Tethering Group Research Articles

Controlled Protein Confinement in Phase-Separated Membranes Tethered onto Micro-Patterned Supports

Phase-separation of lipid membranes into liquid-disordered (ld) and liquid-ordered (lo) domains has been recognized as a key principle for the functional organization of the plasma membrane. In classic model systems such as GUVs, the spatial organization of phase separated membranes is a stochastic, time-dependent process, which depends on the lipid composition and often leads to a complete coalescence of the lipid phases. We have here established an approach for a spatial control of lipid phase separation in tethered polymer-supported membranes (PSM). On a dense poly(ethylene glycol) polymer brush functionalized with hydrophobic tethers, contiguous, highly fluid PSM were obtained by means of fusion of SUVs.1 Free diffusion of lipids and reconstituted transmembrane proteins in these PSM was confirmed by FRAP, FCS and single molecule tracking. Strikingly, phase separation of ternary lipid mixtures (DOPC/SM/cholesterol) in PSM into ld and lo phases was dependent on the properties of the anchoring group. We exploited these features for assembly of lo domains into defined structures using micropatterned tethers. Within isolated micropatterns, ld and lo phases self-assembled into stable, reproducible membrane architectures. By binary micro-patterning of different tethering groups into complementary areas, ternary lipids mixtures separated into lo and ld phases controlled by the geometry of the underlying tethers. Transmembrane proteins reconstituted in these phase-separated PSM strictly partitioned into the ld phase. Hence, the lo phase could be used for confining transmembrane proteins into microscopic and submicroscopic domains. The permeability of these barriers for lipids and proteins and thus their exchange between adjacent ld compartments can be globally and locally controlled by the temperature. These features have been exploited for probing interactions and diffusion of a transmembrane receptor in the context of ld and lo phases.1) Roder, F.; et al. Anal Chem 2011, 83, 6792-6799.

N-Halamine Biocidal Coatings via a Layer-by-Layer Assembly Technique

Two N-halamine copolymer precursors, poly(2,2,6,6-tetramethyl-4-piperidyl methacrylate-co-acrylic acid potassium salt) and poly(2,2,6,6-tetramethyl-4-piperidyl methacrylate-co-trimethyl-2-methacryloxyethylammonium chloride) have been synthesized and successfully coated onto cotton fabric via a layer-by-layer (LbL) assembly technique. A multilayer thin film was deposited onto the fiber surfaces by alternative exposure to polyelectrolyte solutions. The coating was rendered biocidal by a dilute household bleach treatment. The biocidal efficacies of tested swatches composed of treated fibers were evaluated against Staphylococcus aureus and Escherichia coli. It was determined that chlorinated samples inactivated both S. aureus and E. coli O157:H7 within 15 min of contact time, whereas the unchlorinated control samples did not exhibit significant biocidal activities. Stabilities of the coatings toward washing and ultraviolet light exposure have also been studied. It was found that the stability toward washing was superior, whereas the UVA light stability was moderate compared to previously studied N-halamine moieties. The layer-by-layer assembly technique can be used to attach N-halamine precursor polymers onto cellulose surfaces without using covalently bonding tethering groups which limit the structure designs. In addition, ionic precursors are very soluble in water, thus promising for biocidal coatings without the use of organic solvents.

Entropy versus tether strain effects on rates of intramolecular 1,3-dipolar cycloadditions of N-alkenylnitrones

Intramolecular 1,3-dipolar cycloadditions of two N-alkenylnitrones are studied by means of density functional theory calculations. Cycloaddition of an acyclic 4-hexenylnitrone led to the expected isoxazolidine in 46% yield, but a 4-cycloheptenylnitrone did not react. Calculations of the transition states for cycloaddition indicate that although the cycloheptenyl nitrone has a more favorable activation entropy, the strain associated with distortion of the tethering groups into the required boat conformation disfavors the reaction of the cyclic substrate over the acyclic substrate by 8.7 kcal/mol.

