Oligo(ethylene glycol)-Functionalized Ratiometric Fluorescent Probe for the Detection of Hydrazine in Vitro and in Vivo.

Hydrazine induced toxicity causes serious harm to the health of humans. The detection of N2H4 in vitro and in vivo has attracted a great deal of attention, especially in the context of fluorescent probes. Although some fluorescent N2H4 probes have been reported, only a few operate in purely aqueous media and, as a result, require the use of organic cosolvents which hinders their use in analysis of real samples. In addition, most of the current N2H4 probes are either "off-on" or "on-off" types, in which it is difficult to eliminate interference from background fluorescence commonly occurring in in vitro and in vivo systems. Furthermore, some probes are unable to differentiate hydrazine from other organic amines. To address the above problems, we developed a novel oligo(ethylene glycol)-functionalized fluorescent probe for the detection of N2H4. The probe, which has a donor-π-acceptor (D-π-A)-type structure, is water-soluble, and it can be utilized to selectively detect N2H4 in both colorimetric and ratiometric mode. Furthermore, the probe is able to differentiate hydrazine from other organic amines and can be used to detect hydrazine vapor and for imaging A549 cells and zebrafish.

The pH-Triggered Triblock Nanocarrier Enabled Highly Efficient siRNA Delivery for Cancer Therapy.

Small interfering RNA (siRNA) therapies have been hampered by lack of delivery systems in the past decades. Nowadays, a few promising vehicles for siRNA delivery have been developed and it is gradually revealed that enhancing siRNA release from endosomes into cytosol is a very important factor for successful delivery. Here, we designed a novel pH-sensitive nanomicelle, PEG-PTTMA-P(GMA-S-DMA) (PTMS), for siRNA delivery. Owing to rapid hydrolysis in acidic environment, PTMS NPs underwent hydrophobic-to-hydrophilic transition in endosomes that enabled combination of proton sponge effect and raised osmotic pressure in endosomes, resulting in vigorous release of siRNAs from endosomes into cytosol. In vitro results demonstrated that PTMS/siRNA complexes exhibited excellent gene silencing effects in several cell lines. Their gene silencing efficiency could reach ~91%, ~87% and ~90% at the N/P ratio of 50/1 in MDA-MB-231, A549 and Hela cells respectively, which were better than that obtained with Lipofectamine 2000. The highly efficient gene silencing was then proven from enhanced siRNA endosomal release, which is mainly attributed to pH-triggered degradation of polymer and acid-accelerated siRNA release. In vivo experiments indicated that NPs/siRNA formulation rapidly accumulated in tumor sites after i.v. injection. Tumor growth was effectively inhibited and ~45% gene knockdown efficacy was determined at the siRRM2 dose of 1mg/kg. Meanwhile, no significant toxicity was observed during the whole treatment. We also found that PTMS/siRNA formulations could lead to significant gene silencing effects in liver (~63%) and skin (~80%) when injected by i.v. and s.c., respectively. This research work gives a rational strategy to optimize siRNA delivery systems for tumor treatments.

An Improved Strategy for the Synthesis of Ethylene Glycol by Oxamate-Mediated Catalytic Hydrogenation.

The present study reports an improved approach for the preparation of ethylene glycol (EG) by using carbon monoxide as C1 chemical by a two-step oxidative carbonylation and hydrogenation sequence. In the first step, oxamates are synthesized through oxidative cross double carbonylation of piperidine and ethanol by using Pd/C catalyst under phosphine ligand-free conditions and subsequently hydrogenated by Milstein's catalyst (carbonylhydrido[6-(di-t-butylphosphinomethylene)-2-(N,N-diethylaminomethyl)-1,6-dihydropyridine]ruthenium(II)). The presented stepwise oxamate-mediated coupling provides the basis for a new strategy for the synthesis of EG by selective upgrading of C1 chemicals.

Macromolecular Hydrogen Sulfide Donors Trigger Spatiotemporally Confined Changes in Cell Signaling.

