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

1.合成Glycomonomer 2- Lactobionamidoethyl甲基丙烯酰胺<ol><li>溶解2克乳糖酸3.0毫升无水甲醇和缓慢下降明智的时尚加无水乙醇直到溶液刚变浑浊，然后通过旋转蒸发除去溶剂。 </li><li>溶解残渣，从步骤1.1，溶在3.0ml无水甲醇中，并再次，慢慢加入无水乙醇直至刚好混浊，然后经由旋转蒸发蒸去溶剂。重复此步骤3次，得到lactobiono -1,5-内酯（1.94克，98％产率）。此产品具有足够的纯度在下面反应中使用。 </li><li> 3.0毫升甲醇中添加1.0克lactobionolactone与N - （2-氨基乙基）甲基丙烯酰胺（AEMA，0.58克）和氢醌单甲醚（MEHQ，1.0毫克），自聚合的抑制剂，2.0毫升甲醇中，接着以1.0毫升三乙胺。在RT搅拌48小时。 </li><li>加20 500ml的去离子H 2澳（DH 2 </子&gt; O）的反应烧瓶中，然后通过蒸发除去甲醇和自由三乙胺通过旋转蒸发浓缩至干。 </li><li>以除去任何剩余的乳糖酸，加20的dh 2 O中的溶液，然后通过阴离子交换柱的水溶液（OH -型 ，10毫米×20毫米）到含有1.0毫克的MEHQ接收烧杯中。 </li><li>除去三乙胺，在步骤1.5中产生的，通过蒸发，通过旋转蒸发浓缩至干。 </li><li>添加20毫升的dh 2 O，并通过缓慢加入阳离子交换树脂（H +型 ）的1毫克等分试样直至不再茚三酮反应性材料是可检测的除去未反应AEMA。监视除去通过取该溶液的1微升等份各树脂加入后，将其应用到一个薄层层析板，然后喷涂板在乙醇溶液中的2％茚三酮。当没有深蓝色的颜色被观察到的开发时，板被加热到90℃保持1分钟，终点已经到达。 </li><lI&gt;通过多孔玻璃漏斗过滤溶液，将滤液转移到一个试管中，冷冻的样品在-80℃下，然后冷冻干燥。 </li><li>从样品中取出的MEHQ通过在甲醇（约0.5毫升）的最低量溶解的冷冻干燥物，然后添加冷的无水丙酮（-20℃，15毫升）以沉淀产物。用烧结玻璃漏斗收集沉淀，通过过滤，然后干燥该沉淀物在干燥器中在真空下，得到2- lactobionamidoethyl甲基丙烯酰胺（LAEMA），为灰白色粉末（0.94克，68％产率）。此产品具有足够的纯度在下面反应中使用。 </li></ol> 2.合成单体2- Gluconamidoethyl甲基丙烯酰胺注：2-gluconamidoethyl甲基的制备（GAEMA），它不具有侧链的糖，改编自发布方法15。 <ol><li>添加2.0克AEMA的溶解在10毫升甲醇中的溶液的D-葡萄糖酸内酯（1.6克）在30毫升甲醇中，并在搅拌下，慢慢地添加1.6毫升三乙胺。 </li><li>在RT搅拌24小时，进行反应。 </li><li>用烧结玻璃漏斗过滤沉淀出的产品，并冲洗沉淀三次，每次10毫升异丙醇中，然后用10毫升无水丙酮。干燥在真空下干燥器沉淀的产物。 </li></ol> 3. R​​AFT Glycopolymer合成<ol><li>以除去抑制剂的MEHQ存在于商业的N - （2-羟基乙基）丙烯酰胺（HEAA），加入1 ml HEAA至2ml微量离心管中，随后加入0.5克三氧化二铝纳米颗粒。离心管中，在300×g离心30秒，并使用顶层HEAA在以下的反应。 </li><li>小心加入32.8毫克LAEMA的（70.0微摩尔），1.7毫克AEMA的（10.5微摩尔）和27.5微升HEAA（270微摩尔），全部溶解在0.4的dh 2 O毫升，以良好的清洁1毫升Schlenk管，从而有一个monom3：77 20 ER摩尔比。 </li><li>在平行的反应中，为了产生对照聚合物，其不具有任何挂件糖，以代替在步骤3.2采用LAEMA的，在反应代替21.4毫克GAEMA的（70.0微摩尔）。 </li><li>到相应的Schlenk管（ 即 3.2或3.3），依次添加含0.53毫克（4-氰基戊酸）-4-二硫代苯甲酸酯（1.9微摩尔，RAFT试剂）和50微升DMF中含有250微克的DMF中的50微升4,4&#39;-偶氮双（4-氰基戊酸）（0.9微摩尔，引发剂）。通过手指轻轻拍打混合。 </li><li>冻结包含在Schlenk管内容采用干冰：乙醇浴（75克干冰在100ml乙醇中），内10-50毫乇施加真空，然后关闭的Schlenk阀并允许溶液缓慢解冻至室温。重复此冷冻撤离冻融循环两次。确保所有的试剂，最后解冻后解散。 </li><li>将舒伦克管插入密封塑料BA克，疏散袋的空气，然后密封。含有Schlenk管袋转移到水浴预热在70℃孵育24小时。 </li><li>在Schlenk管中的溶液小心地转移到准备好的透析袋（MWCO = 3500），和透析针对卫生署2 O（10×2升）24小时，改变的dh 2 O每隔一小时的前8个小时。以下透析，从透析管转移样品到一试管中，冷冻的样品在-80℃下，然后冻干它。 </li></ol>注意：得到的统计的聚甲基丙烯酰胺/丙烯酰胺（PMA）含有侧链4-ö-β-D-吡喃半乳糖基-D-葡糖酰胺（lactobionamide）（来自步骤3.2）或D-葡糖酰胺（来自步骤3.3）共聚物，分别是获得。为便于讨论，这两个glycopolymers简写为PMA-LAEMA和PMA-GAEMA分别。 4.后期修饰Glycopolymers与荧光团</P&gt; <ol><li>溶解5.0毫克含有〜0.9微摩尔的伯胺官能团的glycopolymer PMA-LAEMA或PMA-GAEMA在0.9磷酸盐缓冲盐水（PBS，0.1M磷酸钠，0.15M氯化钠，pH值7.5）中的溶液，分别</li><li>慢慢在100μlDMF中添加0.6毫克的羧基琥珀酰亚胺基酯向快速搅拌下的解决方案。轻轻搅拌该反应16小时，在黑暗中在室温。 </li><li>虽然避光，装载样品放入事先准备好的透析管（MWCO = 3500），并针对透析卫生署2 O（2升）16小时，更换透析液每隔一小时前8小时。以下透析，从透析管转移样品到一试管中，冷冻的样品在-80℃下，然后冻干它。 </li></ol>注意：按照冻干，荧光glycopolymers PMA-LAEMA荧光素和PMA-GAEMA荧光素，分别被获得。 在甘氨酸5表征共聚物<ol><li>确定数均分子量（M n）时，重均分子量（M w）的与glycopolymers的上装有凝胶渗透色谱法（GPC）的软件，其GPC商用HPLC系统的分散度（M W / M n）的列适合分子量的兴趣，和折射率检测器，用0.1M的Tris / 0.1M的氯化钠缓冲液（pH 7）作为洗脱剂以0.6ml / min的14的流速。使用聚乙二醇标准作为分子量标准（分子量：200-1,200,000克/摩尔）。 </li><li>量化glycopolymers 16内伯胺官能团的实际浓度。根据公布的方法分析17总碳水化合物的含量合成glycopolymers的。 </li><li>通过核磁共振光谱执行14结构组成和glycomonomers LAEMA纯度测试，GAEMA和glycopolymers PMA-LAEMA，PMA-GAEMA在D 2 O中。 </li></ol>合成Glycopolymers与6.结合凝集素试验涂琼脂糖珠<ol><li>添加1.5毫升PBS中至50μl悬浮液的鸡冠刺桐凝集素（ECL）包被的琼脂糖珠，离心机在300×g离心1分钟，并小心地取出和处理上清液。重复此步骤两次，然后重悬珠子在0.5ml PBS中。 </li><li>加入3 PMA-LAEMA荧光素或PMA-GAEMA荧光素（阴性对照）在6微升PBS至珠粒悬浮微克，孵育混合物，在黑暗中，在室温进行1小时。 </li><li>用1.5毫升PBS清洗3次，并重新悬浮珠0.2毫升的PBS洗涤混合物。加载一个等分试样（4微升）到一个井上的免疫荧光显微镜载玻片（聚四氟乙烯涂层），盖上盖玻片，并使用FITC滤波用荧光显微镜观察（激发波长：467-498纳米，发射波长：513- 566纳米）和10X客观的检查T的结合他荧光glycopolymers与珠14。 </li></ol>

