Name: facile and efficient preparation of tri-component fluorescent glycopolymers via raft-controlled polymerization
Uploaded: 2015-06-19T14:00:00
Duration: 10 min 54 s
Description: Watch this Scientific Journal Video about Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization at JoVE.com

This work was supported by the Experiment Station Chemical Laboratories of the University of Missouri, and by the Cystic Fibrosis Association of Missouri.

1. Synthesis of Glycomonomer 2-Lactobionamidoethyl Methacrylamide
<ol>
	<li>Dissolve 2 g of lactobionic acid in 3.0 ml of anhydrous methanol and slowly add absolute ethanol in a drop wise fashion until the solution just turns cloudy, then remove the solvents via rotoevaporation.</li>
	<li>Dissolve the residue, from step 1.1, in 3.0 ml anhydrous methanol and, once again, slowly add absolute ethanol until just cloudy, then evaporate the solvents via rotoevaporation. Repeat this step 3 times to obtain lactobiono-1,5-lactone (1.94 g, 98% yield). This product is of sufficient purity to be used in the following reactions.</li>
	<li>Add 1.0 g of lactobionolactone in 3.0 ml of methanol to N-(2-aminoethyl) methacrylamide (AEMA, 0.58 g) and hydroquinone monomethyl ether (MEHQ, 1.0 mg), an inhibitor of self-polymerization, in 2.0 ml of methanol, followed by 1.0 ml of triethylamine. Stir at RT for 48 hr.</li>
	<li>Add 20 ml of deionized H2O (dH2O) to the reaction flask, then remove the methanol and free triethylamine by evaporation to dryness via rotoevaporation.</li>
	<li>To remove any remaining lactobionic acid, add 20 ml of dH2O, then pass the aqueous solution through an anion exchange column (OH- form, 10 mm x 20 mm) into a receiving beaker containing 1.0 mg of MEHQ.</li>
	<li>Remove triethylamine, produced in Step 1.5, by evaporating to dryness via rotoevaporation.</li>
	<li>Add 20 ml of dH2O and remove unreacted AEMA by slowly adding 1 mg aliquots of cation exchange resin (H+ form) until no ninhydrin reactive materials are detectable. Monitor the removal by taking 1 &#181;l aliquots of the solution after each resin addition, applying it to a thin-layer chromatography plate, then spraying the plate with a 2% ninhydrin in ethanol solution. When no deep blue color is observed to develop when the plate is heated to 90 &#176;C for 1 min, the end point has been reached.</li>
	<li>Filter the solution through a fritted glass funnel, transfer the filtrate to a test tube, freeze the sample at -80 &#176;C, and then lyophilize.</li>
	<li>Remove MEHQ from the sample by dissolving the freeze-dried material in a minimum amount of methanol (~0.5 ml), then add cold anhydrous acetone (-20 &#176;C, 15 ml) to precipitate the product. Collect the precipitate by filtration using a fritted glass funnel, then dry the precipitate in a desiccator under vacuum to obtain 2-lactobionamidoethyl methacrylamide (LAEMA) as an off-white powder (0.94 g, 68% yield). This product is of sufficient purity to be used in the following reactions.</li>
</ol>
2. Synthesis of Monomer 2-Gluconamidoethyl Methacrylamide
Note: The preparation of 2-gluconamidoethyl methacrylamide (GAEMA), which does not possess a pendant sugar, was adapted from a published method15.
<ol>
	<li>Add 2.0 g of AEMA dissolved in 10 ml of methanol to a solution of D-gluconolactone (1.6 g) in 30 ml of methanol and, with stirring, slowly add 1.6 ml of triethylamine.</li>
	<li>Stir the reaction at RT for 24 hr.</li>
	<li>Filter the precipitated product using a fritted glass funnel and rinse the precipitate three times with 10 ml each of isopropanol, then wash with 10 ml of dry acetone. Dry the precipitated product in a desiccator under vacuum.</li>
</ol>
3. RAFT Glycopolymer Synthesis
<ol>
	<li>To remove the inhibitor MEHQ present in commercial N-(2-hydroxyethyl) acrylamide (HEAA), add 1 ml of HEAA to a 2 ml microcentrifuge tube, followed by adding 0.5 g of aluminum oxide nanoparticles. Centrifuge the tube at 300 x g for 30 sec, and use the top layer HEAA in the following reaction.</li>
	<li>Carefully add 32.8 mg of LAEMA (70.0 &#181;mol), 1.7 mg of AEMA (10.5 &#181;mol) and 27.5 &#181;l of HEAA (270 &#181;mol), all dissolved in 0.4 ml of dH2O, to a well-cleaned 1 ml Schlenk tube, thus having a monomer molar ratio of 20:3:77.</li>
	<li>In a parallel reaction, in order to produce control polymers that do not possess any pendant sugar, in lieu of using LAEMA in step 3.2, substitute 21.4 mg of GAEMA (70.0 &#181;mol) in the reaction.</li>
	<li>To the respective Schlenk tube (i.e., 3.2 or 3.3), sequentially add 50 &#181;l of DMF containing 0.53 mg of (4-cyanopentanoic acid)-4-dithiobenzoate (1.9 &#181;mol, RAFT agent), and 50 &#181;l of DMF containing 250 &#181;g of 4,4&#8242;-azobis-(4-cyanovaleric acid) (0.9 &#181;mol, initiator). Gently mix by finger tapping.</li>
	<li>Freeze the contents contained in the Schlenk tube employing a dry ice:ethanol bath (75 g dry ice in 100 ml ethanol), apply a vacuum to within 10-50 mTorr, then close the Schlenk valve and permit the solution to slowly thaw to RT. Repeat this freeze&#8211;evacuate&#8211;thaw cycle twice more. Ensure that all reagents are dissolved after the last thaw.</li>
	<li>Place the Schlenk tube into a sealable plastic bag, evacuate the bag&#8217;s air, and then seal it. Transfer the bag containing the Schlenk tube to a water bath preheated at 70 &#176;C and incubate for 24 hr.</li>
	<li>Carefully transfer the solution in the Schlenk tube to a prepared dialysis bag (MWCO = 3,500), and dialyze against dH2O (10 x 2 L) for 24 hr, changing the dH2O every hour for the first 8 hr. Following dialysis, transfer the sample from the dialysis tubing to a test tube, freeze the sample at -80 &#176;C, and then lyophilize it.</li>
