Oligoethylene Glycol Linker Research Articles

Real-time Imaging of Nascent DNA in Live Cells by Monitoring the Fluorescence Lifetime of DNA-Incorporated Thiazole Orange-Modified Nucleotides.

A modified 2'-deoxycytidine triphosphate derivative (dCTOTP) bearing thiazole orange moiety tethered via an oligoethylene glycol linker was designed and synthesized. The nucleotide was found to incorporate into DNA by DNA polymerases in vitro as well as in live cells. Upon incorporation of dCTOTP into DNA, the thiazole orange moiety exhibited a fluorescence lifetime that differed significantly from the non-incorporated (i.e. free and non-covalently intercalated) forms of dCTOTP. When dCTOTP was delivered into live U-2 OS cells using a synthetic nucleoside triphosphate transporter, it allowed us to distinguish and monitor in real time the cells that were actively synthesizing DNA from the very first moments after treatment. We anticipate that this probe can be used to study chromatin organization and dynamics.

Real‐time Imaging of Nascent DNA in Live Cells by Monitoring the Fluorescence Lifetime of DNA‐Incorporated Thiazole Orange‐Modified Nucleotides

AbstractA modified 2′‐deoxycytidine triphosphate derivative (dCTOTP) bearing a thiazole orange moiety tethered via an oligoethylene glycol linker was designed and synthesized. The nucleotide was incorporated into DNA by DNA polymerases in vitro as well as in live cells. Upon incorporation of dCTOTP into DNA, the thiazole orange moiety exhibited a fluorescence lifetime that differed significantly from the non‐incorporated (i.e. free and non‐covalently intercalated) forms of dCTOTP. When dCTOTP was delivered into live U‐2 OS cells using a synthetic nucleoside triphosphate transporter, it allowed us to distinguish and monitor cells that were actively synthesizing DNA in real time, from the very first moments after the treatment. We anticipate that this probe could be used to study chromatin organization and dynamics.

Rational design of highly stabilized and selective adrenomedullin analogs.

The peptide hormone adrenomedullin (ADM) consists of 52 amino acids with a disulfide bond and an amidated C-terminus. Due to the vasodilatory and cardioprotective effects, the agonistic activity of the peptide on the adrenomedullin 1 receptor (AM1 R) is of high pharmacological interest. However, the wild-type peptide shows low metabolic stability leading to rapid degradation in the cardiovascular system. Previous work by our group has identified proteolytic cleavage sites and demonstrated stabilization of ADM by lipidation, cyclization, and N-methylation. Nevertheless, these ADM analogs showed reduced activity and subtype selectivity toward the closely related calcitonin gene-related peptide receptor (CGRPR). Here, we report on the rational development of ADM derivatives with increased proteolytic stability and high receptor selectivity. Stabilizing motifs, including lactamization and lipidation, were evaluated regarding AM1 R and CGRPR activation. Furthermore, the central DKDK motif of the peptide was replaced by oligoethylene glycol linkers. The modified peptides were synthesized by Fmoc/t-Bu solid-phase peptide synthesis and receptor activation of AM1 R and CGRPR was measured by cAMP reporter gene assay. Peptide stability was tested in human blood plasma and porcine liver homogenate and analyzed by RP-HPLC and MALDI-ToF mass spectrometry. Combination of the favorable lactam, lipidation, ethylene glycol linker, and previously described disulfide mimetic resulted in highly stabilized analogs with a plasma half-life of more than 144 h. The compounds display excellent AM1 R activity and wild-type-like selectivity toward CGRPR. Additionally, dose-dependent vasodilatory effects of the ADM derivatives lasted for several hours in rodents. Thus, we successfully developed an ADM analog with long-term in vivo activity.

