Monomeric Ligands Research Articles

Preparation and characterization of bonded silica hydride intermediate from triethoxysilane and dimethylmethoxysilane using supercritical carbon dioxide and dioxane as reaction medium

This research examines bonding methodology, surface coverage and silanol conversion efficiencies on the preparation of silica hydride (SiH) intermediate from triethoxysilane (TES) and dimethylmethoxysilane (DMMS) using sc-CO2 and dioxane as reaction solvent. Under sc-CO2 reaction conditions (at temperature and pressure of 100°C, 414bar, respectively and 3h reaction time), the surface coverages of SiH (evaluated from %C obtained from elemental analysis) prepared with DMMS (3.39μmol/m2) and TES (4.46μmol/m2) increased by 2- and 4-folds respectively, when compared to reaction performed in dioxane (2.66μmol/m2, SiH, DMMS and 0.69μmol/m2, SiH, TES). The relatively higher surface coverage of SiH from TES over DMMS generated in sc-CO2 is due to the inherent trialkoxy moiety of the TES that favours siloxane crosslinkage, forming polymeric surface attachments to yield a higher ligand density than the monomeric DMMS ligand. A conversion efficiency of ∼84.4% of SiH prepared from TES in sc-CO2 estimated from 29Si CP/MAS NMR analysis is comparable to TES silanization in dioxane or toluene. Moreover, silica hydride (SiH) conversion efficiency of ca. 42.4% achieved for the hydride intermediate prepared from DMMS in sc-CO2 is more superior to 33.3% efficiency obtained in dioxane. The differences in conversion efficiencies is attributed to the ability of sc-CO2 being able to access silica pores that are inaccessible in organic solvents. Bonded silica hydride from TES, DMMS prepared in sc-CO2 were characterized using elemental analysis, thermogravimetric analysis (TGA), BET surface area, Fourier transform infrared (FI-IR) and solid state NMR spectroscopy. Silica hydride technology/chemical functionalization of silica in sc-CO2 avoid extended purification steps (i.e. filtration and washing), generation of waste organic solvent and the need of costly or energy consuming drying processing with improved modification efficiency.

Influence of Spacer–Receptor Interactions on the Stability of Bivalent Ligand–Receptor Complexes

Experiments show that a ligand-receptor complex formed by binding a bivalent ligand (D) in which the two ligating units are joined covalently by a flexible polymeric spacer (S) can be orders of magnitude more stable than the corresponding complex formed with monomeric ligands. Although molecular models rationalizing this "enhancement effect" have been proffered, they ignore spacer-receptor (S-R) interactions, which can substantially influence the relative stability of complexes. Here, the results of a computational study designed to assess the impact of S-R interactions in the prototypic bivalent complex are presented and compared to results of experiments. The S-R interactions mimicking general features of biological systems are modeled by contoured R surfaces with hills (or depressions) at the binding sites. In the fictitious limit of vanishing S-R interactions, the enhancement is pronounced, as observed in experiments. For strictly repulsive S-R interactions (hard R surface), the enhancement vanishes, or even reverses. This is particularly the case if the R surface is convex (i.e., rising between the binding sites), while the enhancement is only moderately reduced if the R surface is concave. Alternatively, a weak S-R attraction close to the R surface can increase the enhancement. It is concluded that large enhancement should be observed only if both features are present: a concave R surface plus a weak S-R attraction. The latter occurs for spacer material such as polyethylene glycol (PEG), which is weakly hydrophobic and thus attracted by protein surfaces. It is shown that the enhancement of bivalent binding can be characterized by a single key parameter, which may also provide guidelines for the design of multivalent complexes with large enhancement effect.

