这份手稿的准备工作得到了科罗拉多州丹佛大学（JMRE）的启动资金的支持。 AF希望得到研究和创意活动奖（RaCAS，CU Denver）的支持。克罗地亚科罗拉多大学研究服务处资助出版费用得到承认。我们要感谢实验室成员Cassandra Herbert女士和Yannick K. Dzowo先生在视频部分的贡献。

1. RNA寡聚核苷酸的固相合成<ol><li> 制备含有亚磷酰胺的溶液（表1）。 <ol><li>计算核苷酸数量，并适合n + 1方程（其中n =核苷酸数目）对应于每个碱基，并填写缺失值的表格。体积= 0.15mL /核苷酸，浓度为0.1M，溶于无水乙腈。 </li><li>将每个亚磷酰胺称量到烘箱干燥的10毫升琥珀色瓶（隔垫顶部琥珀色394阿米申13毫米ID×20毫米OD顶部）中，并使用含有干燥剂的干燥器立即置于真空下。 </li><li>用干燥氩气填充干燥器，取出瓶子并立即用相应量的乙腈（无水）稀释，然后再固定在仪器上。 注意:确保使用气密注射器，并始终保持无水的气氛。 </li><li>从瓶子上取下隔垫并放在m上通过瓶子更换功能。 注意:该方法需要在合成之前补充每个溶液0.15mL（因此在上述n + 1方程中的过量体积）引出线。 </li></ol></li><li> 使用软件设置序列和固相合成（ 图3 ）。 <ol><li>单击软件图标（ 例如 ，OligoNet 1.0.1），然后选择仪器名称&gt;确定 。通过File&gt; New Synthesis&gt; Order创建一个新的综合。 </li><li>填写日期runid为;选择仪器;序列名称序列（5&#39;-to-3&#39;end）。在|循环下，分配先前创建的方法（ 图3 ）。选择结束程序（留下与CPG树脂结合的寡核苷酸）&gt;手册。 </li><li>选择DMT Off&gt;（TCA处理在最后一步获得5&#39;-OH基团合成结束） ;保存文件&gt;另存为并提供实验的名称。 </li><li>发送文件开始综合订单&gt;发送订单到合成器并选择列 1-4 。打开合成器窗口，选择三位一体监视器|选择功能|三位一体&gt;监控每一步 。 </li><li>根据需要合成的寡核苷酸数量，重复上述步骤（请注意，所有订单必须具有相同的方法， 即相同的耦合时间和事件顺序）。 </li><li>开始合成Synthesizer&gt;准备开始 。将所需的3&#39;末端柱放在仪器上指示的位置。在仪器上单击开始&gt;否 （用于ABI准备）。 </li><li>一旦合成完成，从仪器中取出色谱柱，放入圆底烧瓶中，然后减压干燥约0.5小时。 注意:建议检查在兰德里是否有深橙色om耦合（步骤1.2.4 - 1.2.7），以避免三位一体监视功能的潜在错误。 </li></ol></li><li> RNA寡核苷酸的脱保护和纯化。 <ol><li>通过扭转黑色盖子将白色树脂的所有（或一半，根据需要或目的））转移到1.6 mL离心管中，打开色谱柱。以1:1的比例加入0.5mL甲胺（40％水溶液）/氨（40％水溶液）。 </li><li>用石蜡膜固定离心管盖，并使用热块将其加热至60°C 1.5 h。 注意:重物可放置在管的顶部，以确保反应管内的氨浓度保持不变。 </li><li>从热块中取出并慢慢冷却至室温。将样品简单离心以旋转内容物，然后将上清液转移到新的离心管中。 </li><li>将试管浸入液氮（或干冰/乙醇浴）中冷冻样品，同时旋转干燥在离心机中减压。 </li><li>将固体重悬于0.4mL三乙胺/ N-甲基吡咯烷酮/三乙胺 - 三氢氟酸（2:2:3比例）溶液中。加热至60℃1.5小时，然后缓慢冷却至室温。 </li><li>加入0.04mL NaOAc溶液（3M，pH 5.5，用HCl调节），然后用移液管尖端轻轻搅拌。加入乙醇（1 mL），冷却至约-70℃（干冰/乙醇浴）15分钟。 </li><li>以15,000rpm和4℃离心10分钟。使用移液管提取等分试样，并在减压下干燥所得颗粒。 </li><li>将所得固体重新悬浮在加载缓冲液（0.2mL，90％甲酰胺，1mM EDTA）中，并混合直到混合物均匀。 </li><li>将悬浮液装载到预先制备的聚丙烯酰胺凝胶上（20％变性，孔尺寸:30cm×1mm） 22 。 </li><li>通过凝胶施加电流，直到溴酚蓝标记位于1 / 2-2 / 3之间凝胶（凝胶尺寸:约21.5厘米×30厘米）。 </li><li>从支架上取出凝胶，将凝胶内容物从玻璃转移到塑料包装（双面覆盖）上，放在覆盖有二氧化硅（含有254nm荧光染料）的薄层色谱（TLC）板上以观察条带使用紫外灯（ λmax = 254nm）。 </li><li>使用标记来描绘上方所在的位置，并使用新的剃须刀切割。