</ol>
Note: The resultant statistical poly-methacrylamide/acrylamide (PMA) copolymers containing pendant 4-O-&#946;-D-galactopyranosyl-D-gluconamide (lactobionamide) (from Step 3.2) or D-gluconamide (from Step 3.3), respectively, are obtained. For convenience of discussion, these two glycopolymers are abbreviated as PMA-LAEMA and PMA-GAEMA, respectively.
4. Post-modification of Glycopolymers with Fluorophores
<ol>
	<li>Dissolve 5.0 mg of glycopolymer PMA-LAEMA or PMA-GAEMA containing ~0.9 &#181;mole of primary amine functional groups in 0.9 ml of phosphate buffered saline (PBS, 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5), respectively.</li>
	<li>Slowly add 0.6 mg of carboxyfluorescein succinimidyl ester in 100 &#181;l of DMF to the solutions with rapid stirring. Gently stir the reactions for 16 hr in the dark at RT.</li>
	<li>While protected from light, load the sample into a prepared dialysis tubing (MWCO = 3,500) and dialyze against dH2O (2 L) for 16 hr, changing the dialysis solution every hour for the first 8 hr. Following dialysis, transfer the sample from the dialysis tubing to a test tube, freeze the sample at -80 &#176;C, and then lyophilize it.</li>
</ol>
Note: Following lyophilization, fluorescent glycopolymers PMA-LAEMA-Fluorescein and PMA-GAEMA-Fluorescein, respectively, are obtained.
5. Characterization of the Glycopolymers
<ol>
	<li>Determine the number average molecular weight&#160;(Mn), the weight average molecular weight (Mw) and the dispersity (Mw/Mn) of the glycopolymers on a commercial HPLC system equipped with gel permeation chromatography (GPC) software, a GPC column suitable for the molecular weight of interest, and a refractive index detector, using 0.1 M Tris/0.1 M sodium chloride buffer (pH 7) as eluent at a flow rate of 0.6 ml/min14. Use polyethylene glycol standards as molecular weight standards (MW: 200-1,200,000 g/mol).</li>
	<li>Quantify the actual concentrations of primary amine functional groups within the glycopolymers16. Analyze the total carbohydrate content of the synthesized glycopolymers according to a published method17.</li>
	<li>Perform tests of structural composition and purity of the glycomonomers LAEMA, GAEMA and glycopolymers PMA-LAEMA, PMA-GAEMA in D2O by NMR spectroscopy14.</li>
</ol>
6. Binding Tests of the Synthetic Glycopolymers with Lectin-coated Agarose Beads
<ol>
	<li>Add 1.5 ml of PBS to 50 &#181;l of suspension of Erythrina crista-galli lectin (ECL)-coated agarose beads, centrifuge at 300 x g for 1 min, and carefully remove and dispose of the supernatant. Repeat this step twice, and then resuspend the beads in 0.5 ml of PBS.</li>
	<li>Add 3 &#181;g of PMA-LAEMA-Fluorescein or PMA-GAEMA-Fluorescein (negative control) in 6 &#181;l of PBS to the beads suspension, and incubate the mixtures, in the dark, at RT for 1 hr.</li>
	<li>Wash the mixtures with 1.5 ml of PBS three times, and resuspend beads in 0.2 ml of PBS. Load an aliquot (4 &#181;l) into a well on an immunofluorescence microscope slide (Teflon-coated), cover with a cover slip, and observe by fluorescence microscopy using a FITC filter (excitation wavelength: 467-498 nm, emission wavelength: 513-566 nm) and a 10X objective to examine the binding of the fluorescent glycopolymers with the beads14.</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+glycopolymers+by+controlled+radical+polymerization+techniques+and+their+applications.">Synthesis of glycopolymers by controlled radical polymerization techniques and their applications.</a> Chembiochem. 13, 2478-2487 (2012).</li><li>Jiang, X., Ahmed, M., Deng, Z., Narain, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinylated+glyco-functionalized+quantum+dots:+Synthesis,+characterization,+and+cytotoxicity+studies.">Biotinylated glyco-functionalized quantum dots: Synthesis, characterization, and cytotoxicity studies.</a> Bioconjugate Chem. 20 (5), 994-1001 (2009).</li><li>Deng, Z., Li, S., Jiang, X., Narain, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Well-defined+galactose-containing+multi-functional+copolymers+and+glyconanoparticles+for+biomolecular+recognition+processes.">Well-defined galactose-containing multi-functional copolymers and glyconanoparticles for biomolecular recognition processes.</a> Macromolecules. 42 (17), 6393-6405 (2009).</li><li>Qin, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galactosylated+N-2-hydroxypropyl+methacrylamide-b-N-3-guanidinopropyl+methacrylamide+block+copolymers+as+hepatocyte-targeting+gene+carriers.">Galactosylated N-2-hydroxypropyl methacrylamide-b-N-3-guanidinopropyl methacrylamide block copolymers as hepatocyte-targeting gene carriers.</a> Bioconjugate Chem. 22 (8), 1503-1512 (2011).</li><li>Albertin, L., Wolnik, A., Ghadban, A., Dubreuil, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+RAFT+polymerization+of+N-acryloylmorpholine,+synthesis+of+an+ABA+triblock+glycopolymer+and+study+of+its+self-association+behavior.">Aqueous RAFT polymerization of N-acryloylmorpholine, synthesis of an ABA triblock glycopolymer and study of its self-association behavior.</a> Macromol. 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Chem. 47 (2), 614-627 (2009).</li><li>Noel, S., Liberelle, B., Robitaille, L., De Crescenzo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+primary+amine+groups+available+for+subsequent+biofunctionalization+of+polymer+surfaces.">Quantification of primary amine groups available for subsequent biofunctionalization of polymer surfaces.</a> Bioconjugate Chem. 22 (8), 1690-1699 (2011).</li><li>Fox, A., Morgan, S. L., Gilbart, J., Biermann, C. J., McGinnis, G. 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The authors have nothing to disclose.