Methacryloyl-GlcNAc Derivatives Copolymerized with Dimethacrylamide as a Novel Antibacterial and Biocompatible Coating

Improving medical implants with functional polymer coatings is an effective way to further improve the level of medical care. Antibacterial and biofilm-preventing properties are particularly desirable in the area of wound healing, since there is a generally high risk of infection, often with a chronic course in the case of biofilm formation. To prevent this we here report a polymeric design of polymer-bound N-acetyl-glucosamine-oligoethylene glycol residues that mimic a cationic, antibacterial, and biocompatible chitosan surface. The combination of easy to use, crosslinkable, thin, potentially 3D-printable polymethacrylate layering with antibacterial and biocompatible functional components will be particularly advantageous in the medical field to support a wide range of implants as well as wound dressings. Different polymers containing a N-acetylglucosamine-methacryloyl residue with oligoethylene glycol linkers and a methacryloyl benzophenone crosslinker were synthesized by free radical polymerization. The functional monomers and corresponding polymers were characterized by 1H, 13C NMR, and infrared (IR) spectroscopy. The polymers showed no cytotoxic or antiadhesive effects on fibroblasts as demonstrated by extract and direct contact cell culture methods. Biofilm formation was reduced by up to 70% and antibacterial growth by 1.2 log, particularly for the 5% GlcNAc-4EG polymer, as observed for Escherichia coli and Staphylococcus aureus as clinically relevant Gram-negative and Gram-positive model pathogens.

Tamoxifen-zinc(II) phthalocyanine conjugates for target-based photodynamic therapy and hormone therapy

Although photodynamic therapy has been extensively studied in recent years and preclinical studies have shown promising results, strategies for enhancing PDT outcomes and reducing side effects still urgently need to be developed. In this study, a series of Tamoxifen-zinc(II) phthalocyanine conjugates have been designed and synthesized. In these “double-headed” conjugates, photodynamic therapy agent zinc(II) phthalocyanine and hormone therapy drug Tamoxifen were combined via oligoethylene glycol linkers. The conjugates show high specificity, and some of them show cytotoxic effects against the MCF-7 cells overexpressed Estrogen receptor, due to the targeting and cytostatic Tamoxifen moiety. Upon illumination, all these conjugates show high cytotoxicity due to the photosensitizing phthalocyanine unit. Their structure-activity relationship was also assessed. The results show that [Formula: see text]-substituted Tamoxifen-zinc(II) phthalocyanine conjugates are highly promising anticancer targeting agents which exhibit additive effects of photodynamic therapy and hormone therapy.

Endogenous Membrane Receptor Labeling by Reactive Cytokines and Growth Factors to Chase Their Dynamics in Live Cells

Summary Cytokine and growth factor receptors localized on cell membranes play key roles in many biological events. Because the dynamic behaviors of receptors are deeply involved in cytokine- and growth-factor-dependent signaling pathways, reliable techniques for real-time imaging of receptors in live cells are strongly desired. Here, we describe a method of covalently labeling and imaging membrane receptors in live cells by using chemically reactive cytokines and growth factors coupled with 4-dimethylaminopyridine (DMAP), an organocatalyst that facilitates acyl transfer. Construction of the DMAP cytokines and growth factors relies on non-covalent coordination chemistry between an oligo-histidine tag and dimeric Ni(II)-nitrilotriacetate in an in situ manner. This supramolecular approach is relatively simple and flexible and can be applied to labeling not only transiently but also endogenously expressed receptors. Moreover, given the benefit of this traceless labeling of the receptors, our technique allows real-time imaging of the ligand-induced dynamics of endogenously expressed receptors in live cells.