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The authors have nothing to disclose.

一种简便，高效的协议进行的RAFT基三组分荧光glycopolymers，具有和不具有侧链的碳水化合物，以及它们在一个凝集素结合试验使用，证明本报告中。该协议开始的glycomonomers LAEMA和GAEMA的准备。通过单步的RAFT控制共聚，glycopolymers具有可重复的产率，预测的单体组合物和低分散​​性，能得到。以下修正后glycopolymers用羧基琥珀酰亚胺酯，将所得各荧光标记glycopolymer的结合是很容易可测试其凝集素结合特异性。 在初始制备的步骤是在随后glycopolymer合成所采用的glycomonomers的，容易获得的乳糖酸和葡糖酸内酯被利用。从理论上讲，任何感兴趣的碳水化合物，从单糖到复杂的寡糖，可以转换器具d，来glycomonomers由缀合的靶糖到伯羟基上的葡萄糖的C6。以下的氧化还原葡萄糖残基，和其后续的脱水内酯，产物然后可以容易地与上AEMA的伯胺反应形成相应的glycomonomer反应。这条路线的进一步的实例可以看出，在最近的一份报告14。应当指出的是，在启动任何聚合步骤之前，MEHQ，一种有效的聚合抑制剂，必须从所有单体和glycomonomer制剂在使用前除去。这可容易的通过使用甲醇的最小量以溶解该具有MEHQ然后立即用丙酮把它在-20℃以沉淀在高收率抑制剂无产品的glycomonomer完成。 至关重要的自由基聚合计划，注重细节和单体纯度的强调。由于是典型在RAFT聚合体系，它由自由基源，在RAFT试剂，单体和溶剂。在这种可视呈现，单步RAFT聚合系统进行说明，着重于生产从反应混合物具有在水溶液中三种不同的单体生成的统计共聚物。两个独立的RAFT介导的反应被呈现，其中一个利用glycomonomer一个拥有一个挂件，非还原性糖类末端（ 即，β-D-半乳糖），以及其他的，具有无界糖残基的多元醇。常见的两种RAFT介导的反应是单体具有在作为间隔分子奇异羟基，而另一个具有一个游离胺为修正后与氨基反应的荧光团。 因为氧在反应混合物中，环境中的存在是有害的RAFT介导的聚合，其去除跟踪水平通过几个冷冻EVA是容易实现cuate - 解冻循环，同时保持在高真空下的Schlenk管反应容器中。 应当指出的是，在反应的不同单体的摩尔比可根据需要进行调整。另外，通过改变RAFT试剂的使用量，所得到的聚合物的长度，可以控制18。然而，在RAFT试剂与引发剂的摩尔比应始终大于二，以确保产品的低分散度。在这些条件下，共聚的演变是稳定，并且该反应的再现性是非常高的。如此说来，这是不可能的，可以得到所有参与单体的统计共聚物内的完全均匀分布，由于它们的不同的聚合速度。表征不同单体的聚合物中的分布仍是非常具有挑战性的。 修正后的方法，在这里提出，是既简单，更amenabl以e为使用更宽选择的荧光标记，比施加到标签glycopolymers 2,11其他协议。这些将包括许多水溶性胺 - 反应性荧光团，量子点，生物素，和其他。合成，标记glycopolymers的结合特异性可容易地核查使用外源凝集素与已知结合亲和力。 PMA-GAEMA属没有挂件糖是一个合适的阴性对照。 Glycopolymers经由这个路线来制备不同的荧光标记物已被成功地用于在的凝集素介导的细菌结合14调查。所提出，此简单而且有效的统计荧光glycopolymers准备应于各种各样glycobiological研究提供的巨大潜力。