A facile and efficient protocol for RAFT-based tri-component fluorescent glycopolymers, with and without a pendant carbohydrate, and their use in a lectin-binding test, is demonstrated in this report. The protocol starts with the preparation of glycomonomers LAEMA and GAEMA. Through a one-step RAFT-controlled copolymerization, glycopolymers with reproducible yield, predictable monomer composition and low dispersity, are obtained. Following post-modification of glycopolymers with carboxyfluorescein succinimidyl ester, the binding of the resulting respective fluorescent-labeled glycopolymer is readily testable for its lectin-binding specificity.
In the initial preparative steps of the glycomonomers that are to be employed in the subsequent glycopolymer syntheses, readily available lactobionic acid and gluconolactone were utilized. In theory, any carbohydrates of interest, from monosaccharides to complex oligosaccharides, can be converted to glycomonomers by conjugating the target sugar onto the primary hydroxyl group on C6 of glucose. Following oxidation of the reducing glucose residue, and its subsequent dehydration to a lactone, the product can then be readily reacted with the primary amine on AEMA to form the corresponding glycomonomer. Further examples of this route can be seen in a recent report14. It should be noted that before initiating any polymerization step, MEHQ, a potent polymerization inhibitor, must be removed from all monomer and glycomonomer preparations just prior to use. This is readily accomplished by using the minimum amount of methanol to dissolve the glycomonomer that possesses MEHQ then immediately treat it with acetone at -20 &#176;C to precipitate the inhibitor-free product in high yield.
Essential in any radical polymerization scheme, attention to detail and monomer purities are emphasized. As is typical of a RAFT polymerization system, it consists of a radical source, a RAFT reagent, a monomer and solvent. In this visualized presentation, a single-step RAFT polymerization system is described that focuses on the production of statistical copolymers generated from a reaction mixture possessing three different monomers in an aqueous solution. Two separate RAFT-mediated reactions are presented in which one utilizes a glycomonomer that possesses a pendant, non-reducing carbohydrate terminus (i.e.,&#160;&#946;-D-galactose), and the other, possessing a polyol with no bound carbohydrate residue. Common to both RAFT-mediated reactions were monomers possessing a singular hydroxyl group that serves as a spacer molecule, and another possessing a free amine for post-modification with an amino-reactive fluorophore.
Since the presence of oxygen in the reaction mixture and environment is detrimental to RAFT-mediated polymerization, its removal to trace levels is readily accomplished via several freeze&#8211;evacuate&#8211;thaw cycles while maintaining the Schlenk tube reaction vessel under high vacuum.
It should be noted that the molar ratio of different monomers in the reaction can be adjusted as needed. Also, by varying the amount of RAFT agent used, the length of the resulting polymers can be controlled18. However, the molar ratio of the RAFT agent to initiator should always be greater than two to assure the low dispersity of the product. Under these conditions, the evolution of the copolymerization is steady, and the reproducibility of the reaction is very high. That being said, it is unlikely that one obtains a totally uniform distribution of all participating monomers within a statistical copolymer, due to their different polymerization speeds. Characterizing the distribution of different monomers within the polymer is still very challenging.
The post-modification method, presented here, is both simpler and more amenable to the use of a wider selection of fluorescent labels, compared to other protocols applied to label glycopolymers2,11. These would include many of the water-soluble amine-reactive fluorophores, quantum dots, biotins, and others. The binding specificities of the synthesized, labeled glycopolymers are readily verifiable using lectins with known binding affinities. PMA-GAEMA possessing no pendant sugar is an appropriate negative control. Glycopolymers with different fluorescent labels prepared via this route have been successfully used in investigations of lectin-mediated bacterial binding14. As presented, this facile and efficient preparation of statistical fluorescent glycopolymers should provide great potential to a wide variety of glycobiological research.