Bodipy Dyes with Tunable Redox Potentials and Functional Groups for Further Tethering: Preparation, Electrochemical, and Spectroscopic Characterization

The preparation, spectroscopic, and electrochemical characterization of a family of 16 new bodipy dyes with tunable redox potentials and versatile functional groups is reported. Electron-withdrawing or -donating groups (Et, H, Cl, or CN) at positions C2 and C6 enabled tuning the redox potentials within a ca. 0.7 eV window without significantly affecting either the HOMO-LUMO gap or the absorption and emission spectra. Hydroxymethyl or formyl groups at the meso (C8) position in turn provided a handle for covalent tethering to receptors and biomolecules of interest, which dispenses with the more commonly used meso-aryl moiety as a means to tag molecules. The dyes can thus be coupled to both electrophiles and nucleophiles. Importantly, it is shown that meso-formyl bodipy dyes are nonemissive and have significantly lower molar extinction coefficients compared to their meso-hydroxymethyl and meso-acetoxymethyl counterparts (which in turn are bright, with emission quantum yields in the range of 0.7-1). The nonemissive meso-formyl bodipy dyes thus provide unique opportunities as fluorogenic probes of nucleophilic attack and as fluorescent labeling agents where uncoupled fluorophores will not contribute to the fluorescence background. Overall, the new bodipy dyes reported here are promising candidates for the preparation of fluorescent sensors relying on photoinduced electron transfer and may find use in a number of fluorescent-labeling protocols.

Effect of tethering chemistry of cationic surfactants on clay exfoliation, electrospinning and diameter of PMMA/clay nanocomposite fibers

We examine the influence of tethering chemistry of cationic surfactants on exfoliation of montmorillonite (MMT) clay dispersed in methyl methacrylate (MMA) followed by in-situ polymerization to form poly(methyl methacrylate) (PMMA) nanocomposites, the effect of exfoliation and clay loading on the rheology of polymer/clay dispersions in dimethyl formamide, and the diameters of nanocomposite fibers formed from these dispersions by electrospinning. Incorporation of an additional reactive tethering group of methacryl functionality significantly improves the intercalation and exfoliation of clays in both in-situ polymerized PMMA nanocomposites and the corresponding electrospun fibers. The proper surfactant chemistry also increases the dispersion stability, extensional viscosity, extent of strain hardening and thus the electrospinnablity of the nanocomposite dispersions, especially at low nanocomposite concentrations. The degree of the enhancement in electrospinnability by clays with proper tethering chemistry is at least the same as or greater than that obtained with three times higher loading level of clay particles without proper tethering chemistry in the nanocomposites. These results suggest a new strategy to produce smaller diameter fibers from very dilute polymer solutions, which are otherwise not electrospinnable, by incorporating a small amount of well-exfoliated clays.

Influence of structure of organic modifiers and polyurethane on the clay dispersion in nanocomposites via in situ polymerization

AbstractInfluence of polyurethane (PU) structure on the nature of PU/clay nanocomposite was studied using varying amounts of trimethylol propane (TMP) as branching agent. The effect of hydroxyl groups in the modifier of organoclays on the structure of PU/clay nanocomposites was studied. Nanocomposites were characterized using wide‐angle X‐ray diffraction measurements (WAXD) and transmission electron microscopy (TEM). The results show that formation of completely exfoliated and well dispersed polyurethane/clay nanocomposites via in situ polymerization, is facilitated by the presence of tethering groups on the clay surface and an ability to form branched and crosslinked structures. Incorporation of long alkyl chains in addition to tethering hydroxyl groups in the modifier structure of the clay did not significantly improve the compatibility of linear PU with the clay. Intercalated thermoplastic polyurethane/clay nanocomposites, prepared using poly(caprolactone diol) as soft segment and isophorone diisocyanate and 1,4‐butanediol as hard segments show increase in storage tensile moduli at temperatures before glass transition temperature when functional groups capable of chemically reacting with the growing polymer chains are present in the clay modifier. This is indicative of improved interaction of the polymer with the clay surface when the modifier has larger number of hydroxyl groups. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

Mechanism of Photolytic Decomposition of N-Halamine Antimicrobial Siloxane Coatings

Generally, antimicrobial N-halamine siloxane coatings can be rehalogenated repetitively upon loss of their biocidal efficacies, a marked advantage over coatings containing other antimicrobial materials. However, the N-halamine materials tend to slowly decompose upon exposure to ultraviolet irradiation as in direct sunlight. In this work the mechanism of photolytic decomposition for the N-halamine siloxanes has been studied using spectroscopic and theoretical methods. It was found that the N-chlorinated coatings slowly decomposed upon UVA irradiation, whereas the unhalogenated coatings did not. Model compound evidence in this work suggests that upon UVA irradiation, the N-Cl bond dissociates homolytically, followed by a Cl radical migration to the alkyl side chain connected to the siloxane tethering group. An alpha and/or beta scission then occurs causing partial loss of the biocidal moiety from the surface of the coated material, thus precluding complete rechlorination. NMR, FTIR, GCMS, and computations at the DFT (U)B3LYP/6-311++G(2d,p) level of theory have been employed in reaching this conclusion.