Hydrogen sulfide (H2S) is involved in a myriad of cell signaling processes that trigger physiological events ranging from vasodilation to cell proliferation. Moreover, disturbances to H2S signaling have been associated with numerous pathologies. As such, the ability to release H2S in a cellular environment and stimulate signaling events is of considerable interest. Herein we report the synthesis of macromolecular H2S donors capable of stimulating cell signaling pathways in both the cytosol and at the cell membrane. Specifically, copolymers having pendent oligo(ethylene glycol) and benzonitrile groups were synthesized, and the benzonitrile groups were subsequently transformed into primary aryl thioamide groups via thionation using sodium hydrosulfide. These thioamide moieties could be incorporated into a hydrophilic copolymer or a block copolymer (i.e., into either the hydrophilic or hydrophobic domain). An electrochemical sensor was used to demonstrate release of H2S under simulated physiological conditions. Subsequent treatment of HEK293 cells with a macromolecular H2S donor elicited a slow and sustained increase in cytosolic ERK signaling, as monitored using a FRET-based biosensor. The macromolecular donor was also shown to induce a small, fast and sustained increase in plasma membrane-localized PKC activity immediately following addition to cells. Studies using an H2S-selective fluorescent probe in live cells confirmed release of H2S from the macromolecular donor over physiologically relevant time scales consistent with the signaling observations. Taken together, these results demonstrate that by using macromolecular H2S donors it is possible to trigger spatiotemporally confined cell signaling events. Moreover, the localized nature of the observed signaling suggests that macromolecular donor design may provide an approach for selectively stimulating certain cellular biochemical pathways.

Nucleophilic Hydroxylation in Water Media Promoted by a Hexa-Ethylene Glycol-Bridged Dicationic Ionic Liquid.

Hexaethylene glycol bis(3-hexaethylene glycol imidazolium) dimesylate ionic liquid (hexaEG-DHIM) was designed and prepared as a highly efficient promoter for the nucleophilic hydroxylation of alkyl halides to the corresponding alcohol products in neat water media. It was observed that hexaEG-DHIM promoter enhanced the nucleophilicity of water significantly in the reaction. In addition, the hexaEG-DHIM could be reused several times without loss of activity. Moreover, the hydroxylation reactions of base-sensitive and/or polar alkyl halide substrates proceeded highly chemoselectively in excellent yields.

Highly efficient conversion of biomass-derived glycolide to ethylene glycol over CuO in water.

The efficient conversion of biomass-derived glycolide into ethylene glycol over CuO in water was investigated. The reaction of glycolide was carried out with 25 mmol Zn and 6 mmol CuO with 25% water filling at 250 °C for 150 min, which yielded the desired ethylene glycol in 94% yield.

A facile and versatile approach to design self-assembled monolayers on glass using thiol-ene chemistry.

This work describes an integrated approach for designing on demand Self-Assembled Monolayers (SAMs) on silicon oxides and particularly glass substrates for cell biology applications. Starting from commercially available compounds, the strategy relies on thiol-ene reaction and provides high quality SAMs exhibiting adhesive and anti-adhesive patterns.

Efficient synthesis of diverse heterobifunctionalized clickable oligo(ethylene glycol) linkers: potential applications in bioconjugation and targeted drug delivery.

Herein we describe the sequential synthesis of a variety of azide-alkyne click chemistry-compatible heterobifunctional oligo(ethylene glycol) (OEG) linkers for bioconjugation chemistry applications. Synthesis of these bioorthogonal linkers was accomplished through desymmetrization of OEGs by conversion of one of the hydroxyl groups to either an alkyne or azido functionality. The remaining distal hydroxyl group on the OEGs was activated by either a 4-nitrophenyl carbonate or a mesylate (-OMs) group. The -OMs functional group served as a useful precursor to form a variety of heterobifunctionalized OEG linkers containing different highly reactive end groups, e.g., iodo, -NH(2), -SH and maleimido, that were orthogonal to the alkyne or azido functional group. Also, the alkyne- and azide-terminated OEGs are useful for generating larger discrete poly(ethylene glycol) (PEG) linkers (e.g., PEG(16) and PEG(24)) by employing a Cu(I)-catalyzed 1,3-dipolar cycloaddition click reaction. The utility of these clickable heterobifunctional OEGs in bioconjugation chemistry was demonstrated by attachment of the integrin (α(v)ß(3)) receptor targeting peptide, cyclo-(Arg-Gly-Asp-D-Phe-Lys) (cRGfKD) and to the fluorescent probe sulfo-rhodamine B. The synthetic methodology presented herein is suitable for the large scale production of several novel heterobifunctionalized OEGs from readily available and inexpensive starting materials.

Temperature-controlled phase-transfer catalysis for ethylene glycol production from cellulose.