Photoresponsive self-assembly of a β-cyclodextrin derivative with an azobenzene terminal group in water

We report the photoresponsive self-assembly of a β-cyclodextrin derivative containing an azobenzene terminal group with an oligoethylene glycol linker (Azo-EG-βCD). Azo-EG-βCD was synthesized, and its aggregation was investigated using several techniques including TEM analysis, DLS and other spectral measurements. In a freshly prepared aqueous solution, Azo-EG-βCD self-assembled into a network-like structure by forming intermolecular host–guest inclusions that transformed into vesicles upon aging because of hydrogen bonding. Transformation from trans-azo to cis-azo using UV light induced the dissociation of azobenzene in the β-CD cavity, and the aggregate transformed into vesicles. The construction of a photoresponsive self-assembly may provide a unimolecular host–guest complexation model in stimuli-responsive host–guest self-assembly.

Inverse relationship between hSHBG affinity for testosterone and hSHBG concentration revealed by surface plasmon resonance

A wide range of human sex hormone-binding globulin (hSHBG) affinity constants for testosterone (KA_hSHBG) has been reported in literature. To bring new insight on the KA_hSHBG value, we implemented a study of the molecular interactions occurring between testosterone and its plasma transport proteins by using surface plasmon resonance.The immobilization on the sensorchip of a testosterone derivative was performed by an oligoethylene glycol linker. For different plasmas with hSHBG concentrations, an assessment of the KA_hSHBG was obtained from a set of sensorgrams and curve-fitting these data. We observed that KA_hSHBG decreased, from at least two decades, when the plasma hSHBG concentration increased from 4.4 to 680 nmol/L. Our study shows a wide biological variability of KA_hSHBG that is related to the hSHBG concentration. These unexpected results may have a physiological significance and question the validity of current methods that are recommended for calculating free testosterone concentrations to evaluate androgen disorders in humans.

Cyclopolymerization of α,ω‐heterodifunctional monomers containing styrene and maleimide moieties

ABSTRACTA series of α,ω‐heterodifunctional monomers with styrene (St) and maleimide moieties bridged by a varied length of oligo‐ethylene glycol (OEG) linkers were synthesized. Cyclopolymerizations of these monomers through reversible addition–fragmentation chain transfer‐mediated alternating radical copolymerization between intramolecular St and maleimide moieties were investigated. For the monomers with three or more ethylene glycol (EG) units, their cyclopolymerizations can be realized properly in low monomer feeding concentrations, affording well‐defined cyclopolymers with crown ether encircled in their main chains. Importantly, the cyclopolymerizations of monomers with six or seven EG units in the presence of KPF6 could be enhanced by the supramolecular effects between the OEG linkers and the potassium metal ion. Thus, the monomer feeding concentration could be largely improved, which may benefit preparation of the cyclopolymers with high degrees of copolymerization. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 330–338

Synthesis and Anticancer Activity of Glucosylated Podophyllotoxin Derivatives Linked via 4β-Triazole Rings

A series of 4β-triazole-linked glucose podophyllotoxin conjugates have been designed and synthesized by employing a click chemistry approach. All the compounds were evaluated for their anticancer activity against a panel of five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480) using MTT assays. Most of these triazole derivatives have good anticancer activity. Among them, compound 35 showed the highest potency against all five cancer cell lines tested, with IC50 values ranging from 0.59 to 2.90 μM, which is significantly more active than the drug etoposide currently in clinical use. Structure-activity relationship analysis reveals that the acyl substitution on the glucose residue, the length of oligoethylene glycol linker, and the 4'-demethylation of podophyllotoxin scaffold can significantly affect the potency of the anticancer activity. Most notably, derivatives with a perbutyrylated glucose residue show much higher activity than their counterparts with either a free glucose or a peracetylated glucose residue.

Preparation and in Vitro Photodynamic Activities of Folate-Conjugated Distyryl Boron Dipyrromethene Based Photosensitizers

Two folate-conjugated diiododistyryl boron dipyrromethenes have been prepared and characterized with various spectroscopic methods. These conjugates exhibit higher photocytotoxicity toward the KB human nasopharyngeal carcinoma cells, which have high expression of folate receptors when compared with the MCF-7 human breast adenocarcinoma cells, which have low expression of folate receptors. The difference in photocytotoxicity for these two cell lines is particularly large for the conjugate with a shorter oligoethylene glycol linker (compound 11a) as a result of its higher cellular uptake and slightly lower aggregation tendency. Its IC50 value toward KB cells (0.06 μM) is 43-fold lower than that for MCF-7 cells, while the difference is only 6-fold for the analogue with a longer linker (compound 11b). The length of the spacer also affects their subcellular localization. While compound 11a shows high affinity toward the endoplasmic reticulum of KB cells, conjugate 11b is mainly localized in the lysosomes.