在过去的二十年里，研究合成glycopolymers经历了缓慢但持续的发展，在研究，包括研究侧重于凝集素识别过程1-3传染机制证明显著的潜力。因为合成glycopolymers具有多价的糖部分表现出高得多的凝集素结合功效相比，单价碳水化合物，它们在糖生物学领域3很大的需求。在临床研究中特别感兴趣的是使用荧光glycopolymers的表征的凝集素介导的细菌可用对人体呼吸道细胞表面和粘膜糖蛋白的碳水化合物结合。早在体外研究中细菌的结合试验中使用可商购的聚丙烯酰胺系glycopolymers。其中一些探头表现出令人鼓舞的结果，但对于，obtainability，和很多到很多差异在这两个POL引发市场担忧ymer分子量和glycoepitope内容。一种经济的在实验室协议被开发这将提供对结构的含量，大小和靶向细菌凝集素合成glycopolymers的纯度令人满意的控制。 在寻 ​​找一个合适的合成方法来glycopolymers，一个相对较新的聚合技术，使用采用可逆加成-断裂链转移（RAFT）试剂4一型受控的自由基聚合的测试。这样的RAFT试剂最近已应用在几glycopolymer制剂5-7。与其他glycopolymer制备协议相比，RAFT介导的聚合中表现出一些优点，包括耐多种单体的结构和反应条件，用水溶液潜在的兼容性，以及所需的聚合产物8,9-低尺寸分散性。值得注意的令人感兴趣的是制备RAFT-BA的协议SED三组分glycopolymers，允许不同的单体，其中每一个可具有不同的功能10-13的组合物的控制。然而，大多数的以前的研究工作要么缺乏异头挂件碳水化合物10，或采用阶梯导致三嵌段共聚物的聚合反应，即包括共价连接的均聚物，这往往用于不同的目的不是统计聚合物，其是共聚物，其中单体的序列残留遵循统计规律9-13。 最近，采用硫羰基硫基RAFT化合物（4-氰基戊酸）-4-二硫代苯甲酸酯在含水环境中，一组的制备RAFT基含有特定挂件糖及其在应用线性三组分统计glycopolymers凝集素介导的细菌结合测试报告14。该方法中，以视觉方式呈现的总体目标，是制备三组分通过RAFT控制共聚统计荧光glycopolymers。因为易于一步法聚合协议的，精细地控制聚合物长度和组合物，以及该反应的高再现性，该协议可以很容易地应用于glycopolymers具有所需结构的其它的RAFT基合成。

<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

合成glycopolymers是各种生化和生物医学研究领域使用的工具和灵活的工具。使用可逆加成 - 断裂链转移控制良好的荧光统计glycopolymers的简便而有效的合成的一例（RAFT）基聚合是证明。该合成开始于由lactobionolactone 和 N的反应得到β -半乳糖-含glycomonomer -2- lactobionamidoethyl甲基丙烯酰胺的制备- （2-氨基乙基）甲基丙烯酰胺（AEMA）。 2- Gluconamidoethyl甲基丙烯酰胺（GAEMA）被用作结构类似物缺乏一个终端β-D-半乳糖苷。下面RAFT介导的共聚合反应涉及三个不同的单体：N - （2-羟乙基）丙烯酰胺作为间隔物，AEMA作为靶进行进一步荧光标记，和glycomonomers。宽容含水系统，在反应中使用的RAFT试剂是（4-氰基戊酸）-4-二硫代苯甲酸酯。低分散度（≤1.32），可预测的共聚物组合物，以及高的聚合再现性的产品中观察到。荧光聚合物是通过修改glycopolymers用羧基琥珀酰亚胺酯靶向伯胺官能团上AEMA获得。所得glycopolymers的凝集素结合特异性通过用涂有特定glycoepitope识别外源凝集素相应琼脂糖珠测试验证。因为易于合成的，严密控制的产品组合物和反应的重现性好，该协议可以被翻译朝向其它制备RAFT基glycopolymers具有特定的结构和组合物，根据需要。