A temperature-controlled phase-transfer catalyst-tungsten acid, which in combination with a robust heterogeneous catalyst Ru/C shows a high activity and exceptional reusability for the one-pot conversion of cellulose to ethylene glycol. This binary system can be reused more than 20 times with ethylene glycol yield over 50%.

Synthesis and properties of caprolactone and ethylene glycol copolymers for neural regeneration.

Copolymer networks from poly(ethylene glycol) methacrylate (PEGMA) and caprolactone 2-(methacryloyloxy) ethyl ester were synthesized and the resulting structure of the copolymer network was characterized by differential scanning calorimetry, thermogravimetry, Fourier transform infrared spectroscopy, equilibrium water gain and dynamic mechanical analysis, results which were employed to conclude about the network structure of the resulting copolymers. The new material is a random copolymer with a good miscibility and increasing hydrophilicity as the PEGMA content increases in the composition. Physical data suggest an excess free volume and synergistic interactions between the lateral chains of both comonomers. Olfactory ensheathing cells were cultured on the different networks, and cell viability and proliferation were assessed by MTS assay. The copolymers with a 30 wt% of PEGMA showed the best results compared with the other compositions in this respect, indicating the relevance for biological performance of a balance of hydrophilic and hydrophobic functionalities in the polymer chain.

Chemoselective fabrication of high density peptide microarray by hetero-bifunctional tetra(ethylene glycol) linker for click chemistry conjugation.

A hetero-bifunctional tetra(ethylene glycol) molecule with silane and azide termini was synthesized, and this molecule was used to prepare azide-derivatized glass surface in one step. The resulting glass surface was available for fabricating peptide microarray by the conjugation with alkyne-containing peptide using click chemistry, which proceeded to the completion at low temperature and in aqueous solution. A high density of peptide on the surface was achieved due to concise overall procedure and highly efficient conjugation reaction. Immobilized peptides were highly bio-functional on the surface, as demonstrated by the ability to detect protease activity. Due to the biologically orthogonal manner of conjugation, peptide conjugated by site-specific immobilization was more accessible by protease than that conjugated by random amide conjugation. This site-specific and high efficient immobilization technique could be expanded to large scale development of biocompatible peptide and protein arrays for use in various applications.

Microcontact printing for creation of patterned lipid bilayers on tetraethylene glycol self-assembled monolayers.

Supported lipid bilayers (SLBs) formed on many different substrates have been widely used in the study of lipid bilayers. However, most SLBs suffer from inhomogeneities due to interactions between the lipid bilayer and the substrate. In order to avoid this problem, we have used microcontact printing to create patterned SLBs on top of ethylene-glycol-terminated self-assembled monolayers (SAMs). Glycol-terminated SAMs have previously been shown to resist absorbance of biomolecules including lipid vesicles. In our system, patterned lipid bilayer regions are separated by lipid monolayers, which form over the patterned hexadecanethiol portions of the surface. Furthermore, we demonstrate that α-hemolysin, a large transmembrane protein, inserts preferentially into the lipid bilayer regions of the substrate.

Temperature sensitivity trends and multi-stimuli sensitive behavior in amphiphilic oligomers.

A series of oligomers, containing oligo(ethylene glycol) (OEG) moieties, with the same composition of amphiphilic functionalities has been designed, synthesized, and characterized on the basis of their temperature-sensitive behavior. The non-covalent amphiphilic aggregates, formed from these molecules, influence their temperature sensitivity. Covalent tethering of the amphiphilic units also has a significant influence on their temperature sensitivity. The lower critical solution temperatures of these oligomers show increasingly sharp transitions with increasing numbers of OEG functional groups, indicating enhanced cooperativity in dehydration of the OEG moieties when they are covalently tethered. These molecules were also engineered to be concurrently sensitive to enzymatic reaction and pH. This possibility was investigated using porcine liver esterase as the enzyme; we show that enzymatic action on the pentamer lowers its temperature sensitivity. The product moiety from the enzymatic reaction also gives the amphiphilic oligomer a pH-dependent temperature sensitivity.

Design and synthesis of monofunctionalized, water-soluble conjugated polymers for biosensing and imaging applications.