Synthesis and biology of oligoethylene glycol linked naphthoxylosides

Proteoglycans (PGs) are important macromolecules in mammalian cells, consisting of a core protein substituted with carbohydrate chains, known as glycosaminoglycans (GAGs). Simple xylosides carrying hydrophobic aglycons can enter cells and act as primers for GAG chain synthesis, independent of the core protein. Previously it has been shown that aromatic aglycons can be separated from the sugar residue by short linkers without affecting the GAG priming ability. To further investigate the effects of the xylose–aglycon distance on the GAG priming ability, we have synthesized xyloside derivatives with 2-naphthyl and 2-(6-hydroxynaphthyl) moieties connected to xylose, directly, via a methylene bridge, or with oligoethylene glycol linkers of three different lengths. The GAG priming ability and the antiproliferative activity of the xylosides, as well as the composition of the xyloside-primed GAG chains were investigated in a matched pair of human breast fibroblasts and human breast carcinoma cells. An increase of the xylose–aglycon distance from 0.24 to 0.37nm resulted in an increased GAG priming ability in both cell lines. Further increase of the xylose–aglycon distance did not result in any pronounced effects. We speculate that by increasing the xylose–aglycon distance, and thereby the surface area of the xyloside, to a certain level would make it more accessible for enzymes involved in the GAG synthesis. The compositions of the primed GAG chains varied with different xylosides, independent of the xylose–aglycon distance, probably due to various affinities for enzymes and/or different cellular uptake. Furthermore, no correlations between the antiproliferative activities, the xylose–aglycon distances, and the amounts or compositions of the GAG chains were detected suggesting involvement of other factors such as fine structure of the GAG chains, effects on endogenous PG synthesis, or other unknown factors for the antiproliferative activity.

Efficient selective dehydration of fructose and sucrose into 5-hydroxymethylfurfural (HMF) using dicationic room temperature ionic liquids as a catalyst

Abstract Dicationic room temperature ionic liquids (RTILs), with short oligo ethylene glycol linkers have been found to be highly active catalyst for the selective dehydration of fructose and sucrose into 5-hydroxymethylfurfural (HMF). The bis ( N -methylimidazolium) cations containing short oligo ethylene glycol linkers and mesylate (CH 3 SO 3 − ) anions based ILs were employed in catalytic amount for sugar dehydration reactions. As a result, 92.3% of HMF yield was obtained from fructose in 40 min with one equivalent of [TetraEG(mim) 2 ][OMs] 2 at 120 °C. While, 67.2% of HMF was achieved from dehydration of sucrose at 120 °C in 150 min using two equivalents of [TetraEG(mim) 2 ][OMs] 2 . Among those dicationic RTILs, [TetraEG(mim) 2 ][OMs] 2 RTIL was observed to be the most efficient catalyst, demonstrated by its ability to achieve 100% conversion and highest yield of HMF. In addition, effect of reaction time, molar ratio, temperature, and co-catalyst effect were discussed. The use of dicationic RTILs as a catalyst in sugar dehydration greatly reduces the equimolar ratio of catalyst and reaction time required for conversion of sugar into HMF.