An efficient and recyclable dendritic catalyst able to dramatically decrease palladium leaching in Suzuki couplings

A series of novel monomeric and dendritic thiazolyl phosphines (generations 1 and 3) was prepared and the activity these ligands conferred to palladium in Suzuki couplings was evaluated. The heteroaryl ligands were revealed to be very efficient and were able to perform the reactions in mild conditions. Besides, their efficiency was compared to that of the corresponding triphenylphosphines. Moreover, the possibility to reuse both families of dendritic ligands was explored and the thiazolyl phosphine-based catalytic systems could be successfully recycled for five consecutive reactions without loss of activity, contrary to their triphenylphosphine counterparts. Remarkably, the palladium leaching was found to be dramatically reduced by using the dendrimer-supported thiazolylphosphines instead of the monomeric ligand and only trace amounts of metal (<0.55 ppm) could be found in the coupling product before any purification.

Regulation of integrin affinity on cell surfaces

Lymphocyte activation triggers adhesiveness of lymphocyte function-associated antigen-1 (LFA-1; integrin α(L)β(2)) for intercellular adhesion molecules (ICAMs) on endothelia or antigen-presenting cells. Whether the activation signal, after transmission through multiple domains to the ligand-binding αI domain, results in affinity changes for ligand has been hotly debated. Here, we present the first comprehensive measurements of LFA-1 affinities on T lymphocytes for ICAM-1 under a broad array of activating conditions. Only a modest increase in affinity for soluble ligand was detected after activation by chemokine or T-cell receptor ligation, conditions that primed LFA-1 and robustly induced lymphocyte adhesion to ICAM-1 substrates. By stabilizing well-defined LFA-1 conformations by Fab, we demonstrate the absolute requirement of the open LFA-1 headpiece for adhesiveness and high affinity. Interaction of primed LFA-1 with immobilized but not soluble ICAM-1 triggers energy-dependent affinity maturation of LFA-1 to an adhesive, high affinity state. Our results lend support to the traction or translational motion dependence of integrin activation.

Cell-Specific Targeting by Heterobivalent Ligands

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.

Recombinant monomeric CD40 ligand for delivering polymer particles to dendritic cells

Dendritic cells (DCs) are considered the most efficient antigen-presenting cells and are therefore ideal targets for in vivo delivery of antigen for vaccines. We are investigating the strategy of using CD40 ligand (CD40L) as a targeting moiety because this protein has the potential to not only target DCs, but also stimulate cell maturation, leading to more potent immune responses. We have shown that a recombinant, monomeric CD40 ligand fusion protein conjugated to polystyrene micro- and nanoparticles led to significantly enhanced uptake by DCs in vitro. This enhancement was observed for particles of both sizes and in both a murine DC cell line and primary DCs. The uptake appeared to be specifically mediated by CD40L binding to CD40 expressed on DCs. Enhanced uptake of nanoparticles in draining lymph nodes of mice was not observed, however, 48 hours after subcutaneous injection. These findings suggest that CD40 ligand may be a potentially useful targeting moiety for delivery of particulate vaccines to DCs, and that further optimization of both CD40L and the polymer carriers is necessary to achieve efficacy in vivo.

CXCL12 Oligomerization Dictates Divergent Signaling and Migratory Responses in Intestinal Epithelial Cells

Chemotactic migration classically exhibits a bell-shaped response over a narrow concentration range. Previous studies have shown the chemokine, CXCL12, exists in a monomer-dimer equilibrium with heightened concentrations resulting in preferential accumulation of homodimeric chemokine. Little is understood how CXCL12 oligomerization induces distinct cellular effects through the same cognent receptor, CXCR4. The objective of these studies is to determine the effects of monomeric and dimeric CXCL12 on colorectal cancer cell migration. HCT116 and HT29 human colonic carcinoma cells were utilized to examine both chemotactic and restitutive migration respectively. Previously engineered preferential monomeric CXCL12(H25R), locked dimer CXCL12(2), and wild-type CXCL12(WT) were utilized in our analyses. Transwell inserts and aspiration wounded monolayers demonstrated that CXCL12(WT) and CXCL12(H25R) induced a strong chemotactic and restitutive migration response. In contrast, CXCL12(2) dimer was unable to elicit cellular migration. In agreement with those data, filamentous actin formation was induced by CXCL12(WT) and CXCL12(H25R) but not dimer. Intracellular calcium flux, a classical hallmark of G-protein coupled receptor signaling, was elicited equally by each oligomeric variant. This response was blocked with addition of CXCR4-specific inhibitors indicating each of the variants engaged the same cognate receptor. Similarly, CXCL12(2) dose-dependently inhibited CXCR4, but not CXCR1, mediated chemotaxis. The lack of migration was subsequently shown using nuclear magnetic resonance analyses to reflect distinct interactions of the monomeric and dimeric ligand for CXCR4 homotypic receptor dimers. These latter data support the notion that double occupancy of dimeric receptor limits migration while single occupancy of the same receptors evokes strong migration responses. Together these data underlie the importance of CXCL12 oligomerization in dictating epithelial migration and suggest the CXCL12 dimer may serve as a potential inhibitor of colorectal cancer metastasis.