In the past two decades, investigations with synthetic glycopolymers have undergone slow but continual development, demonstrating significant potential in examining infectious mechanisms that include research which focuses on lectin recognition processes1-3. Since synthetic glycopolymers possessing multivalent sugar moieties exhibit much higher lectin-binding efficacies, as compared to monovalent carbohydrates, they are of great demand in the glycobiology field3. Of particular interest in clinical research is the use of fluorescent glycopolymers to characterize the lectin-mediated bacterial binding with carbohydrates available on human respiratory cell surfaces and mucous glycoprotein. Early in vitro studies employed commercially available polyacrylamide-based glycopolymers in bacterial binding tests. Several of these probes showed promising results, but raised concerns regarding, obtainability, and lot-to-lot variances in both polymer molecular weight and glycoepitope content. An economical in-laboratory protocol was developed which would provide for a satisfactory control of structure content, size, and purity of synthetic glycopolymers targeting bacterial lectins.
In search for a suitable synthetic approach to glycopolymers, a relatively new polymerization technique was tested using a type of controlled radical polymerization that employed reversible addition-fragmentation chain-transfer (RAFT) agents4. Such RAFT reagents have recently been used in a few glycopolymer preparations5-7. Compared with other glycopolymer preparation protocols, RAFT-mediated polymerizations demonstrate several advantages, including the tolerance to a variety of monomer structures and reaction conditions, potential compatibility with aqueous solutions, and low size dispersity of the desired polymeric products8,9. Of notable interest are protocols for preparation of RAFT-based tri-component glycopolymers, allowing control of compositions of different monomers, each of which may have distinct functions10-13. However, most of the previous research endeavors either lacked anomeric pendant carbohydrates10, or employed stepped polymerizations resulting in tri-block copolymers, that consist of covalently linked homopolymers, which often serve different purposes than statistical polymers which are copolymers in which the sequence of monomer residues follow a statistical rule9-13.
Recently, employing the thiocarbonylthio RAFT compound (4-cyanopentanoic acid)-4-dithiobenzoate in an aqueous environment, the preparation of a group of RAFT-based linear tri-component statistical glycopolymers containing specific pendant sugars and their application in lectin-mediated bacterial binding tests was reported14. The overall goal of this method, presented in a visual manner, is to prepare tri-component statistical fluorescent glycopolymers via RAFT-controlled copolymerization. Because of the ease of the one-step polymerization protocol, the fine control over the polymer length and compositions, and the high reproducibility of the reaction, this protocol can be readily applied to other RAFT-based syntheses of glycopolymers with desired structures.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4">Reagent</td>
		</tr>
		<tr>
			<td>Lactobionic acid</td>
			<td>Sigma-Aldrich</td>
			<td>153516</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Gluconolactone&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>G2164</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(2-hydroxyethyl) acrylamide (HEAA)</td>
			<td>Sigma-Aldrich</td>
			<td>697931</td>
			<td></td>
		</tr>
		<tr>
			<td>Orange II sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>O8126</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroquinone monomethyl ether (MEHQ)</td>
			<td>Sigma-Aldrich</td>
			<td>54050</td>
			<td>Polymerization inhibitor</td>
		</tr>
		<tr>
			<td>N-(2-aminoethyl) methacrylamide hydrochloride (AEMA)</td>
			<td>Polysciences, Inc</td>
			<td>24833-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Fisher Scientific</td>
			<td>BP-616</td>
			<td></td>
		</tr>
		<tr>
			<td>Anion-exchange resin IRN-78 hydroxide-form, 80 mesh</td>
			<td>Sigma-Aldrich</td>
			<td>10343-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Cation-exchange resin 50Wx8, 200 mesh</td>
			<td>Sigma-Aldrich</td>
			<td>217514</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum oxide, ~150 mesh&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A1522</td>
			<td>Type WN-6, Neutral, Activity Grade Super I</td>
		</tr>
		<tr>
			<td>Ninhydrin</td>
			<td>Sigma-Aldrich</td>
			<td>N4876</td>
			<td>An ethanol solution of 0.2 % ninhydrin was used in the test</td>
		</tr>
		<tr>
			<td>4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>722995</td>
			<td>RAFT agent</td>
		</tr>
		<tr>
			<td>4,4&#8242;-Azobis(4-cyanovaleric acid)</td>
			<td>Sigma-Aldrich</td>
			<td>11588</td>
			<td>Polymerization initiator</td>
		</tr>
		<tr>
			<td>Carboxyfluorescein succinimidyl ester&#160;</td>
			<td>Life Technologies</td>
			<td>C1157</td>
			<td></td>
		</tr>
		<tr>
			<td>Erythrina Cristagalli lectin coated agarose bead</td>
			<td>Vector Laboratorie</td>
			<td>AL-1143&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Solvent</td>
		</tr>
		<tr>
			<td>dH2O</td>
			<td></td>
			<td></td>
			<td>Produced by Barnstead water purification system, 18 megOhm-cm</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>A461-4</td>
			<td>ACS grade or better</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A454-4</td>
			<td>ACS grade or better</td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818-100</td>
			<td>ACS grade or better</td>
		</tr>
		<tr>
			<td>Dimethylformamide</td>
			<td>Sigma-Aldrich</td>
			<td>22705</td>
			<td>ACS grade or better</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>A929-4</td>
			<td>ACS grade or better</td>
		</tr>
		<tr>
			<td colspan="4">Equipment</td>
		</tr>
		<tr>
			<td>Dialysis membrane (MWCO: 3,500)</td>
			<td>Spectrum Labs</td>
			<td>132720</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol analytical standard standard</td>
			<td>Sigma-Aldrich</td>
			<td>O2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Schlenk tube, 1 mL</td>
			<td>Quark Glass</td>
			<td></td>
			<td>Customized</td>
		</tr>
		<tr>
			<td>TSK-GEL G4000 PWxl&#160;</td>
			<td>Tosoh Bioscience&#160;</td>
			<td>8022</td>
			<td>Used for GPC analysis of the glycopolymers</td>
		</tr>
		<tr>
			<td>Empower 3 with GPC/SEC package</td>
			<td>Waters Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Waters Alliance HPLC system&#160;</td>
			<td>Waters Corporation</td>
			<td></td>
			<td>Equipped with refractive index detector (Waters 2414) and fluorescence detector (Waters 2475)</td>
		</tr>
		<tr>
			<td>Avance III 800 MHz NMR Spectrometer</td>
			<td>Brucker Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BX43 fluorescence microscope</td>
			<td>Olympus Corporation</td>
			<td></td>
			<td>Used with FITC filter in the glycopolymer binding test</td>
		</tr>
		<tr>
			<td>Rotavap / Rotoevaporator</td>
			<td>Heidolph</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fritted disc funnel</td>
			<td>Fisher Scientific</td>
			<td>10-310-109</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Labconco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immunofluorescence microscope slide</td>
			<td>Polysciences</td>
			<td>18357-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Revco Ultima Plus -80C Freezer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Vacuum Bag and Hand Pump</td>
			<td>Ziploc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Pump, Direct Drive, Maxima C Plus</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Gauge</td>
			<td>Sargent-Welch</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