Bivalent molecular probes for dopamine D2-like receptors

Merging two arylamidoalkyl substituted phenylpiperazines as prototypical recognition elements for dopamine D2-like receptors by oligoethylene glycol linkers led to a series of bivalent ligands. These dimers were investigated in comparison to their monomeric analogues for their dopamine D2long, D2short, D3 and D4 receptor binding. Radioligand binding experiments revealed strong bivalent effects for some para-substituted benzamide derivatives. For the D3 subtype, the target compounds 32, 34 and 36 showed an up to 70-fold increase of affinity and a substantial enhancement of subtype selectivity when compared to the monovalent analogue 24. Analysis of the binding curves displayed Hill slopes very close to one indicating that the bivalent ligands displace 1equiv of radioligand. Obviously, the two pharmacophores occupy an orthosteric and an allosteric binding site rather than adopting a receptor-bridging binding mode.

Effect of oligoethylene glycol moieties in porous silicon surface functionalisation on protein adsorption and cell attachment

We report on the synthesis of two carboxy-functional alkene compounds as modifiers of porous silicon (pSi) surface chemistry by means of thermal hydrosilylation. Both alkene compounds have hydrophobic, aliphatic carbon segments, with terminal carboxylic acid functionality appended through a thioether linkage. In one of the alkene linkers, we incorporated a short oligoethylene glycol (OEG) moiety to explore its low-fouling properties when attached to porous silicon. We examined surface stability of the surface-modified porous silicon in aqueous milieu. Albumin and fibronectin were adsorbed and, using carbodiimide chemistry, covalently immobilised to linker-modified porous silicon, and the propensity for mammalian cells to attach to these surfaces investigated. Surface chemistry was characterised by infrared spectroscopy and the stability of porous silicon in an aqueous milieu was investigated by interferometric reflectance spectroscopy (IRS). Surfaces functionalised with the alkene linker containing OEG displayed greater resistance to the adsorption of albumin. This linker also facilitated higher levels of covalent protein immobilisation to the functionalised pSi surface. Higher levels of cell attachment were observed on pSi surfaces with fibronectin covalently immobilised onto the OEG linker, than for fibronectin covalently immobilised on the non-OEG linker.

New oligoethylene glycol linkers for the surface modification of an ultra-high frequency acoustic wave biosensor

This work describes the application of acoustic wave technology for the real-time and label-free detection of biotin–avidin interactions. Biosensing surfaces are constructed onto unelectroded piezoelectric quartz discs as functionalizable mixed self-assembled monolayers (SAM) produced from previously unreported linker and diluent molecules. Biotinthiol can subsequently be immobilized for detection purposes in a straightforward and coupling-free manner. Specific and non-specific adsorptions of avidin are measured at ultra-high frequencies (1.06 and 0.82 GHz) with an electromagnetic piezoelectric acoustic sensor (EMPAS) using micromolar avidin-spiked buffer solutions. These biosensing surfaces, especially the oligoethylene glycol SAM-based variety, display high specificity for avidin, with moderate to excellent reproducibility. This preliminary work constitutes the first application of SAM chemistry and EMPAS technology in the bioanalytical field.

Matrix effects on an antigen immobilized format for competitive enzyme immunoassay of salivary testosterone

Matrix interferences in salivary testosterone enzyme immunoassays are important in the development of direct ELISA. An alternative format for sensitive enzyme immunoassay of testosterone was developed using immobilization of the antigen as part of a protein conjugate using oligoethylene glycol linker to project the antigen, with color development via enzyme labeled secondary antibody. This technique gave the required sensitivity for detection of testosterone from male saliva with a limit of detection (LOD) of 8.9 pg/mL (31 pmol/L). Application of the immunoassay directly in human saliva gave suppression of binding signals for the samples, indicating clear matrix interference with antibody binding. A wide variety of treatments were used in an attempt to overcome this effect, including use of both synthetic saliva and stripped human saliva for standard preparation, use of bovine serum albumin (BSA) to reduce non-specific binding of contaminants, pH adjustment of samples and/or standards and use of different antibodies. None of these techniques proved effective, causing either substantial suppression or enhancement of signal, and dilution was not possible because of the very low physiological concentrations. Complete removal of the saliva medium by chemical extraction was the only technique studied that could overcome this problem. These issues have not been explored previously for direct ELISA of salivary testosterone using alternative assay formats and have implications for the design of small molecule plate-based immunoassays in this medium.