Regulation of the Gene Encoding the Intestinal Bile Acid Transporter ASBT by CDX Transcription Factors

Chemotactic migration classically exhibits a bell-shaped response over a narrow concentration range. Previous studies have shown the chemokine, CXCL12, exists in a monomer-dimer equilibrium with heightened concentrations resulting in preferential accumulation of homodimeric chemokine. Little is understood how CXCL12 oligomerization induces distinct cellular effects through the same cognent receptor, CXCR4. The objective of these studies is to determine the effects of monomeric and dimeric CXCL12 on colorectal cancer cell migration. HCT116 and HT29 human colonic carcinoma cells were utilized to examine both chemotactic and restitutive migration respectively. Previously engineered preferential monomeric CXCL12(H25R), locked dimer CXCL12(2), and wild-type CXCL12(WT) were utilized in our analyses. Transwell inserts and aspiration wounded monolayers demonstrated that CXCL12(WT) and CXCL12(H25R) induced a strong chemotactic and restitutive migration response. In contrast, CXCL12(2) dimer was unable to elicit cellular migration. In agreement with those data, filamentous actin formation was induced by CXCL12(WT) and CXCL12(H25R) but not dimer. Intracellular calcium flux, a classical hallmark of G-protein coupled receptor signaling, was elicited equally by each oligomeric variant. This response was blocked with addition of CXCR4-specific inhibitors indicating each of the variants engaged the same cognate receptor. Similarly, CXCL12(2) dose-dependently inhibited CXCR4, but not CXCR1, mediated chemotaxis. The lack of migration was subsequently shown using nuclear magnetic resonance analyses to reflect distinct interactions of the monomeric and dimeric ligand for CXCR4 homotypic receptor dimers. These latter data support the notion that double occupancy of dimeric receptor limits migration while single occupancy of the same receptors evokes strong migration responses. Together these data underlie the importance of CXCL12 oligomerization in dictating epithelial migration and suggest the CXCL12 dimer may serve as a potential inhibitor of colorectal cancer metastasis.

Dependence of TCR triggering on ligand three-dimensional freedom (109.19)

Abstract T lymphocytes (T cells) recognize antigens through the binding of T cell receptors (TCRs) to peptide ligands presented by MHC molecules (pMHCs) on antigen presenting cells (APCs). The mechanism of how pMHC-TCR binding triggers a signal from TCR is still unclear. One unique aspect of TCR triggering is that surface-anchored pMHC triggers TCR highly efficiently in a two dimensional setting, while soluble monomeric pMHC ligands with full three dimensional freedom have no activity. Here, we investigate the dependence of TCR triggering on the 3D freedom of pMHC ligands using highly flexible hetero-bifunctional poly(ethylene glycol) (PEG) polymer linkers. Fluorescence resonance energy transfer assay showed increased 3D freedom of ligands with increased linker length. As measured by IL2 production, T cells responded equally well to pMHCs anchored on surfaces with 0.56 nm, 3.1 nm and 12.7 nm linkers. T cell response started to decrease, however, when the linker was 22 nm long, and decreased further for the 32 nm linker. pMHCs anchored with the 48 nm linker still stimulated T cells IL2 production, although with only about one tenth the potency. Therefore, pMHC ligands with 3D freedom of up to 22 nm are well tolerated by TCR, but larger 3D freedom gradually reduces potency. Our findings argue against the kinetic segregation of TCR triggering but are in support of receptor deformation, a model of TCR triggering that tolerates increased ligand 3D freedom.