SERS detection of indirect viral DNA capture using colloidal gold and methylene blue as a Raman label

An indirect capture model assay using colloidal Au nanoparticles is demonstrated for surface enhanced Raman scattering (SERS) spectroscopy detection of DNA. The sequence targeted for capture was derived from the West Nile Virus (WNV) RNA genome and selected on the basis of exhibiting minimal secondary structure formation. Upon incubation with colloidal Au, hybridization complexes containing the WNV target sequence, a complementary capture oligonucleotide conjugated to a strong tethering group and a complementary reporter oligonucleotide conjugated to methylene blue (MB), a Raman label, anchors the resultant ternary complex to Au nanoparticles and positions MB within the required sensing distance for SERS enhancement. The subsequent elicitation of surface enhanced plasmon resonance by laser excitation provides a spectral peak signature profile that is capture-specific and characteristic of the Raman spectrum for MB. Detection sensitivity is in the submicromolar range and was shown to be highest for thiol, and less so for amino, modifications at the 5′ terminus of the capture oligonucleotide. Finally, using Quartz Crystal Microbalance-Dissipation as a tool for modeling ternary complex binding to Au surfaces, quantitative measurements of surface mass coverage on Au plated sensor crystals established a positive correlation between levels of ternary complex adsorption and their correspondent levels of SERS signal intensification. Adapted to a compact Raman spectrometer, which is designed for analyte detection in capillary tubes, this assay provides a rapid, mobile and cost effective alternative to expensive spectroscopic instrumentation, which is often restricted to analytical laboratories.

Preparation of a new crown ether-based chiral stationary phase containing thioester linkage for the liquid chromatographic separation of enantiomers

A new chiral stationary phase (CSP) containing thioester linkages was prepared by bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to mercaptopropylsilica gel. The chiral recognition ability of the new CSP was found to be greater than that of the previously reported CSP containing amide linkages in the resolution of the various α-amino acids that were tested, except for that of Met, Ser and Thr. In the resolution of racemic amines and amino alcohols, the new CSP was always better than the one containing amide linkages in terms of the separation factors ( α) and the resolutions ( R S). Given the identical elution orders on the two CSPs, it was concluded that the chiral recognition mechanism is not affected by the change of the linkage type. In addition, the new CSP was found to be quite stable under the acidic mobile phase conditions that were utilized, indicating that the thioester linkage is useful as a tethering group.

Molecular Dynamics Simulation of Transmembrane Helix Dissociation

In a typical single-molecule force spectroscopy (SMFS) experiment with a transmembrane protein (TM), a protein surface loop or tail is grabbed by the atomic force microscope cantilever and helices unwind as the TM is pulled in a direction perpendicular to the plane of the membrane. This process is unrelated to the insertion of helical segments from the translocon into the bilayer in a direction parallel to the plane of the membrane. We now use molecular dynamics simulations (MD) to consider a SMFS experiment in which TMs are pulled apart in the plane of the lipid bilayer, so that the helix-helix interactions can be studied. Parallel-versus perpendicular-pulling energy. We calculated the work of separating the helices of the glycophorin A dimer in a sodium dodecyl sulfate (SDS) micelle. Pulling perpendicular to the membrane plane requires about four times the work as parallel pulling to separate the helices. Point pulling. Using MD, we pulled Ala 82 of glycophorin A in a direction parallel to the membrane plane. For 150 ps, the two helices separated smoothly, essentially as rigid rods. However, both Ile 76 side chains remain in van der Waals contact, locking the dimer together. Further pulling unfolds the backbone around Ala 82, until the Ile 76 groups separate after 600 ps. Similar results were observed for a two-helix fragment of bacteriorhodopsin in an SDS micelle. Pulling on Ile 52, the helices separate smoothly for 150 ps. However, during this motion, Leu 13 and Leu 61 remain locked in van der Waals contact. We conclude that SMFS of helical membrane proteins pulled in the direction of the membrane plane would provide valuable information about the interactions that stabilize integral membrane proteins. Experimental implementation will require development of tethering groups compatible with bicelles.

Tethered indoles as functionalizable ligands for the estrogen receptor

To create ligands for the estrogen receptor that contain pendant groups for tethering to a poly(amido)amine (PAMAM) dendrimer, we have explored a class of N-substituted 2-phenyl indoles. Attachment of tethers of different length and chemical nature to this non-steroidal indole scaffold gave high affinity ligand-tether conjugates that can be easily functionalized. To further explore the utility of this system, an indole-conjugated dendrimer was prepared and evaluated as an estrogen receptor ligand.

