino acid sequences can serve as adequate linkers as long as they contain a minimum of 12 residues between the first transmembrane segment and the FIW triplet. Thus, neither a specific distribution of charge on the linker nor a specific structure in the linker is required to support the inactivation process.

Development of new linkers for the formation of aliphatic C-H bonds on polymeric supports

Abstract Two polymeric linkers, 8 and 11, were prepared in high yields. Attachment of an alkyl group to these linkers was facile, and their cleavage from the MeO-PEG polymer support was accomplished by desulfurization using Raney nickel to yield the new carbon-hydrogen bond product 6. The protocol reported herein allows efficient preparation of new polymeric linkers as well as their possible application to combinatorial libraries.

Automated chemical synthesis: from resins to instruments.

Solid-phase organic synthesis is now a prevalent activity in drug discovery. In keeping with this keen interest is the need to develop reliable automated synthesis instrumentation as well as polymeric supports and linkers suitable for the full range of organic synthesis applications. In this paper, we review our activities in the development of new and enabling tools for automated chemical synthesis, including the following: (i) new solid supports such as ArgoGel (PS-PEG-based) and Argo-X203 (PS-based); and (ii) the Nautilus 2400 system, a fully closed and inert automated chemistry development workstation. Selected chemistry optimization and synthesis examples performed on the Nautilus and new solid supports will be described.