facile and efficient preparation of tri-component fluorescent glycopolymers via raft-controlled polymerization

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and efficient synthesis of well-controlled fluorescent statistical glycopolymers using reversible addition-fragmentation chain-transfer (RAFT)-based polymerization is demonstrated. The synthesis starts with the preparation of &#946;-galactose-containing glycomonomer 2-lactobionamidoethyl methacrylamide obtained by reaction of lactobionolactone and N-(2-aminoethyl) methacrylamide (AEMA). 2-Gluconamidoethyl methacrylamide (GAEMA) is used as a structural analog lacking a terminal &#946;-galactoside. The following RAFT-mediated copolymerization reaction involves three different monomers: N-(2-hydroxyethyl) acrylamide as spacer, AEMA as target for further fluorescence labeling, and the glycomonomers. Tolerant of aqueous systems, the RAFT agent used in the reaction is (4-cyanopentanoic acid)-4-dithiobenzoate. Low dispersities (&#8804;1.32), predictable copolymer compositions, and high reproducibility of the polymerizations were observed among the products. Fluorescent polymers are obtained by modifying the glycopolymers with carboxyfluorescein succinimidyl ester targeting the primary amine functional groups on AEMA. Lectin-binding specificities of the resulting glycopolymers are verified by testing with corresponding agarose beads coated with specific glycoepitope recognizing lectins. Because of the ease of the synthesis, the tight control of the product compositions and the good reproducibility of the reaction, this protocol can be translated towards preparation of other RAFT-based glycopolymers with specific structures and compositions, as desired.

