Hydrophilic Ligands Research Articles

Synthesis and physicochemical characterization of new squalenoyl amphiphilic gadolinium complexes as nanoparticle contrast agents

A family of novel amphiphilic gadolinium chelates was successfully obtained by coupling the hydrophilic DOTA ligand [1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecane] to squalenoyl moieties. Thanks to the self-assembling properties of their squalenoyl lipophilic moieties, all these derivatives were able to form, without any adjuvant, micellar or liposome-like supramolecular nanoassemblies, endowed with high relaxivities (r(1) = 15-22 mM(-1) s(-1) at 20 MHz and 37 °C). The remarkably high payloads of Gd(3+) ions reached 10 to 17 wt %. Moreover, one of these derivatives interacted with human serum albumin (HSA) forming mixed micelles, which induced a remarkable increase in relaxivity. Liposome-like structures were obtained when the Gd(3+) complex of DOTA was coupled to two squalene units. These liposomal structures were characterized by a high loading of Gd(3+) (about 74,000 gadolinium ions per particle of 100 nm). The supramolecular architecture of these nano-objects has been investigated by electron microscopy and small-angle X-ray scattering. Squalenoylation of gadolinium derivatives offers a platform to conceive contrast agents (CAs) in mild conditions (no toxic solvents, no surfactants, no energy input). These new amphiphilic gadolinium chelates could also find potential applications in theranostics, by forming mixed systems with other squalenoylated drugs, or to delineate blood vessels owing to the interaction with HSA.

Solvothermal synthesis of InP quantum dots and their enhanced luminescent efficiency by post-synthetic treatments

InP quantum dots (QDs) were solvothermally synthesized by using a greener phosphorus source of P(N(CH 3) 2) 3 instead of highly toxic P(TMS) 3 widely used, and subsequently subjected to a size-sorting processing. While as-grown QDs showed an undetectably low emission intensity, post-synthetic treatments such as photo-etching, photo-radiation, and photo-assisted ZnS shell coating gave rise to a substantial increase in emission efficiency due to the effective removal and passivation of surface states. The emission efficiency of the photo-etched QDs was further enhanced by a consecutive UV photo-radiation, attributable to the photo-oxidation at QD surface. Furthermore, a relatively thick ZnS shell on the surface of InP QDs that were surface-modified with hydrophilic ligands beforehand was photochemically generated in an aqueous solution at room temperature. The resulting InP/ZnS core/shell QDs, emitting from blue to red wavelengths, were more efficient than the above photo-treated InP QDs, and their luminescent properties (emission bandwidth and quantum yield) were comparable to those of InP QDs synthesized with P(TMS) 3. Structural, size, and compositional analyses on InP/ZnS QDs were also conducted to elucidate their core/shell structure.

In vitro antitumour activity of water soluble Cu(I), Ag(I) and Au(I) complexes supported by hydrophilic alkyl phosphine ligands

Hydrophilic, monocationic [M(L)4]PF6 complexes (M=Cu or Ag; L: thp=tris(hydroxymethyl)phosphine, L: PTA=1,3,5-triaza-7-phosphaadamantane, L: thpp=tris(hydroxypropyl)phosphine) were synthesized by ligand exchange reaction starting from [Cu(CH3CN)4]PF6 or AgPF6 precursors at room temperature in the presence of an excess of the relevant phosphine. The related [Au(L)4]PF6 complexes (L=thp, PTA or thpp) were synthesized by metathesis reactions starting from [Au(L)4]Cl at room temperature in the presence of equimolar quantity of TlPF6. The three series of complexes [M(L)4]PF6 were tested as cytotoxic agents against a panel of several human tumour cell lines also including a defined cisplatin resistant cell line. These investigations have been carried out in comparison with the clinically used metallodrug cisplatin and preliminary structure–activity relationships are presented. The best results in terms of in vitro antitumour activity were achieved with metal-thp species and, among the coinage metal complexes, copper derivatives were found to be the most efficient drugs. Preliminary studies concerning the mechanism of action of these [M(L)4]PF6 species pointed to thioredoxin reductase as one of the putative cellular targets of gold and silver complexes and provided evidence that copper derivatives mediated their cytotoxic effect through proteasome inhibition.