将含有RNA（无塑料包装）的凝胶放入50mL锥形管中，并用玻璃棒压碎成小块。 </li><li>将凝胶残留物悬浮于NaCl水溶液中。溶液（2mM和1mM EDTA），并将悬浮液在37℃下摇动12小时。将锥形管离心10分钟。 </li><li>通过使用反相C18色谱柱脱盐。 <ol><li>使用10 mL注射器，通过以下方式洗涤，准备墨盒: 乙腈（10mL） H 2 O（两次，10mL） 5mM NH4氯化钠溶液（3mL） 含有RNA的溶液，注意不要倒入任何凝胶残留物。 </li><li>用H 2 O洗涤（3次，10 mL）。 </li><li>使用60％的水溶液从柱中洗脱。甲醇溶液（3mL） </li></ol></li><li>在减压下浓缩并重新溶解于无RNase的水（0.3mL） </li><li>准备稀释溶液（10μL），并在UV-vis仪器（ 如 Nanodrop）上沉积1μL，以测量紫外 - 可见光谱（200-450 nm）。 </li><li>使用啤酒朗伯定律来确定所得溶液的浓度: <img alt="公式1" src="/files/ftp_upload/56189/56189eq1.jpg" /> 其中A =获得的吸光度; ε =计算摩尔消光系数; c =浓度，对于1mm的路径长度， l = 0.1。 </li><li>计算寡核苷酸的摩尔消光系数;在这里用在线计算器计算使用DINAMelt软件（http://unafold.ra.albany.edu/?q=dinamelt）23 </li></ol></li><li> 通过MALDI-TOF进行浓度测定和表征。 <ol><li>将样品加载到MALDI平板上，使用装有C18端头的移液管末端进行脱盐，并点出每个寡核苷酸。 <ol><li>用50％乙腈（10μLx 2）清洗尖端。用0.1％三氟乙酸（TFA;10μL×2）平衡末端。加载样品（通常为100-150 pmol）。 </li><li>用0.1％TFA（10μLx 2），然后用水（10μLx 2）清洗末端。 </li><li>将样品洗脱到含有所需基质的溶液中;我们使用10μL25mM-2,4,6-三羟基苯乙酮一水合物（THAP），10mM柠檬酸铵和300mM氟化铵在50％乙腈中的溶液。 </li><li>通过沉积0.9μL（随后空气干燥）直接点到MALDI板上，并根据需要重复该过程。 注意:所有光谱均在反射器正模式下获得。 </li></ol></li></ol></li></ol> 2. 通过 CD进行RNA结构分析<ol><li> CD的解决方案的准备。 <ol><li>制备含有修饰的RNA [3μM]，NaCl [10mM]，磷酸钠缓冲液[10mM，pH7.2]和MgCl 2 [5mM]的0.25mL。样品准备好分析，特别是如果它代表单分子转换。 </li><li>如果目标是分析双相结构或双分子转换，则此时添加补体（如果适用，为1摩尔当量），或继续下面的步骤。 </li><li>将样品放在热块上，预热至90°C，关闭热量以控制缓慢冷却至室温（典型值为2-4h）。 </li></ol></li><li> 光谱采集 <ol><li>制备空白溶液（NaCl 10mM，磷酸钠10mM pH 7.3，MgCl 2 5mM），转移toa微量比色皿（1cm路径长度，250μL最小体积），并置于仪器内的样品更换器的保持器位置。 </li><li>将含有RNA的样品转移到另一个微量比色皿中，并置于样品更换器中。如果测量热变性转变，请小心地添加一层油，而不会中断水层，并使用一块特氟龙胶带固定比色杯盖。 </li><li>打开氮气罐以提供将位于仪器上的空气浮子移动到大约的流量。打开冷却器。 </li><li>打开仪器并打开软件Spectra Manager图标，然后打开采集窗口光谱测量 。 </li><li>在采集前用氮气吹扫仪器5分钟。 </li><li>使用以下参数测量&gt;参数获取光谱，并按如下所示调整参数: <ol><li>在常规下 ，选择:扫描200-350 nm;频道CD和HT:数据间距为0.1nm;扫描速度为100 nm / min;带宽为1nm;累计数量为5。 </li><li>在电池单元下 ，选择20°C。在控制下 ，选择快门自动打开和关闭。在信息下 ，选择名称，浓度，运算符。 </li><li>在数据下 ，浏览到所需的文件夹。单击确定 。 </li></ol></li><li>获取光谱测量&gt;样品测量 ，相应地识别样品转换器1-6中的比色杯的位置，单击确定 。 </li></ol></li><li> 如果进行热变性转变: <ol><li>关闭采集软件文件&gt;退出并打开新程序可变温度测量&gt;测量&gt;参数 。 </li><li>应用以下参数记录热转换，可根据需要进行调整: <ol><li>在温度下 ，选择开始温度:4°C;间隔:0.2; TArget:90°C;梯度:1℃/ min;等待0秒。在开始/结束下，根据需要选择启动条件和结束条件。 </li><li>在一般情况下 ，选择3个通道，CD / HT / Abs;带宽:1nm;波长:270nm（或根据需要）。在控制下 ，选择无。在" 信息"下 ，选择名称，浓度，运算符。 </li><li>在数据下 ，浏览到所需的文件夹。单击确定 。 </li></ol></li><li>将样品冷却至4°C，然后在此温度下等待5 min，然后再进行光谱测量。 </li></ol></li><li> 光谱数据处理。 <ol><li>使用软件上的功能，从目标采集中减去空白频谱，以考虑从使用的缓冲系统产生的背景信号: 光谱分析&gt;文件&gt;打开 。 </li><li>一旦打开了空白文件和光谱文件，请在View 1（屏幕左侧）下拖动View 2。 </li><li>点击Pr加权&gt;减法&gt;确定或交换数据&gt;确定 。 </li><li>将数据提取为ASCII文件: 文件&gt;导出并选择ASCII 。 </li><li>使用软件绘制相应的光谱。在信噪比低于预期的情况下，数据平滑是可选的。可以通过应用平滑数据选项来应用此转换。 </li></ol></li><li> 热变性转变的数据处理。 <ol><li>从JASCO文件中提取数据作为ASCII文件。 </li><li>绘制椭圆率作为温度的函数。通常，根据正在检查的波长，需要平滑数据。在一些情况下，使用210nm波长的图形显示出需要平滑的曲线之间的较大变化。然而，根据样品和浓度，使用原始数据进行了一些计算。 </li><li>计算热值固化转变（T m ）值，获得曲线的一阶导数。最大值或最小值是该参数的指示。如上一步，有些情况需要平滑数据以获得最准确的值。 注意:实验通常一式三份进行，这导致测量的相应平均值和标准偏差。 </li></ol></li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+properties+and+thermal+stability+of+oligonucleotides+of+RNA+containing+7,8-dihydro-8-hydroxyadenosine.">Biophysical properties and thermal stability of oligonucleotides of RNA containing 7,8-dihydro-8-hydroxyadenosine.</a> Biopolymers. 103 (3), 167-174 (2015).</li><li>Maniatis, T., Fritsch, E. F., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. 14, 173-185 (1987).</li><li>Markham, N. R., Zuker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UNAFold:+software+for+nucleic+acid+folding+and+hybridization.">UNAFold: software for nucleic acid folding and hybridization.</a> Methods Mol Biol. 453, 3-31 (2008).</li><li>Breslow, R., Huang, D. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+metal+ions+including+Mg2++and+lanthanides+on+the+cleavage+of+ribonucleotides+and+RNA+model+compounds.">Effects of metal ions including Mg2+ and lanthanides on the cleavage of ribonucleotides and RNA model compounds.</a> Proc Natl Acad Sci U S A. 88 (10), 4080-4083 (1991).</li><li>Blumberg, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+a+ribonuclease-free+environment.">Creating a ribonuclease-free environment.