glycomonomer的合成 乳糖酸被本文用作glycomonomers的制备的一个例子。使用在LAEMA 11合成的初步报告的方法，不同的收益率在准备与纯度不理想，观察。使用阳离子和阴离子交换树脂的改性的纯化方法，除去提供稳定的产品产率和高纯度的未反应的起始原料，它是由1 H和13 C-NMR光谱（ 图1）证实。 RAFT glycopolymer合成和修改后的glycopolymers与荧光 与此相反，通过台阶的RAFT聚合制备的嵌段glycopolymers，这一步共聚协议提供在整个聚合物主链均匀glycomonomer分布。这里示出的glycopolymers含有20摩尔％GL的ycomonomer，77摩尔％HEAA作为间隔物，和3摩尔％AEMA的作为后修饰（参见图2）。1 H和13 C-NMR光谱法的靶确认PMA-LAEMA和PMA-GAEMA的结构（ 图3和4）。如图5所示 ，当对没有RAFT合成的glycopolymer的GPC洗脱曲线作图，二者PMA-LAEMA和PMA-GAEMA具有低分散度，证明了RAFT方法的功效。正如预期的那样，PMA-GAEMA有Mn为比PMA-LAEMA由于PMA-GAEMA缺乏挂件糖较小。碳水化合物和伯胺官能团的RAFT glycopolymers的内容的分析表明，单体在产物glycopolymers的比率为与开始在RAFT介导的聚合反应（ 表1）使用的单体的化学计量比是一致的。这标志着单体compositio的严格控制ns的合成glycopolymers，作为设计的。 伯胺官能基团与活化的荧光团反应是在蛋白质标记一种广泛使用的技术。这种技术就是在这里采用与羧基标签纯化glycopolymers。以下修正后，荧光聚合物获得（ 图6）。通过GPC分析法检测反应中的荧光素标记的聚合物的不降解（数据未显示）。 结合合成glycopolymers测试与外源凝集素涂层琼脂糖珠 以评估合成glycopolymers的凝集素结合特异性，凝集素包被的琼脂糖珠与已知的糖结合特异性使用。 鸡冠刺桐凝集素（ECL），在实验中使用的，具有朝向β-D-半乳糖苷的结合特异性。 图7A清楚地表明，PMA-LAEMA-FLuorescein，其中包含β-D-半乳糖苷作为侧碳水化合物，表现出很强的与ECL凝集素结合。与此相反，负结合于glycopolymer PMA-GAEMA荧光素，它不具有一个侧挂糖的ECL， 如图7B所示 。该结果例证了合成荧光glycopolymer的结合效力和亲和力。 <img alt="图1" src="/files/ftp_upload/52922/52922fig1.jpg" /> 图1.分配1 H-（a）和13 C-NMR（B）光谱（D 2 O）为LAEMA。（这个数字已经被修改Wang 等 14） <a href="https://www.jove.com/files/ftp_upload/52922/52922fig1large.jpg" target="_blank">请点击这里查看本一个更大的版本图。</a> &lt;P类=“j​​ove_content”FO：保持together.within页=“总是”&gt; <img alt="图2" src="/files/ftp_upload/52922/52922fig2.jpg" /> 图2.含β半乳糖苷作为吊坠糖荧光glycopolymer PMA-LAEMA的合成示意图。 <a href="https://www.jove.com/files/ftp_upload/52922/52922fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52922/52922fig3.jpg" /> 图3.分配1 H-（A）和13 C-NMR（B）光谱（D 2 O）的PMA-LAEMA glycopolymer。（这个数字已经被修改Wang 等 14） <a href="https://www.jove.com/files/ftp_upload/52922/52922fig3large.jpg" target="_blank">辩诉交易</a>SE点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/52922/52922fig4.jpg" /> 图4.分配1 H-（A）和13 C-NMR（B）光谱（D 2 O）的PMA-GAEMA。（这个数字已经被修改Wang 等 14） <a href="https://www.jove.com/files/ftp_upload/52922/52922fig4large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <img alt="图5" src="/files/ftp_upload/52922/52922fig5.jpg" /> RAFT基PMA-GAEMA和PMA-LAEMA图5.凝胶渗透色谱痕迹使用和不使用RAFT试剂制备的。与此相反的PMA-LAEMA而不RAFT试剂（蓝色）中制备，RAFT-<html>基于PMA-LAEMA（绿色）具有低得多的分散度（M W / M n）的。筏基PMA-GAEMA（红色）和PMA-LAEMA有类似的GPC曲线，但前者具有较小的Mn为由于没有任何挂件糖。 <a href="https://www.jove.com/files/ftp_upload/52922/52922fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52922/52922fig6.jpg" /> 白色非标记glycopolymer（左管）相比图6 PMA-LAEMA前和修正后用荧光团之后。（A）中 ，荧光素标记的PMA-LAEMA显示强的黄色（右管）。 （B）在紫外线，非标记的PMA-LAEMA（左管，1毫克/毫升在PBS中）是黑暗，并与无荧光呈现，而荧光素标记的PMA-LAEMA（右管，1毫克/毫升在PBS中）示出了强烈的绿色荧光。：//www.jove.com/files/ftp_upload/52922/52922fig4large.jpg“目标=”_空白“&gt;点击此处查看该图的放大版本。 <img alt="图7" src="/files/ftp_upload/52922/52922fig7.jpg" /> 图7. 鸡冠刺桐凝集素（ECL）涂层琼脂糖珠结合含glycopolymersβ-D-半乳糖，而不是那些不具备一个挂件糖。（A）PMA-LAEMA荧光素（3微克）表现出很强的结合与ECL ，而在（B）的 PMA-GAEMA荧光素，其具有无挂件β-D-半乳糖苷残基，显示出与凝集素包被的珠无结合。 比例尺= 100微米。 表1.定向值的合成参数和glycopolymers的实际组成。一）定位值，值期望的产品; B）DP，聚合度; C）NA，无法使用。 <a href="https://www.jove.com/files/ftp_upload/52922/52922fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</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"> DP b </td><td style="width:70px"> 分散度 </td><td style="width:120px"> glycomonomers的实际内容 摩尔％ </td><td> 伯胺的实际内容 摩尔％ </td></tr><tr><td> 指定值 </td><td> 100 </td><td> &lt;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> NAÇ </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.

通过RAFT控制聚合轻便和高效制备三组分荧光Glycopolymers

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

9.6K 次观看.  University of Missouri. 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.

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

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.

内部自组装脂质颗粒的简易制备碳纳米管稳定

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

科研

化学

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.

的4-取代喹唑啉衍生物简便制备

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.

使用Photoredox催化剂蠕虫状胶束的简便合成可见光介导的分散聚合

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.

一个两亲性共聚物的阴离子聚合通过π-π堆积作用稳定嵌段共聚物胶束的制备

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.

热可逆交联橡胶通过的Diels-Alder反应的化学制备与性能

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.

功能性降冰片烯的催化聚合插入一种简单高效的协议

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.

在环境环境中制备具有或不具有介孔MCM-41的超细氢氧化铝颗粒

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.

（2<em&gt;ž</em&gt;，4<em&gt;电子</em&gt;） - 通过电子缺陷烯烃与烯丙基乙酸酯的烯化反应制备二烯酰胺

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.

由烃类和氟碳油组成的同液滴的自由基聚合制备空心聚苯乙烯颗粒和微胶囊

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.

一种高效的植物多嵌合体荧光融合蛋白的协同表达

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