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane*

β(2)-adrenergic receptors (β(2)-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β(2)-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β(2)-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β(2)-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β(2)-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β(2)-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals.

Contributors

Limited knowledge of the molecular evolution of deep-sea fish proteomes so far suggests that a few widespread residue substitutions in cytosolic proteins binding hydrophilic ligands contribute to resistance to the effects of high hydrostatic pressure (HP). Structure-function studies with additional protein systems, including membrane bound proteins, are essential to provide a more general picture of adaptation in these extremophiles. We explored molecular features of HP adaptation in proteins binding hydrophobic ligands, either in lipid bilayers (cytochrome P450 1A - CYP1A) or in the cytosol (the aryl hydrocarbon receptor - AHR), and their partners P450 oxidoreductase (POR) and AHR nuclear translocator (ARNT), respectively. Cloning studies identified the full-length coding sequence of AHR, CYP1A and POR, and a partial sequence of ARNT from Coryphaenoides armatus, an abyssal gadiform fish thriving down to 5000 m depth. Inferred protein sequences were aligned with many non-deep-sea homologs to identify unique amino acid substitutions of possible relevance in HP adaptation. Positionally unique substitutions of various physicochemical properties were found in all four proteins, usually at sites of strong-to-absolute residue conservation. Some were in domains deemed important for protein-protein interaction or ligand binding. In addition, some involved removal or addition of beta-branched residues; local modifications of beta-branched residue patterns could be important to HP adaptation. In silico predictions further suggested that some unique substitutions might substantially modulate the flexibility of the polypeptide segment in which they are found. Repetitive motifs unique to the abyssal fish AHR were predicted to be rich in glycosylation sites, suggesting that post-translational changes could be involved in adaptation as well. Recombinant CYP1A and AHR showed functional properties (spectral characteristics, catalytic activity and ligand binding) that demonstrate proper folding at 1 atm, indicating that they could be used as deep-sea fish protein models to further evaluate protein function under pressure. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone".

Quantum Dot Peptide Biosensors for Monitoring Caspase 3 Proteolysis and Calcium Ions

The nanoscale size and unique optical properties of semiconductor quantum dots (QDs) have made them attractive as central photoluminescent scaffolds for a variety of biosensing platforms. In this report we functionalize QDs with dye-labeled peptides using two different linkage chemistries to yield Förster resonance energy transfer (FRET)-based sensors capable of monitoring either enzymatic activity or ionic presence. The first sensor targets the proteolytic activity of caspase 3, a key downstream effector of apoptosis. This QD conjugate utilized carbodiimide chemistry to covalently link dye-labeled peptide substrates to the terminal carboxyl groups on the QD's surface hydrophilic ligands in a quantitative manner. Caspase 3 cleaved the peptide substrate and disrupted QD donor-dye acceptor FRET providing signal transduction of enzymatic activity and allowing derivation of relevant Michaelis-Menten kinetic descriptors. The second sensor was designed to monitor Ca2+ ions that are ubiquitous in many biological processes. For this sensor, Cu+-catalyzed [3 + 2] azide-alkyne cycloaddition was exploited to attach a recently developed azide-functionalized CalciumRuby-Cl indicator dye to a cognate alkyne group present on the terminus of a modified peptide. The labeled peptide also expressed a polyhistidine sequence, which facilitated its subsequent metal-affinity coordination to the QD surface establishing the final FRET sensing construct. Adding exogenous Ca2+ to the sensor solution increased the dyes fluorescence, altering the donor-acceptor emission ratio and manifested a dissociation constant similar to that of the native dye. These results highlight the potential for combining peptides with QDs using different chemistries to create sensors for monitoring chemical compounds and biological processes.