Ultrasensitive detection of testosterone using conjugate linker technology in a nanoparticle-enhanced surface plasmon resonance biosensor

A rationally designed oligoethylene glycol linker conjugate to testosterone was synthesised and covalently immobilized on a surface plasmon resonance (SPR) biosensor surface. The sensing surface was stable for more than 330 binding and regeneration cycles allowing a high degree of re-use. This surface was then used in the development of an ultrasensitive immunobiosensor system for testosterone in buffer utilizing both secondary antibody and gold nanoparticle signal enhancement. The mechanism for the increased sensitivity results from increased binding mass and a gold plasmon coupling effect. The addition of a secondary antibody with an attached gold nanoparticle increased the signal sensitivity of the assay 12.5-fold compared with primary antibody alone. In the enhanced format the assay had limits of detection (LOD) of 3.7pgml−1 with standard in running buffer, and 15.4pgml−1 in a stripped human saliva matrix. This immunobiosensor system has sufficient sensitivity to measure testosterone across the broad physiologically relevant range in male saliva (29–290pgml−1) in under 13min allowing monitoring of testosterone in near real-time.

RNA-Mediated Synthesis of Palladium Nanoparticles on Au Surfaces

RNA catalysts for the shape-controlled synthesis of Pd particles from the precursor complex trisdibenzylideneacetone dipalladium ([Pd2(DBA)3] were recently discovered in our laboratory (J. Am. Chem. Soc. 2005, 127, 17814-17818). In the work described here, RNA codes for hexagonal Pd platelets and Pd cubes were covalently immobilized on gold surfaces and evaluated for their activity toward particle synthesis. When coupled to gold via oligoethylene glycol linkers, both RNA sequences were able to catalyze the formation of Pd particles with the same shape control previously observed in solution. For low surface coverages, the average distance between RNA molecules on the surface was estimated at ca. 300 nm, yet large (e.g., dimensions of hundreds of nanometers) Pd hexagons and cubes still formed. This surprising result suggests that a single RNA molecule may be sufficient for nucleating and controlling the shapes of these particles. Finally, the use of surface-bound RNA as a tool for directing the orthogonal synthesis of materials on surfaces was demonstrated. Patterning the RNA code for Pd hexagons next to the code for Pd cubes, followed by incubation in a solution containing [Pd2(DBA)3], resulted in the spontaneous formation of spatially distinct spots of hexagonal and cubic particles.

Sensitivity enhancement of surface plasmon resonance biosensing of small molecules

Surface plasmon resonance (SPR) biosensor formats using gold nanoparticle or protein signal amplification for the sensitive assay of small molecules were developed using progesterone as a model compound. Progesterone was immobilized to a dextran surface in the Biacore biosensor through in situ covalent immobilization using an oligoethylene glycol linker attached to the 4 position of the steroid. This surface produced stable antibody binding for in excess of 1100 assay cycles. Using this surface, assays were developed for progesterone using 10- and 20-nm gold–streptavidin labels attached to biotinylated monoclonal antibody in both label prebinding and sequential binding formats. Prelabeling formats gave no signal enhancement but produced assays with limits of detection of 143 pg/ml, compared with approximately 1 ng/ml in previous studies. Sequential binding formats gave signal enhancements of 2.2-fold over the monoclonal antibody and a limit of detection of 23.1 pg/ml. It was found that secondary antibody labeling gave 8.1-fold signal enhancements and a limit of detection of 20.1 pg/ml, whereas use of secondary antibody–25 nm gold complexes provided more signal enhancement (13-fold) and a further improvement in limit of detection of 8.6 pg/ml.