</a> Methods Enzymol. 152 (2), 20-24 (1987).</li><li>AbouHaidar, M. G., Ivanov, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-enzymatic+RNA+promoted+by+the+combined+catalytic+activity+of+buffers+and+magnesium+ions.">Non-enzymatic RNA promoted by the combined catalytic activity of buffers and magnesium ions.</a> Z Naturforsch C. 54 (7-8), 542-548 (1999).</li><li>Farrell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RNA Methodologies (4th Ed): A laboratory guide for isolation and characterization. 2, 45-80 (2010).</li></ol>

作者没有什么可以披露的。

该手稿的目的是作为领域研究人员，初学者或专家的指导，成功实现或增强DNA或RNA寡核苷酸的合成。所描述的方法侧重于使用通过标准亚磷酰胺化学的自动化DNA / RNA合成仪的固相合成。该报告描述了RNA十二聚体的合成，纯化和表征的逐步描述。此外，使用CD来识别二级结构基序和热变性转变。 重要的是要注意，这项工作可以适应其他情况， 即固相合成中的各种品牌的设备，修改，保护基团和/或试剂。因此，改变在该过程中涉及的许多参数中的一个或多个或通过调节反应条件可以实现。一世此外，本文提供的详细结构分析（通过CD）预计将为该领域的研究人员提供一种替代方法，通常通过 UV-vis进行类似的分析。 以确定亚磷酰胺的固相合成的群众计算是基于以下所示的序列，并制备和在相同的运行纯化的四种寡核苷酸（加下划线的位置表示一个2&#39;-O- -thiophenylmethyl修饰的存在） 。所有计算和值都包含在表1中 1 5&#39; - [CUA CGG AAU CAU] -3&#39; 2 5&#39; - [CUA CGG A AU CAU] -3&#39; 3 5&#39; - [CUA CG G A AU CAU] -3&#39; 4 5&#39; - [AUG AUU CCG UAG] -3&#39; 也许处理RNA最重要的方面在于其对核糖核酸酶降解的敏感性，在水溶液中和在金属离子24的存在下进行水解。因此，无卤素条件必须随时执行25 ，如下所述:1）所有水在焦碳酸二乙酯（0.1％w / v，DEPC）存在下进行高压灭菌; 2）将所有玻璃器皿进行高压灭菌，在烘箱（150℃，过夜）中烘烤，并用DEPC处理的水冲洗; 3）所有的管和移液器吸头是从制造商以无RNase的形式购买的; 4）任何时候都使用手套，在指定的护罩上进行工作;和5）所有表面和设备不断擦拭可以从各种制造商购买的RNase去污溶液。根据使用频率，将所有纯化的RNA分成小部分并储存在-20℃或-80℃，而未切割或未纯化的树脂在20℃下储存。正如我们以前指出的， 16 ，寡核苷酸以10-24个核苷酸长度的大小显示出比其它报道26更高的稳定性。因此，当处理较长的RNA时，读者参考其他存储程序27 。 在自动化合成中逐步产率（通过去离子化步骤计算）在97-100％之间，并且表现出良好的偶联效率，特别是已经被修饰的那些。从树脂切割后得到的总产率为30-75％，去保护和纯化（ 通过凝胶电泳），其对应于〜300-750nmol的分离的寡核苷酸。合理地，掺入修饰物并不影响RNA链的总产率。然而，虽然这些范围可以被认为是可接受的量，但大于〜50个核苷酸的寡核苷酸的合成（以前的，未发表在我们实验室的数据）可能受到逐步收益率低于98％的显着影响。因此，如果需要大于50个核苷酸的链，必须采取某些预防措施， 例如 ，将乙腈的来源改变为更高的质量，将亚磷酰胺暴露于大气中的时间最小化，使仪器将亚磷酰胺稀释成一套每次使用新瓶规范的亚磷酰胺时，使用HPLC纯化代替电泳分析，和/或使用温和的去保护条件。 所有寡核苷酸的质谱（MALDI-TOF MS）在ABI 4800 Plus MALDI-TOF / TOF质谱仪中以正模式进行。在图5中示出3 -使用本文和光谱ON 1的实施例中描述的方法制备所有的样品。 所有的实验通常以三重态进行美食。