Synthesis of glycomonomer
Lactobionic acid was used herein as an example for the preparation of glycomonomers. Using methods in the initial report on the synthesis of LAEMA11, varied yields in the preparation with unsatisfactory purity were observed. The modified purification method using cation and anion exchange resins to remove unreacted starting material offered stable product yield and high purity, which is confirmed by 1H and 13C NMR spectroscopy (Figure 1).
RAFT glycopolymer synthesis and post-modification of glycopolymers with fluorophores
In contrast to the block-glycopolymers prepared through stepped RAFT polymerizations, this one-step copolymerization protocol provides a uniform glycomonomer distribution throughout the polymer backbone. The glycopolymers shown here contain 20 mol% of glycomonomer, 77 mol% of HEAA as a spacer, and 3 mol% of AEMA as a target for post-modifications (see Figure 2). 1H- and 13C-NMR spectroscopy confirmed the structures of PMA-LAEMA and PMA-GAEMA (Figures 3 and 4). As shown in Figure 5, when plotted against the GPC elution profiles of the glycopolymer synthesized without RAFT, both PMA-LAEMA and PMA-GAEMA have low dispersities, proving the efficacy of the RAFT approach. As expected, PMA-GAEMA has a Mn smaller than that of PMA-LAEMA due to PMA-GAEMA&#8217;s lack of a pendant sugar. Analysis of the carbohydrates and primary amine functional groups content of the RAFT glycopolymers revealed that the ratio of monomers in the product glycopolymers is consistent with the stoichiometric ratio of starting monomers employed in the RAFT-mediated polymerization reaction (Table 1). This signifies a tight control of the monomer compositions in the synthesized glycopolymers, as designed.
Reaction of primary amine functional groups with activated fluorophores is a widely-used technique in protein labeling. This technique was employed here to label purified glycopolymers with carboxyfluorescein. Following post-modification, fluorescent polymers were obtained (Figure 6). No degradation of the fluorescein-labeled polymers in the reaction was detected by GPC analysis (data not shown).
Binding tests of the synthetic glycopolymers with lectin-coated agarose beads 
To assess the lectin-binding specificity of the synthesized glycopolymers, lectin-coated agarose beads with known carbohydrate binding specificity was used. Erythrina crista-galli lectin (ECL), employed in the experiments, has a binding specificity towards &#946;-D-galactoside. Figure 7A clearly demonstrates that PMA-LAEMA-Fluorescein, which contains &#946;-D-galactoside as a pendant carbohydrate, exhibited strong binding with the ECL lectin. In contrast, the negative binding to the ECL of the glycopolymer PMA-GAEMA-Fluorescein, which does not possess a pendant sugar, is shown in Figure 7B. This result exemplifies the binding effectiveness and affinity of the synthesized fluorescent glycopolymer.
<img alt="Figure 1" src="/files/ftp_upload/52922/52922fig1.jpg" /> 
Figure 1. Assigned 1H- (a) and 13C-NMR (b) spectra (D2O) for LAEMA. (This figure has been modified from Wang et al.14) <a href="https://www.jove.com/files/ftp_upload/52922/52922fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52922/52922fig2.jpg" /> 
Figure 2. Schematic illustration of the synthesis of fluorescent glycopolymer PMA-LAEMA containing &#946;-galactoside as the pendant sugar. <a href="https://www.jove.com/files/ftp_upload/52922/52922fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52922/52922fig3.jpg" /> 
Figure 3. Assigned 1H- (A) and 13C-NMR (B) spectra (D2O) for PMA-LAEMA glycopolymer. (This figure has been modified from Wang et al.14) <a href="https://www.jove.com/files/ftp_upload/52922/52922fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52922/52922fig4.jpg" /> 
Figure 4. Assigned 1H- (A) and 13C-NMR (B) spectra (D2O) for PMA-GAEMA. (This figure has been modified from Wang et al.14) <a href="https://www.jove.com/files/ftp_upload/52922/52922fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52922/52922fig5.jpg" /> 
Figure 5. Gel permeation chromatography traces of RAFT-based PMA-GAEMA and PMA-LAEMA prepared with and without using RAFT agent. In contrast to the PMA-LAEMA prepared without RAFT agent (blue), RAFT-based PMA-LAEMA (green) has a much lower dispersity (Mw/Mn). RAFT-based PMA-GAEMA (red) and PMA-LAEMA have similar GPC profiles, but the former has a smaller Mn due to the absence of any pendant sugars. <a href="https://www.jove.com/files/ftp_upload/52922/52922fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52922/52922fig6.jpg" /> 
Figure 6. PMA-LAEMA before and after post-modification with fluorophore. (A) Compared with white non-labeled glycopolymer (left tube), fluorescein-labeled PMA-LAEMA shows a strong yellow color (right tube). (B) Under UV, non-labeled PMA-LAEMA (left tube, 1 mg/ml in PBS) is dark and presents with no fluorescence, whereas fluorescein-labeled PMA-LAEMA (right tube, 1 mg/ml in PBS) shows strong green fluorescence. <a href="https://www.jove.com/files/ftp_upload/52922/52922fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/52922/52922fig7.jpg" /> 
Figure 7. Erythrina crista-galli lectin (ECL)-coated agarose beads bind &#946;-D-galactoside containing glycopolymers, and not those not possessing a pendant sugar. (A) PMA-LAEMA-Fluorescein (3 &#181;g) demonstrated strong binding with ECL, whereas in (B) PMA-GAEMA-Fluorescein, which possesses no pendant &#946;-D-galactoside residue, showed no binding with the lectin-coated beads. Scale bar = 100 &#181;m.
Table 1. Targeting valuesa of synthetic parameters and actual compositions of the glycopolymers. a) Targeting values, values that are desired of the products; b) DP, degree of polymerization; c) NA, not available. <a href="https://www.jove.com/files/ftp_upload/52922/52922fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody style="vertical-align:middle;text-align:center">
		<tr>
			<td style="width:120px"></td>
			<td style="width:40px">DPb&#160;</td>
			<td style="width:70px">Dispersity</td>
			<td style="width:120px">Actual content of glycomonomers 
			mol %</td>
			<td>Actual content of primary amine 
			mol %</td>
		</tr>
		<tr>
			<td>Targeting values</td>
			<td>100</td>
			<td>&#60; 1.3</td>
			<td>20</td>
			<td>3</td>
		</tr>
		<tr>
			<td>PMA-LAEMA</td>
			<td>99</td>
			<td>1.26</td>
			<td>19</td>
			<td>3.2</td>
		</tr>
		<tr>
			<td>PMA-GAEMA</td>
			<td>89</td>
			<td>1.32</td>
			<td>NAc</td>
			<td>2.7</td>
		</tr>
	</tbody>
</table>

Watch this Scientific Journal Video about Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization at JoVE.com

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

An efficient, three-step synthesis of RAFT-based fluorescent glycopolymers, consisting of glycomonomer preparation, copolymerization, and post-modification, is demonstrated. This protocol can be used to prepare RAFT-based statistical glycopolymers with desired structures.

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and ...

facile-efficient-preparation-tri-component-fluorescent-glycopolymers

Research

JoVE Journal

Chemistry

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Facile Preparation of 4-Substituted Quinazoline Derivatives

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted 2-aminobenzophenones and thiourea in the presence of dimethyl sulfoxide (DMSO). This is a unique complementary reaction system in which thiourea undergoes thermal decomposition to form carbodiimide and hydrogen sulfide, where the former reacts with 2-aminobenzophenone to form 4-phenylquinazolin-2(1H)-imine intermediate, whilst hydrogen sulfide reacts with DMSO to give methanethiol or other sulfur-containing molecule which then functions as a complementary reducing agent to reduce 4-phenylquinazolin-2(1H)-imine intermediate into 4-phenyl-1,2-dihydroquinazolin-2-amine. Subsequently, the elimination of ammonia from 4-phenyl-1,2-dihydroquinazolin-2-amine affords substituted quinazoline derivative. This reaction usually gives quinazoline derivative as a single product arising from 2-aminobenzophenone as monitored by GC/MS analysis, along with small amount of sulfur-containing molecules such as dimethyl disulfide, dimethyl trisulfide, etc. The reaction usually completes in 4-6 hr at 160 &#186;C in small scale but may last over 24 hr when carried out in large scale. The reaction product can be easily purified by means of washing off DMSO with water followed by column chromatography or thin layer chromatography.