Polyvalent Display and Packing of Peptides and Proteins on Semiconductor Quantum Dots: Predicted Versus Experimental Results

Quantum dots (QDs) are loaded with a series of peptides and proteins of increasing size, including a <20 residue peptide, myoglobin, mCherry, and maltose binding protein, which together cover a range of masses from <2.2 to approximately 44 kDa. Conjugation to the surface of dihydrolipoic acid-functionalized QDs is facilitated by polyhistidine metal affinity coordination. Increasing ratios of dye-labeled peptides and proteins are self-assembled to the QDs and then the bioconjugates are separated and analyzed using agarose gel electrophoresis. Fluorescent visualization of both conjugated and unbound species allows determination of an experimentally derived maximum loading number. Molecular modeling utilizing crystallographic coordinates or space-filling structures of the peptides and proteins also allow the predicted maximum loadings to the QDs to be estimated. Comparison of the two sets of results provides insight into the nature of the QD surface and reflects the important role played by the nanoparticle's hydrophilic solubilizing surface ligands. It is found that for the larger protein molecules steric hindrance is the major packing constraint. In contrast, for the smaller peptides, the number of available QD binding sites is the principal determinant. These results can contribute towards an overall understanding of how to engineer designer bioconjugates for both QDs and other nanoparticle materials.

Highly soluble multifunctional MnO nanoparticles for simultaneous optical and MRI imaging and cancer treatment using photodynamic therapy

Superparamagnetic MnO nanoparticles were functionalized using a hydrophilic ligand containing protoporphyrin IX as photosensitizer. By virtue of their magnetic properties these nanoparticles may serve as contrast enhancing agents for magnetic resonance imaging (MRI), while the fluorescent target ligand protoporphyrin IX allows simultaneous tumor detection and treatment by photodynamic therapy (PDT). Caki-1 cells were incubated with these nanoparticles. Subsequent exposure to UV light lead to cell apoptosis due to photoactivation of the photosensitizer conjugated to the nanoparticles. This method offers great diagnostic potential for highly proliferative tissues, including tumors. In addition, it is an efficient platform that combines the advantages of a biocompatible photosensitizer with the possibility for MRI monitoring due to the magnetic properties of the highly soluble functionalized manganese oxide nanoparticles.

3P095 親水性結合部位を有したタンパク質-リガンド間の結合自由ネルギーにおける水和エントロピーの寄与(蛋白質-計測・解析の方法論,第48回日本生物物理学会年会)

3P095 親水性結合部位を有したタンパク質-リガンド間の結合自由ネルギーにおける水和エントロピーの寄与(蛋白質-計測・解析の方法論,第48回日本生物物理学会年会)

Determination of phosphatidylcholine in soybean lecithin samples by high performance liquid chromatography on titania

High performance liquid chromatography (HPLC) on titania column was performed for phosphatidylcholine (PC) separation and analysis. To fully understand the retention mechanism of PC on titania, the effect of mobile phase type, %acetonitrile (%ACN), phosphate buffer concentration, buffer pH and flow rate were all studied. Separation depended on the eluent conditions and PC retention was attributed to the cooperation of hydrophilic interaction and ligand exchange. PC in soybean lecithin products was completely separated from other components within 6 min and quantitative analysis was achieved by an external standard method, with a correlation coefficient (R2) of 0.9947. The method precision and detection limit of PC were 3.40% and 0.2 μg, respectively, with the recovery ranging from 91% to 101%. Moreover, PC and glycerol monostearate standard mixture was successfully isolated by the proposed method, which underlined the selective affinity of titania for phosphorylated lipids. With the superiority of simplicity, rapidity and accuracy, this HPLC method based on titania is a competent method for the determination of PC in lecithin samples with prospects in the field of organo-phosphates analysis.