Metal-containing polyurethanes from tetradentate Schiff bases: synthesis, characterization, and biocidal activities

N,N′-bis(salicylidene)thiosemicarbazide Schiff base has been synthesized by the reaction of thiosemicarbazide with salicylaldehyde and then reacted with formaldehyde to generate phenolic groups, resulting in the formation of Schiff-base monomeric ligand. It was further incorporated with transition metals, Mn+2, Co+2, Ni+2, Cu+2, and Zn+2, to form Schiff-base metal complex, which was then polymerized with toluene 2,4-diisocyanate to form metal-chelated polyurethanes. Monomeric ligand, its metal complexes, and its metal polychelates were characterized and compared by elemental analysis, FT-IR, 1H NMR, thermal, and biocidal activities to evaluate the enhancement in physical and chemical properties on coordination with metal and on polymerization. SEM images of ligand and polymer metal complexes showed changes in surface morphology, while electronic spectra of polymer metal complexes were used to predict the geometry. Antimicrobial activities were determined by using agar-diffusion method with Staphylococcus aureus, Escherichia coli, Bacillus subtilis (bacteria), Aspergillus niger, Candida albicans, and Aspergillus flavus (yeast). The polymeric ligand had varied antibacterial and antifungal activities, enhanced after chelation and polymerization. Comparative results show that coordination of metal to the ligand enhances its physical and chemical properties which were meliorated on polymerization.

Decoy Strategies: The Structure of TL1A:DcR3 Complex

Decoy Receptor 3 (DcR3), a secreted member of the Tumor Necrosis Factor (TNF) receptor superfamily, neutralizes three different TNF ligands: FasL, LIGHT, and TL1A. Each of these ligands engages unique signaling receptors which direct distinct and critical immune responses. We report the crystal structures of the unliganded DcR3 ectodomain and its complex with TL1A, as well as complementary mutagenesis and biochemical studies. These analyses demonstrate that DcR3 interacts with invariant backbone and side-chain atoms in the membrane-proximal half of TL1A which supports recognition of its three distinct TNF ligands. Additional features serve as antideterminants that preclude interaction with other members of the TNF superfamily. This mode of interaction is unique among characterized TNF:TNFR family members and provides a mechanistic basis for the broadened specificity required to support the decoy function of DcR3, as well as for the rational manipulation of specificity and affinity of DcR3 and its ligands.

New hydroxypyridinone-functionalized sepharoses as sorbing agents for hard metal ions

Two new polymeric matrices functionalized with 3-hydroxy-4-pyridinone chelating units (HP-NH-SEPH and HP-CNH-SEPH) have been prepared and studied for their chelating ability towards a set of metal ions (e.g. Fe(III), Al(III), and Th(IV)). Both matrices demonstrated excellent ability to complex these metal ions, but HP-NH-SEPH evidenced higher chelating capacity than HP-CNH-SEPH. The corresponding metal-complex gels presented high stability in the pH range 3–7, and their chelating capacity followed the order, Fe(III)≈Th(IV)>Al(III), in agreement with previously reported thermodynamics of the corresponding monomeric ligand–metal complexes in aqueous solution. These functionalized supports also showed capacity to be regenerated and reused. Thus, there are good perspectives for potential environmental and medical applications of these new metal sorbents.