Further ‘tethered’ Ru(II) catalysts for asymmetric transfer hydrogenation (ATH) of ketones; the use of a benzylic linker and a cyclohexyldiamine ligand

The synthesis and characterisation of two new Ru(II) catalysts for the asymmetric transfer hydrogenation (ATH) of ketones is described. In the case of 4, the novelty lies in the use of a benzyl tethering group between the asymmetric ligand part (TsDPEN) and the η 6-arene ring, which increases the complex rigidity. For 5, the use of a cyclohexyldiamine as a chiral ligand is described for the first time. In the ATH of ketones in formic acid/triethylamine, alcohols with ees of up to 97% were formed.

Optimizing the Photocurrent Efficiency of Dye-Sensitized Solar Cells through the Controlled Aggregation of Chalcogenoxanthylium Dyes on Nanocrystalline Titania Films

Chalcogenoxanthylium dyes were characterized as sensitizers of nanocrystalline titania in dye-sensitized solar cells (DSSCs). Four series of dyes were characterized: 2,7-bis(dimethylamino)-9-(2-thienyl-5-carboxy)chalcogenoxanthylium dyes (1-E, where E = O, S, Se); 2,7-bis(dimethylamino)-9-(3-thienyl-2-carboxy)chalcogenoxanthylium dyes (2-E, where E = S, Se); a 2,7-bis(dimethylamino)-9-(2-thienyl)selenoxanthylium dye (3-Se); 4-Se, a constrained analog of 1-Se. The orientation and aggregation state of the dyes were controlled by varying the position of the surface-attachment group relative to the xanthylium core. Series 1 dyes and 4-Se underwent H-aggregation on titania surfaces, whereas series 2 dyes adsorbed in amorphous monolayers. (3-Se did not adsorb appreciably to titania films, due to the lack of a tethering group.) The H-aggregated dyes exhibited broader absorption bands, increased light-harvesting efficiencies, and improved photoelectrochemical performance compared to the dyes which adsorbed in amo...

ChemInform Abstract: Synthetic Application of Intramolecular Cyanoboration on the Basis of Removal and Conversion of a Tethering Group by Palladium‐Catalyzed Retro‐Allylation.

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Designing Stable Redox-Active Surfaces: Chemical Attachment of an Osmium Complex to Glassy Carbon Electrodes Prefunctionalized by Electrochemical Reduction of an In Situ-Generated Aryldiazonium Cation

The production of stable redox-active layers on electrode surfaces can lead to improvements in electronic device design. Enhanced stability can be achieved by pretreatment of electrode surfaces to provide surface chemical functional groups for covalent tethering of redox complexes. Herein, we describe pretreatment of glassy carbon electrodes to provide surface carboxylic acid groups by electro-reduction of an in situ-generated aryl diazonium salt from 3-(4-aminophenyl)propionic acid. This surface layer is characterized by attenuated total reflection infrared spectroscopy, atomic force microscopy, and electrochemical blocking studies. The surface carboxylic acid generated is then used to tether an osmium complex, [Os(2,2'-bipyridyl)2(4-aminomethylpyridine)Cl]PF6, to provide a covalently bound redox-active monolayer, E(0) ' of 0.29 V (vs Ag/AgCl in phosphate buffer, pH 7.4), on the pretreated glassy carbon electrode. The layer proves stable to pH, temperature, and storage conditions, retaining electroactivity for at least 6 months.

Synthetic Application of Intramolecular Cyanoboration on the Basis of Removal and Conversion of a Tethering Group by Palladium-Catalyzed Retro-Allylation

A new synthetic strategy, involving utilization of a tethered intramolecular reaction with a removable tether, was demonstrated by the intramolecular cyanoboration-retro-allylation sequence.

Resolution of β‐amino acids on a high performance liquid chromatographic doubly tethered chiral stationary phase containing N–CH3 amide linkage based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid

A doubly tethered chiral stationary phase (CSP) containing N-CH(3 )amide linkage based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was applied to the resolution of various beta-amino acids. The chiral recognition behaviors for the resolution of beta-amino acids on the doubly tethered CSP were consistent with those on the corresponding singly tethered CSP while the chiral recognition ability of the doubly tethered CSP was generally greater in terms of both the separation (alpha) and the resolution factors (R(S)) than that of the corresponding singly tethered CSP. From these results, it was concluded that attaching the second tethering group to silica gel through a carbon atom of the first tethering group of the CSP improves the chiral recognition ability for the resolution of beta-amino acids without any change in the chiral recognition mode. The retention factors (k(1)) on the doubly tethered CSP were larger than those on the corresponding singly tethered CSP and these retention factors were found to be controllable with the variation of the type and the content of the organic and/or acidic modifier in the aqueous mobile phase without significant change in the separation and the resolution factors.