Layer-by-layer assembly of freestanding thin films with homogeneously distributed upconversion nanocrystals

We report a facile and highly-controlled approach to fabricating freestanding upconversion multilayer thin films containing homogeneously distributed lanthanide-doped nanocrystals. Citrate-coated NaYF4:17%Yb, 3%Er nanocrystals were synthesized using a single-phase high-boiling-point-solvent method, followed by ligand exchange. These hydrophilic upconversion nanocrystals were dispersed in freestanding multilayer polyelectrolyte thin films by layer-by-layer assembly over a sacrificial layer. We found that the nanocomposite multilayer thin films possess outstanding mechanical stability and exhibit NIR-to-visible upconversion luminescence. The effect of the hydrophilic ligand exchange on the upconversion properties of these nanocrystals was explored by characterizing the time evolution of upconversion emission following pulsed NIR excitation. It is found that the ligand-exchange process modestly reduces the intrinsic upconversion efficiency of the nanocrystals relative to the as-synthesized oleic acid coated product. Thin films with NIR-to-visible upconversion properties may be suitable for a variety of optical-device and sensing applications.

Anticancer Therapeutics That Target Selenoenzymes: Synthesis, Characterization, in vitro Cytotoxicity, and Thioredoxin Reductase Inhibition of a Series of Gold(I) Complexes Containing Hydrophilic Phosphine Ligands

Gold(I) complexes bearing water-soluble phosphine ligands, including 1,3,5-triaza-7-phosphaadamantane (PTA), 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane (DAPTA), and sodium triphenylphosphine trisulfonate (TPPTS), in combination with thionate ligands, were screened for their antiproliferative activities against human ovarian cancer cell lines A2780 either sensitive or resistant to cisplatin. In addition, the compounds were screened for their inhibition of mammalian thioredoxin reductases (TrxR), enzymes that are overexpressed in many tumor cells and contribute to drug resistance. The gold(I)-phosphine complexes efficiently inhibited cytosolic and mitochondrial TrxRs at concentrations that did not affect the related oxidoreductase glutathione reductase (GR). Additional complementary information on the enzyme metallation process and potential gold binding sites was obtained through the application of a specific biochemical assay using a thiol-tagging reagent, BIAM (biotin-conjugated iodoacetamide).

Inside Cover: Anticancer Therapeutics That Target Selenoenzymes: Synthesis, Characterization, in vitro Cytotoxicity, and Thioredoxin Reductase Inhibition of a Series of Gold(I) Complexes Containing Hydrophilic Phosphine Ligands (ChemMedChem 1/2010)

The inside cover picture shows the X-ray structure of human thioredoxin reductase dimer (TrxR; PDB: 2J3N; ribbon representation), with the catalytic site residues Sec and Cys highlighted (cyan surface). Enzyme activity assays and biochemical studies identified the Sec–Cys dyad as essential for TrxR inhibition by water-soluble AuI–phosphine complexes. For more details, see the Full Paper by M. Laguna, P. J. Dyson, et al. on p. 96 ff.

Hydrophilic ligands derived from glucose: Synthesis, characterization and in vitro cytotoxic activity on cancer cells of Pt(II) complexes

Abstract Aiming to contribute to the design of new antitumoral drugs, we synthesized new hydrophilic Pt(II) complexes of general formula [PtCl 2 ( N , N ′)] containing nitrogen bidentate amine–imine and di-imine ligands derived from glucose. Some chemical properties were discussed. The X-ray molecular structure of [PtCl 2 (α- d -glucopyranoside-methyl-6-deoxy-6(2-(methylimino)methyl)pyridine) ( D ) was reported. [PtCl 2 (β- d -glucopyranosylimine- N -(2-pyridinylmethyl))] ( A ), which is well-soluble both in organic solvents and in water, was tested for cytotoxicity.