A comparison study between polymeric ligand and monomeric ligand for oligopeptide adsorption

This work aimed to compare two types of affinity ligands, i.e. polymeric and monomeric ligands, by investigating their adsorption affinity, capacity and selectivity to oligopeptide. The peptide NH 2-VVRGCTWW-COOH (VW-8) was chosen as the target adsorbate, while histidine (His), aspartic acid (Asp), and leucine (Leu) were selected as the ligands, respectively. For each kind of ligand, both monomeric (M) and polymeric (P) forms were introduced onto the Sepharose matrix respectively to obtain the corresponding adsorbents. Both affinity tests using isothermal titration calorimetry (ITC) and adsorption capacities using static adsorption experiments indicated that the adsorbents with polymeric ligands (MX-P) exhibited better adsorption ability for VW-8 than the adsorbents with monomeric ligands (MX-M). In particular, the MX-PHis exhibited its affinity constant of 2.39 × 10 6 M −1 and its adsorption capacity of 77.4 mg/g for VW-8, which was approximately 8–10 times higher than that of MX-MHis. Such distinct adsorption abilities between polymeric and monomeric ligands were interpreted based on nuclear magnetic resonance (NMR) and ITC data, and the results indicated that such better characters of polymeric ligands were ascribed to their good flexibility which facilitated the cooperative effects as well as the accessibility of ligands to the peptide. Additionally, the selective adsorption experiments indicated that all the adsorbents with polymeric ligands exhibited good selectivity to the peptide VW-8.

Systematic mutation and thermodynamic analysis of central tyrosine pairs in polyspecific NKG2D receptor interactions

The homodimeric, activating natural killer cell receptor NKG2D interacts with multiple monomeric ligands polyspecifically, yet without central conformational flexibility. Crystal structures of multiple NKG2D–ligand interactions have identified the NKG2D tyrosine pair Tyr 152 and Tyr 199 as forming multiple specific but diverse interactions with MICA and related proteins. Here we systematically altered each tyrosine to tryptophan, phenylalanine, isoleucine, leucine, valine, serine, and alanine to measure the effect of mutation on affinity and thermodynamics for binding a range of similar ligands: MICA, the higher-affinity ligand MICB, and MICdesign, a high-affinity version of MICA that shares all NKG2D contact residues with MICA. Affinity and residue size were related: tryptophan could often substitute for tyrosine without loss of affinity; loss of the tyrosine hydroxyl through mutation to phenylalanine was tolerated more at position 152 than 199; and the smallest residues coincide with lowest affinities in general. NKG2D mutant van’t Hoff binding thermodynamics generally show that substitution of other residues for tyrosine causes a moderate positive or flat van’t Hoff slope consistent with moderate loss of binding enthalpy. One set of NKG2D mutations caused MICA to adopt a positive van’t Hoff slope corresponding to absorption of heat, and another set caused MICB to adopt a negative slope of greater heat release than wild-type. MICdesign shared one example of the first set with MICA and one of the second set with MICB. When the NKG2D mutation affinities were arranged according to change in nonpolar surface area and compared to results from specific antibody–antigen and protein–peptide interactions, it was found that hydrophobic surface loss in NKG2D reduced binding affinity less than reported in the other contexts. The hydrophobic effect at the center of the NKG2D binding appears more similar to that at the periphery of an antibody–antigen binding site than at its center. Therefore the polyspecific NKG2D binding site is more tolerant of structural alteration in general than either an antibody–antigen or protein–peptide binding site, and this tolerance may adapt NKG2D to a broad range of protein surfaces with micromolar affinity.

Physical Basis behind Achondroplasia, the Most Common Form of Human Dwarfism

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that plays an important role in long bone development. The G380R mutation in FGFR3 transmembrane domain is known as the genetic cause for achondroplasia, the most common form of human dwarfism. Despite many studies, there is no consensus about the exact mechanism underlying the pathology. To gain further understanding into the physical basis behind the disorder, here we measure the activation of wild-type and mutant FGFR3 in mammalian cells using Western blots, and we analyze the activation within the frame of a physical-chemical model describing dimerization, ligand binding, and phosphorylation probabilities within the dimers. The data analysis presented here suggests that the mutation does not increase FGFR3 dimerization, as proposed previously. Instead, FGFR3 activity in achondroplasia is increased due to increased probability for phosphorylation of the unliganded mutant dimers. This finding has implications for the design of targeted molecular treatments for achondroplasia.