An NMR Chiral Solvating Agent for the Chiral Recognition of the Two Enantiomers of N-(3,5-Dinitrobenzoyl)-α-amino Acids

Two enantiomers of chiral drugs often show different pharmaceutical activities. Consequently, the study on the stereochemical dependence of the pharmacokinetics and the pharmaceutical activities of chiral drugs is important. Determination of the enantiomeric composition of chiral drugs is, therefore, essential. Enantiomeric composition of chiral compounds has been determined most widely by highperformance liquid chromatographic chiral separations on chiral stationary phases (CSPs). Another important method of determining the enantiomeric composition of chiral compounds is the NMR techniques utilizing chiral solvating agent (CSA). The two transient diastereomeric adducts formed between a CSA and each of the two enantiomers of chiral analytes might induce anisochronous NMR resonances and consequently the enantiomeric composition of chiral analytes can be easily assessed from the NMR spectrum. Most CSAs, which have been developed and successfully utilized in the determination of the enantiomeric composition of chiral compounds by NMR spectroscopy, contain aromatic functional group(s) to induce magnetic anisotropic influence and to invoke π-π interaction with analytes. Derivatives of optically active trans-1,2-diaminocyclohexane has also been utilized as NMR CSAs and these CSAs have also been designed to contain aromatic functional group(s) in order to induce magnetic anisotropic influence and to invoke π-π interaction with analytes. Consequently, CSAs derived from optically active trans-1,2diaminocyclohexane have been utilized for the determination of enantiomeric composition of chiral compounds containing aromatic functional group(s). For example, CSA 1 and CSA 2 derived from (1R,2R)-1,2-diaminocyclohexane shown in Figure 1 have been successfully applied to the determination of enantiomeric composition of chiral carboxylic acids by NMR spectroscopy. Recently, we found that a liquid chromatographic chiral stationary phase (CSP 3, Figure 1) derived from optically active (1S,2S)-1,2-diaminocyclohexane is quite successful for the resolution of N-(3,5-dinitrobenzoyl)-α-amino acids even though the CSP does not contain any aromatic functional group, but contains only a simple primary amino group and a ureide tethering group. From these results, we inferred that CSA 4, which has the same structure as the chiral selector of CSP 3, might show nonequivalences for the two enantiomers of N-(3,5-dinitrobenzoyl)-α-amino acids by NMR spectroscopy. CSA 4 was prepared simply by treating (1S,2S)-1,2diaminocyclohexane with ethyl isocyanate. CSA 4 thus prepared was used to see the H NMR chemical shift nonequivalence of the two enantiomers of N-(3,5-dinitrobenzoyl)phenylglycine. As shown in Figure 2, the chemical shift nonequivalence of the two enantiomers of N-(3,5-dinitrobenzoyl)phenylglycine was observed at the NMR peaks corresponding to the protons on the 3,5-dinitrophenyl ring (denoted by a in Figure 1). In addition the chemical shift nonequivalence of the two enantiomers of N-(3,5-dinitrobenzoyl)phenylglycine was observed to be greatest when the ratio of CSA 4 and N-(3,5-dinitrobenzoyl)phenylglycine is 2:1 at ambient temperature (22 ± 0.5 C). The H NMR experimental results for the chemical shift nonequivalences of the two enantiomers of five N-(3,5dinitrobenzoyl)-α-amino acids in the presence of two equivalents of CSA 4 are summarized in Table 1. As shown

F‐Block N‐Heterocyclic Carbene Complexes

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Preparation and application of a new doubly tethered chiral stationary phase containing NCH3 amide linkage based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid

A new doubly tethered chiral stationary phase (CSP 5) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was developed by attaching the second tethering group to silica gel through a carbon atom of the first tethering group of the corresponding singly tethered CSP (CSP 2) containing an N-CH3 tertiary amide linkage, which was previously developed in our laboratory, in order to enhance the CSP stability without the loss of chiral recognition efficiency. The new CSP was quite effective in the resolution of various racemic alpha-amino acids, amines, and amino alcohols, and the chiral recognition efficiency of the new CSP was even greater than that of the corresponding singly tethered CSP especially in terms of the resolution factors (RS). The stability of the new CSP was greater than that of the corresponding singly tethered CSP. The chromatographic resolution behaviors of the new CSP were generally consistent with those of the corresponding singly tethered CSP.