Enhanced human bone marrow stromal cell affinity for modified poly( l-lactide) surfaces by the upregulation of adhesion molecular genes

To enhance and regulate cell affinity for poly ( l-lactic acid) (PLLA) based materials, two hydrophilic ligands, poly (ethylene glycol) (PEG) and poly ( l-lysine) (PLL), were used to develop triblock copolymers: methoxy-terminated poly (ethylene glycol)- block-poly ( l-lactide)– block-poly ( l-lysine) (MPEG- b-PLLA- b-PLL) in order to regulate protein absorption and cell adhesion. Bone marrow stromal cells (BMSCs) were cultured on different composition of MPEG- b-PLLA- b-PLL copolymer films to determine the effect of modified polymer surfaces on BMSC attachment. To understand the molecular mechanism governing the initial cell adhesion on difference polymer surfaces, the mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analysed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). It was found that down regulation of adhesion molecules was responsible for the impaired BMSC attachment on PLLA surface. MPEG- b-PLLA- b-PLL copolymer films improved significantly the cell adhesion and cytoskeleton expression by upregulation of relevant molecule genes significantly. Six adhesion genes (CDH1, ITGL, NCAM1, SGCE, COL16A1, and LAMA3) were most significantly influenced by the modified PLLA surfaces. In summary, polymer surfaces altered adhesion molecule gene expression of BMSCs, which consequently regulated cell initial attachment on modified PLLA surfaces.

A Gadolinium‐Binding Cyclodecapeptide with a Large High‐Field Relaxivity Involving Second‐Sphere Water

A new cyclodecapeptide incorporating two prolylglycine sequences as beta-turn inducers and bearing four side chains with acidic carboxyl groups for cation complexation has been prepared. Structural analysis in water by (1)H NMR spectroscopy and CD shows that this template adopts a conformation suitable for the complexation of lanthanide ions Ln(3+), with its carboxyl groups oriented on the same face of the peptide scaffold. Luminescence titrations show that mononuclear Ln-PA complexes are formed with apparent stability constants of log beta(110) approximately 6.5 (pH 7). The high-field water relaxivity values arising from the Gd-PA complex at 200-500 MHz have been interpreted with molecular parameters determined independently. The experimentally determined water relaxivities are undoubtedly 30% higher than the expected values for this complex with two inner-sphere (IS) water molecules and a medium-range rotational correlation time (tau(R) = 386 ps (+/-10%)). This led us to propose the existence of a large second-sphere (2S) contribution to the relaxivity caused by the interaction of water molecules with the hydrophilic peptide ligand by hydrogen-bonding.

Water‐Soluble Ultra Small Paramagnetic or Superparamagnetic Metal Oxide Nanoparticles for Molecular MR Imaging

AbstractA simple and general one‐pot synthesis for water‐soluble ligand‐coated ultra small paramagnetic or superparamagnetic metal oxide nanoparticles with average particle diameters (dav.) that range from 1–3 nm has been developed and used in molecular magnetic resonance (MR) imaging. The iron oxide, manganese oxide, and gadolinium oxide nanoparticles are coated with various hydrophilic and biocompatible ligands. We found that the ligand‐coated ultra small gadolinium oxide nanoparticles are the superior candidates for use as T1 MRI contrast agents. (© Wiley‐VCH Verlag GmbH &amp; Co. KGaA, 69451 Weinheim, Germany, 2009)

Experimental approach for an in vitro toxicity assay with non-aggregated quantum dots