Macrocyclic Gd3+ Chelates Attached to a Silsesquioxane Core as Potential Magnetic Resonance Imaging Contrast Agents: Synthesis, Physicochemical Characterization, and Stability Studies

Two macrocyclic ligands, 1,4,7,10-tetraazacyclododecane-1-glutaric-4,7,10-triacetic acid (H(5)DOTAGA) and the novel 1,4,7,10-tetraazacyclododecane-1-(4-(carboxymethyl)benzoic)-4,7,10-triacetic acid (H(5)DOTABA), were prepared and their lanthanide complexes (Ln = Gd(3+), Y(3+)) attached to an amino-functionalized T(8)-silsesquioxane. The novel compounds Gadoxane G (GG) and Gadoxane B (GB) possess eight monohydrated lanthanide complexes each, as evidenced by multinuclear ((1)H, (13)C, (29)Si) NMR spectroscopy and high resolution mass spectrometry (HR-MS). Pulsed-field gradient spin echo (PGSE) diffusion (1)H NMR measurements revealed hydrodynamic radii of 1.44 nm and global rotational correlation times of about 3.35 ns for both compounds. With regard to potential MRI contrast agent applications, a variable-temperature (17)O NMR and (1)H nuclear magnetic relaxation dispersion (NMRD) study was carried out on aqueous solutions of the gadolinium(III) complexes of the Gadoxanes and the corresponding monomeric ligands to yield relevant physicochemical properties. The water exchange rates of the inner-sphere water molecules are all very similar (k(ex)(298) between (5.3 +/- 0.5) x 10(6) s(-1) and (5.9 +/- 0.3) x 10(6) s(-1)) and only slightly higher than that reported for the gadolinium(III) complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (H(4)DOTA) (k(ex)(298) = 4.1 x 10(6) s(-1)). Despite their almost identical size and their similar water exchange rates, GB shows a significantly higher longitudinal relaxivity than GG over nearly the whole range of magnetic fields (e.g., 17.1 mM(-1) s(-1) for GB and 12.1 mM(-1) s(-1) for GG at 20 MHz and 25 degrees C). This difference arises from their different local rotational correlation times (tau(lR)(298) = 240 +/- 10 ps and 380 +/- 20 ps, respectively), because of the higher rigidity of the phenyl ring of GB as compared to the ethylene spacer of GG. A crucial feature of these novel compounds is the lability of the silsesquioxane core in aqueous media. The hydrolysis of the Si-O-Si moieties was investigated by (29)Si NMR and PGSE diffusion (1)H NMR spectroscopy, electrospray ionization mass spectrometry (ESI-MS), as well as by relaxivity measurements. Although frozen aqueous solutions (pH 7.0) of GG and GB can be stored at -28 degrees C for at least 10 months without any decomposition, with increasing temperature and pH the hydrolysis of the silsesquioxane core was observed (e.g., t(1/2) = 15 h at pH 7.4 and 55 min at pH 8.1 for GG at 37 degrees C). No change in relaxivity was detected within the first 3 h, since the hydrolysis of the initial Si-O-Si moieties has no influence on the rotational correlation time. However, the further hydrolysis of the silsesquioxane core leads to smaller fragments and therefore to a decrease in relaxivity.

Peptide-MHC heterodimers show that thymic positive selection requires a more restricted set of self-peptides than negative selection

T cell selection and maturation in the thymus depends on the interactions between T cell receptors (TCRs) and different self-peptide–major histocompatibility complex (pMHC) molecules. We show that the affinity of the OT-I TCR for its endogenous positively selecting ligands, Catnb-H-2Kb and Cappa1-H-2Kb, is significantly lower than for previously reported positively selecting altered peptide ligands. To understand how these extremely weak endogenous ligands produce signals in maturing thymocytes, we generated soluble monomeric and dimeric peptide–H-2Kb ligands. Soluble monomeric ovalbumin (OVA)-Kb molecules elicited no detectable signaling in OT-I thymocytes, whereas heterodimers of OVA-Kb paired with positively selecting or nonselecting endogenous peptides, but not an engineered null peptide, induced deletion. In contrast, dimer-induced positive selection was much more sensitive to the identity of the partner peptide. Catnb-Kb–Catnb-Kb homodimers, but not heterodimers of Catnb-Kb paired with a nonselecting peptide-Kb, induced positive selection, even though both ligands bind the OT-I TCR with detectable affinity. Thus, both positive and negative selection can be driven by dimeric but not monomeric ligands. In addition, positive selection has much more stringent requirements for the partner self-pMHC.