A protocol for facile preparation of 4-substituted quinazoline derivatives from 2-aminobenzophenones, thiourea and dimethyl sulfoxide is presented.

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted ...

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by &#960;-&#960; Stacking Interactions

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE). Characterization of the copolymer revealed a narrow molecular distribution (PDI &#60; 1.03) and confirmed the degree of polymerization of mPEG122-b-(PheGE)15. The critical micelle concentration of the copolymer was evaluated using an established fluorescence method with the aggregation behavior evaluated by dynamic light scattering and transmission electronic microscopy. The potential of the copolymer for use in drug delivery applications was evaluated in a preliminary manner including in vitro biocompatibility, loading and release of the hydrophobic anti-cancer drug doxorubicin (DOX). A stable micelle formulation of DOX was prepared with drug loading levels up to 14% (wt%), drug loading efficiencies &#62; 60% (w/w) and sustained release of drug over 4 days under physiologically relevant conditions (acidic and neutral pH, presence of albumin). The high drug loading level and sustained release is attributed to stabilizing &#960;-&#960; interactions between DOX and the core-forming block of the micelles.

Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by &amp;#960;-&amp;#960; Stacking Interactions

The key steps of living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE) are described. The resulting block copolymer micelles (BCMs) were loaded with doxorubicin 14% (wt%) and sustained release of drug over 4 days under physiologically relevant conditions was obtained.

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl ...

The Preparation and Properties of Thermo-reversibly Cross-linked Rubber Via Diels-Alder Chemistry

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ethylene-propylene rubber, grafted with maleic anhydride, is thermo-reversibly cross-linked in two steps. The pending anhydride moieties are first modified with furfurylamine to graft furan groups to the rubber backbone. These pendant furan groups are then cross-linked with a bis-maleimide via a Diels-Alder coupling reaction. Both reactions can be performed under a broad range of experimental conditions and can easily be applied on a large scale. The material properties of the resulting Diels-Alder cross-linked rubbers are similar to a peroxide-cured ethylene/propylene/diene rubber (EPDM) reference. The cross-links break at elevated temperatures (&#62; 150 &#176;C) via the retro-Diels-Alder reaction and can be reformed by thermal annealing at lower temperatures (50-70 &#176;C). Reversibility of the system was proven with infrared spectroscopy, solubility tests and mechanical properties. Recyclability of the material was also shown in a practical way, i.e., by cutting a cross-linked sample into small parts and compression molding them into new samples displaying comparable mechanical properties, which is not possible for conventionally cross-linked rubbers.

A simple two-step approach involving rubber modification and cross-linking yields fully reworkable, elastic rubber products.

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ...

A Simple and Efficient Protocol for the Catalytic Insertion Polymerization of Functional Norbornenes

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic nature of the monomer is conserved. The resulting polymer, polynorbornene, has a very high glass transition temperature, Tg, and interesting optical and electrical properties. However, the polymerization of functional norbornenes by this mechanism is complicated by the fact that the endo substituted norbornene monomer has, in general, a very low reactivity. Furthermore, the separation of the endo substituted monomer from the exo monomer is a tedious task. Here, we present a simple protocol for the polymerization of substituted norbornenes (endo:exo ca. 80:20) bearing either a carboxylic acid or a pendant double bond. The process does not require that both isomers be separated, and proceeds with low catalyst loadings (0.01 to 0.02 mol%). The polymer bearing pendant double bonds can be further transformed in high yield, to afford a polymer bearing pendant epoxy groups. These simple procedures can be applied to prepare polynorbornenes with a variety of functional groups, such as esters, alcohols, imides, double bonds, carboxylic acids, bromo-alkyls, aldehydes and anhydrides.

We describe the catalytic insertion polymerization of 5-norbornene-2-carboxylic acid and 5-vinyl-2-norbornene to form functional polymers with a very high glass transition temperature.

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic ...

Facile Preparation of Ultrafine Aluminum Hydroxide Particles with or without Mesoporous MCM-41 in Ambient Environments