Engineered nanoparticles are increasingly used in consumer products. While the potential of these products hold great promise, it is not known what potential toxic effects these nanomaterials may have on human health. There is a need to develop affordable, systematic, short-term in vitro assays aimed at allowing rapid assessment of potential toxicity. The method reported in this paper describes a system in which the intestinal lining is mimicked (Caco-2 human intestinal cell line) and provides an environment in which quantum dots (QDs), and possibly other nanomaterials, can be applied. Transepithelial electrical resistance (TEER) measurements assessed whether the epithelial integrity was breached because of QD exposure. QDs were suspended in calcium/magnesium-free phosphate buffered saline to study non-aggregated QDs. To maintain cell integrity, normal cell culture conditions were retained below the epithelium to provide necessary nutrients and ions. Toxicity studies completed here show that the nanosized QDs coated with hydrophilic thioglycolate capping ligands purchased for these experiments caused disruption in the epithelium monolayer and cell death at 0.1 mg/L of QDs. This toxicity was caused by the nano-size of the QDs rather than the cadmium ions or the sodium thioglycolate capping ligands. Aggregated QDs did not cause toxicity as measured by TEER.

Structural Basis of the Broad Specificity of a General Odorant-Binding Protein from Honeybee

General odorant-binding proteins (GOBPs) are believed to transport a wide range of volatile hydrophobic molecules across the aqueous sensillum lymph toward olfactory receptors in insects. GOBPs are involved in the first step of odorant recognition, which has a great impact in agriculture and in insect-mediated human disease control. We report here the first structural study of a GOBP, the honeybee ASP2, in complex with a small hydrophilic ligand. The overall fold of the NMR structure of ASP2 consists of the packing of six alpha-helices creating an internal cavity and closely resembles that of the related pheromone-binding proteins (PBPs). The predominantly hydrophobic internal cavity of ASP2 provides additional possible interactions (pi-stacking, electrostatic contact) for ligand binding. We also show that the internal cavity of ASP2 has the ability to bind ligands of different structures and properties, including a hydrophobic component of the floral scent [2-isobutyl-3-methoxypyrazine (IBMP)] and a small hydrophilic ligand. We further demonstrate that IBMP binds ASP2 with two stable alternative conformations inside the ASP2 binding pocket. The (15)N NMR relaxation study suggests that significant backbone mobility occurs at the ligand entry site at the millisecond rate, which likely plays a role in the recognition and the uptake-release mechanism of ligands by ASP2. We propose that the broad ligand specificity of GOBPs compared with PBPs is conferred by the cumulative effects of weak nonspecific protein-ligand interactions and of enhanced protein internal dynamics at the ligand entry site.

Synthesis of Polymerizable Cyclodextrin Derivatives for Use in Adhesion-Promoting Monomer Formulations.

The synthesis of the cyclodextrin derivatives reported herein was assisted by extensive literature research together with structure-property relationships derived from three-dimensional molecular modeling. These studies led to the hypothesis that many of the 21 hydroxyl groups on beta-cyclodextrin molecules could be derivatized to form a closely related family of analogous chemical compounds containing both polymerizable groups and hydrophilic ionizable ligand (substrate-binding) groups, each attached via hydrolytically-stable ether-linkages. The vinylbenzylether polymerizable groups should readily homopolymerize and also copolymerize with methacrylates. This could be highly useful for dental applications because substantially all contemporary dental resins and composites are based on methacrylate monomers. Due to hydrophilic ligands and residual hydroxyl groups, these cyclodextrin derivatives should penetrate hydrated layers of dentin and enamel to interact with collagen and tooth mineral. Analyses indicated that the diverse reaction products resulting from the method of synthesis reported herein should comprise a family of copolymerizable molecules that collectively contain about 30 different combinations of vinylbenzyl and hexanoate groups on the various molecules, with up to approximately seven of such groups combined on some of the molecules. Although the hypothesis was supported, and adhesive bonding to dentin is expected to be significantly improved by the use of these polymerizable cyclodextrin derivatives, other efforts are planned for improved synthetic methods to ensure that each of the reaction-product molecules will contain at least one copolymerizable moiety. The long-term objective is to enable stronger and more durable attachments of densely cross-linked polymers to hydrated hydrophilic substrates. Capabilities for bonding of hydrolytically stable polymers to dental and perhaps other hydrous biological tissues could provide widespread benefits.