Multivalent dendrimeric and monomeric adenosine agonists attenuate cell death in HL-1 mouse cardiomyocytes expressing the A 3 receptor

Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A 1 and A 2A adenosine receptors (ARs), but not of A 2B and A 3ARs. Activation of the heterologously expressed human A 3AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4 h exposure to H 2O 2 (750 μM) but not in untransfected cells. The A 3 agonist IB-MECA (EC 50 3.8 μM) and the non-selective agonist NECA (EC 50 3.9 μM) protected A 3 AR-transfected cells against H 2O 2 in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendrimeric conjugate of a N 6-chain-functionalized adenosine agonist was synthesized and its mass indicated an average of 60 amide-linked nucleoside moieties out of 256 theoretical attachment sites. It non-selectively activated the A 3AR to inhibit forskolin-stimulated cAMP formation (IC 50 66 nM) and, similarly, protected A 3-transfected HL-1 cells from apoptosis-inducing H 2O 2 with greater potency (IC 50 35 nM) than monomeric nucleosides. Thus, a PAMAM conjugate retained AR binding affinity and displayed greatly enhanced cardioprotective potency.

Palladium Nanoparticles Captured in Microporous Polymers: A Tailor-Made Catalyst for Heterogeneous Carbon Cross-Coupling Reactions

A new strategy based on polymerization-induced phase separation (PIPS) techniques was proposed for fabricating palladium nanoparticles (PdNPs) captured in a microporous network polymer. Pd(OAc)(2) was premixed with a monomer having a poly(amidoamine)-based dendrimer ligand, and subsequently this was thermally polymerized with an excess amount of ethylene glycol dimethacrylate under PIPS conditions. In this system, the formation of PdNPs occurred concurrently with the polymer synthesis in a one-pot process, even with no additional reducing reagent. The resultant microporous polymer was found to have a mesoporosity; the nitrogen sorption analysis gave a specific-surface area of 511 m(2) g(-1), an average pore diameter of 9.9 nm, and a total pore volume of 1.01 mL g(-1). The TEM images of the polymer revealed that the created PdNPs were very small with a diameter of mainly ca. 2.0 nm; the high-resolution images were lattice-resolvable, showing the crystalline nature of the PdNPs (Pd(111) facets). Catalytic performances of the PdNP-containing microporous polymers were investigated for a heterogeneous Suzuki-Miyaura reaction of 4'-bromoacetophenone and phenylboronic acid in water. In the presence of 10(-2) molar equiv of the polymer, the reaction efficiently proceeded at 80 degrees C and gave the desired product, 4-acetylbiphenyl, in >90% yield after 2 h. On the basis of the ICP-AES analysis, the Pd content released into the solution phase was estimated to be only 0.27% of the initial charge. Thereby, this polymer was successfully recovered by simple filtration and reused with only a minimal loss of activity (yield >90% even at the eighth run). When the catalytic reaction was examined with a low amount of the polymer catalyst, the turnover number (TON) reached 8.5 x 10(4) while maintaining a good yield. Finally, the dendrimer template effect of the polymer catalyst was discussed by referring to the catalytic performances of a control polymer prepared with nonintegrated ligand monomers.

Design and Synthesis of Highly Potent and Plasma‐Stable Dimeric Inhibitors of the PSD‐95–NMDA Receptor Interaction

On the double: Dimerization of monomeric peptide ligands towards the PDZ domains of the protein PSD-95 (postsynaptic density 95) leads to potent inhibitors of protein–protein interactions with stability in blood plasma. Optimization of the length of the polyethylene glycol linker results in unprecedented affinity for inhibitors of the PDZ1-2 domain (see picture).