Water-soluble conjugated polymers with controlled molecular weight characteristics, absence of ionic groups, high emission quantum yields, and end groups capable of selective reactions of wide scope are desirable for improving their performance in various applications and, in particular, fluorescent biosensor schemes. The synthesis of such a structure is described herein. 2-Bromo-7-iodofluorene with octakis(ethylene glycol) monomethyl ether chains at the 9,9'-positions, i.e., compound 4, was prepared as the reactive premonomer. A high-yielding synthesis of the organometallic initiator (dppe)Ni(Ph)Br (dppe = 1,2-bis(diphenylphosphino)ethane) was designed and implemented, and the resulting product was characterized by single-crystal X-ray diffraction techniques. Polymerization of 4 by (dppe)Ni(Ph)Br can be carried out in less than 30 s, affording excellent control over the average molecular weight and polydispersity of the product. Quenching of the polymerization with [2-(trimethylsilyl)ethynyl]magnesium bromide yields silylacetylene-terminated water-soluble poly(fluorene) with a photoluminescence quantum efficiency of 80%. Desilylation, followed by copper-catalyzed azide-alkyne cycloaddition reaction, yields a straightforward route to introduce a wide range of specific end group functionalities. Biotin was used as an example. The resulting biotinylated conjugated polymer binds to streptavidin and acts as a light-harvesting chromophore to optically amplify the emission of Alexa Fluor-488 chromophores bound onto the streptavidin. Furthermore, the biotin end group makes it possible to bind the polymer onto streptavidin-functionalized cross-linked agarose beads and thereby incorporate a large number of optically active segments.

Protein resistant oligo(ethylene glycol) terminated self-assembled monolayers of thiols on gold by vapor deposition in vacuum.

Protein resistant oligo(ethylene glycol) (OEG) terminated self-assembled monolayers (SAMs) of thiols on gold are commonly used for suppression of nonspecific protein adsorption in biology and biotechnology. The standard preparation for these SAMs is the solution method (SM) that involves immersion of the gold surface in an OEG solution. Here the authors present the preparation of 11-(mercaptoundecyl)-triethylene glycol [HS(CH(2))(11)(OCH(2)CH(2))(3)OH] SAMs on gold surface by vapor deposition (VD) in vacuum. They compare the properties of SAMs prepared by VD and SM using x-ray photoelectron spectroscopy (XPS), polarization modulation infrared reflection absorption spectroscopy, and surface plasmon resonance measurements. VD and SM SAMs exhibit similar packing density and show a similar resistance to the nonspecific adsorption of various proteins (bovine serum albumin, trypsin, and myoglobin) under physiological conditions. A very high sensitivity of the OEG SAMs to x-ray radiation is found, which allows tuning their protein resistance. These results show a new path to in situ engineering, analysis, and patterning of protein resistant OEG SAMs by high vacuum and ultrahigh vacuum techniques.

High-purity discrete PEG-oligomer crystals allow structural insight.

To great (monodisperse) lengths: An improved synthesis of purer ethylene glycol (EG) oligomers allows access to 16- and 32-mers pure enough for multiple incorporation, and also to the longest (48-mer) discrete EG oligomer yet reported. The high purity enables the first crystallizations and hence the first glimpses of secondary 3(10)-helical PEG structures.

Chemoselective capture of glycans for analysis on gold nanoparticles: carbohydrate oxime tautomers provide functional recognition by proteins.

Nanoparticles functionalized with glycans are emerging as powerful solid-phase chemical tools for the study of protein-carbohydrate interactions using nanoscale properties for detection of binding events. Methods or reagents that enable the assembly of glyconanoparticles from unprotected glycans in two consecutive chemoselective steps with meaningful display of the glycan are highly desirable. Here, we describe a novel bifunctional reagent that 1) couples to glycans by oxime formation in solution, 2) aids in purification through a lipophilic trityl tag, and 3) after deprotection then couples to gold nanoparticles through a thiol. NMR studies revealed that these oximes exist as both the open-chain and N-glycosyl oxy-amine tautomers. Glycan-linker conjugates were coupled through displacement of ligands from preformed, citrate-stabilized gold nanoparticles. Recognition of these glycans by proteins was studied with a lectin, concanavalin A (ConA), in an aggregation assay and with a processing enzyme and glucoamylase (GA). We demonstrate that the presence of the N-glycosyl oxy-amines clearly enables functional recognition in sharp contrast to the corresponding reduced oxy-amines. This concept is then realized in a novel reagent, which should facilitate nanoglycobiology by enabling the operationally simple capture of glycans and their biologically meaningful display.