An aqueous suspension of nanogibbsite was synthesized via the titration of aluminum aqua acid [Al(H2O)6]3+ with L-arginine to pH 4.6. Since the hydrolysis of aqueous aluminum salts is known to produce a wide array of products with a wide range of size distributions, a variety of state-of-the-art instruments (i.e., 27Al/1H NMR, FTIR, ICP-OES, TEM-EDX, XPS, XRD, and BET) were used to characterize the synthesis products and identification of byproducts. The product, which was comprised of nanoparticles (10-30 nm), was isolated using gel permeation chromatography (GPC) column technique. Fourier transform infrared (FTIR) spectroscopy and powder X-ray diffraction (PXRD) identified the purified material as the gibbsite polymorph of aluminum hydroxide. The addition of inorganic salts (e.g., NaCl) induced electrostatic destabilization of the suspension, thereby agglomerating the nanoparticles to yield Al(OH)3 precipitate with large particle sizes. By utilizing the novel synthetic method described here, Al(OH)3 was partially loaded inside the highly ordered mesoporous framework of MCM-41, with average pore dimensions of 2.7 nm, producing an aluminosilicate material with both octahedral and tetrahedral Al (Oh/Td = 1.4). The total Al content, measured using energy-dispersive X-ray spectrometry (EDX), was 11% w/w with a Si/Al molar ratio of 2.9. A comparison of bulk EDX with surface X-ray photoelectron spectroscopy (XPS) elemental analysis provided insight into the distribution of Al within the aluminosilicate material. Furthermore, a higher ratio of Si/Al was observed on the external surface (3.6) as compared to the bulk (2.9). Approximations of O/Al ratios suggest a higher concentration of Al(O)3 and Al(O)4 groups near the core and external surface, respectively. The newly developed synthesis of Al-MCM-41 yields a relatively high Al content while maintaining the integrity of the ordered silica framework and can be used for applications where hydrated or anhydrous Al2O3 nanoparticles are advantageous.

An ultrafine aluminum hydroxide nanoparticle suspension was prepared via the controlled titration of [Al(H2O)]3+ with L-arginine to pH 4.6 with and without cage-effect confinement within mesoporous channels of MCM-41.

An aqueous suspension of nanogibbsite was synthesized via the titration of aluminum aqua acid [Al(H2O)6]3+ with L-arginine to pH 4.6. Since the hydrolysis ...

Facile Preparation of (2Z,4E)-Dienamides by the Olefination of Electron-deficient Alkenes with Allyl Acetate

Direct cross-coupling between two alkenes via vinylic C-H bond activation represents an efficient strategy for the synthesis of butadienes with high atomic and step economy. However, this functionality-directed cross-coupling reaction has not been developed, as there are still limited directing groups in practical use. In particular, a stoichiometric amount of oxidant is usually required, producing a large amount of waste. Due to our interest in novel 1,3-butadiene synthesis, we describe the ruthenium-catalyzed olefination of electron-deficient alkenes using allyl acetate and without external oxidant. The reaction of 2-phenyl acrylamide and allyl acetate was chosen as a model reaction, and the desired diene product was obtained in 80% isolated yield with good stereoselectivity (Z,E/Z,Z = 88:12) under optimal conditions: [Ru(p-cymene) Cl2]2 (3 mol %) and AgSbF6 (20 mol %) in DCE at 110 &#186;C for 16 h. With the optimized catalytic conditions in hand, representative &#945;- and/or &#946;-substituted acrylamides were investigated, and all reacted smoothly, regardless of aliphatic or aromatic groups. Also, differently N-substituted acrylamides have proven to be good substrates. Moreover, we examined the reactivity of different allyl derivatives, suggesting that the chelation of acetate oxygen to the metal is crucial for the catalytic process. Deuterium-labeled experiments were also conducted to investigate the reaction mechanism. Only Z-selective H/D exchanges on acrylamide were observed, indicating a reversible cyclometalation event. In addition, a kinetic isotope effect (KIE) of 3.2 was observed in the intermolecular isotopic study, suggesting that the olefinic C-H metalation step is probably involved in the rate-determining step.

Facile Preparation of 2Z,4E-Dienamides by the Olefination of Electron-deficient Alkenes with Allyl Acetate

The ruthenium-catalyzed olefination of electron-deficient alkenes with allyl acetate is described here. By using aminocarbonyl as a directing group, this external oxidant-free protocol has high efficiency and good stereo- and regioselectivity, opening a novel synthetic route to (Z,E)-butadiene skeletons.

Direct cross-coupling between two alkenes via vinylic C-H bond activation represents an efficient strategy for the synthesis of butadienes with high ...

Preparation of Hollow Polystyrene Particles and Microcapsules by Radical Polymerization of Janus Droplets Consisting of Hydrocarbon and Fluorocarbon Oils

In this article, we have demonstrated a method for producing hollow particles and microcapsules using oil droplets consisting of hydrocarbon oil (styrene) and fluorocarbon oil (perfluoro-n-octane, PFO) in aqueous surfactant (sodium dodecylsulfate, SDS) solutions. Since fluorocarbon oils are immiscible with hydrocarbon oils, the two oils are separated. Emulsions are prepared by stirring styrene/PFO/aqueous SDS solution mixtures at 80 &#176;C. The type of emulsions and the morphology of droplets in the emulsions are observed by light microscope and scanning confocal fluorescence microscope. It is found that oil droplets with Janus-type morphologies consisting of mutually immiscible styrene and PFO are formed in aqueous SDS solutions. Polystyrene particles are fabricated by radical polymerization of the ternary mixtures of styrene/PFO/aqueous SDS solution at 80 &#176;C. The morphologies of the polystyrene are confirmed by scanning electron microscopy and scanning transmission electron microscopy observations. These observations show the preparation of hollow polystyrene particles with a single hole on the surface. To our knowledge, this method is a novel strategy using the immiscibility of hydrocarbon and fluorocarbon oils. The hollow particles can also be applied to the preparation of microcapsules.

A protocol for the fabrication of hollow polymer particles and microcapsules by radical polymerization using emulsions consisting of styrene, perfluoro-n-octane, and aqueous SDS (sodium dodecylsulfate) solution is presented.

In this article, we have demonstrated a method for producing hollow particles and microcapsules using oil droplets consisting of hydrocarbon oil (styrene) ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...