所有光谱和实验装置在配备有矩形6电池座的分光偏振计上进行。将所有玻璃器皿用RNase去除溶液洗涤，并用无RNase的水彻底冲洗。每次测量后都丢弃所有样品。重要的是要指出，应该高度关注高压电压（HT是仪器测量的参数）。这可以指示信号中的饱和度，从而对数据的准确性和有效性构成威胁。该参数取决于样品，应保持在任何给定时间内信号不超过500 mV。之前和ON 1的杂交后得到的CD光谱范例- 3被示出在图6中 。 此外，增加钠盐（和其他缓冲系统）中的浓度，或使用其他缓冲系统， 例如 HEPES或MOPS，导致220nm以下的噪声增加。因此，由于210nm处的带对于形成A型双链体是特别重要的，钠离子中的最大浓度保持在〜10mM。 我们还表明，CD的使用提供了与从紫外光谱获得的参数相同的参数。总之，我们描述和说明合成，纯化和表征修饰和未修饰的RNA寡核苷酸的过程。

固相合成，以获得DNA的寡核苷酸/ RNA是一个功能强大的工具，已经使用亚磷酰胺构建模块4自1970年代以来1，2，3提供在各种领域中的应用的几个。其广泛的影响的例子包括:其在标记的影响（ 通过点击化学反应）5，结构探测6，和反义技术的图7，以及其生物机制8,9，源阐明作为遗传物质10，和的研究各种天然的和/或化学修饰11，12，许多others.The修改中，我们在这里使用代表了我们的努力，以获得含有RNA的寡核苷酸的第一步光活性探针，能够对这一重要生物聚合物的结构和功能进行时间​​控制。 具有序列5&#39; - [CUA CG G A AU CAU] -3&#39;/ 5&#39; - [AUG AUU CCG UAG] -3&#39;的RNA十二烷基化物的合成（下划线位置表示C2&#39;- O-硫代苯基甲基修饰）构成了本研究的重点。选择序列以使RNA链的量化和测量成为单链，或者作为其相应的双链体结构（没有其他二级结构被预测为热力学稳定的）。 CD用于建立结构参数， 即双相形成和热变性转变。 合成 获得这些寡核苷酸的总体程序如图1所示，并遵循逐步的方法:自动化固相合成→Deprotection→净化→定量→表征。 图2显示了该过程中必需的单体单元。 RNA的固相合成类似于DNA，因为它基于亚磷酰胺化学（ 图2左侧）和在G，A和C上用于亲核环状胺的碱不稳定保护基团， 例如 ，乙酰基，苯甲酰基，苯氧基乙酰基， 叔丁基或N ， N-二甲基甲酰胺（ 图2 ，右）。由于存在C2&#39;-OH基团（缺乏脱氧寡核苷酸生物聚合物），RNA需要考虑的另一个方面是额外的步骤，必须并入其中以保护和随后的脱亲维护该亲核位置。在这方面，硅基保护基由于其作为双正交部分的潜力而变得有吸引力的策略（特异性的在氟化物存在下脱保护）， 叔丁基二甲基甲硅烷基（TBDMS）和三异丙基甲硅烷氧基甲基 （TOM）作为流行选择（ 图2左下方）。 在这项工作中，自动合成在使用标准亚磷酰胺化学的DNA / RNA合成仪上进行。仪器上的制造商设置包括使用商业版本的亚磷酰胺用于DNA的自动稀释步骤，或者用户稀释的选项。然而，我们决定称量RNA亚磷酰胺并手动稀释，因为:1）RNA的规范亚磷酰胺的价格较高（在某些情况下高达50倍）; 2）经修饰的亚磷酰胺通常少量得到;和3）使用自动稀释步骤（由制造商设定）时浪费的材料的量很大。此外，我们使用:1）市售的固体支持物（ 例如 ，CPG），其含有受保护的核碱基以用作3&#39;-末端;和2）在C2&#39;- O位置用TBDMS基团保护的商业亚磷酰胺（规范核碱基）。合成步骤的详细列表在图3和表1中提供 ，以及针对RNA合成调整的步骤的进一步描述和评论。此外， 图4示出了在选择"三位一体监测器"选项之后对于每个步骤观察到的逐步产量，其定量从每个去离子化步骤释放的三苯甲基。 值得注意的是，在我们的经验中，限制因素是获得含有所需改性的亚磷酰胺。也就是说，开发一种允许在选定地点纳入修改的综合方法。在本报告中，我们专注于掺入修饰的核苷酸，我们已经建立了相应的合成方法，即C2&#39;- O-硫代苯甲基。该组的尺寸小，并且不以任何方式影响固相合成。由于已经报道了将该基团并入RNA的寡核苷酸中，连同结构和热力学参数4 ，本文将描述导致经修饰的亚磷酰胺的有机合成的任何方面。 脱保护，纯化和表征 环外胺和β-氰基乙基的去保护在与CPG-树脂切割相同的步骤中发生。我们应用了在AMA水溶液存在下加热所得树脂的常用条件，然后在氟离子存在下裂解C2&#39;- O-甲硅烷基，然后通过凝胶纯化电泳。虽然这些已经成为在许多情况下的标准条件，其是不稳定的碱性条件或氟离子可能要求更温和的条件13，14， 例如 ，碳酸甲醇/钾（甲醇/ K 2 CO 3），或丁胺修改。因此，在相应的亚磷酰胺上必须有一组不同的保护基团。此外，鉴于我们以前使用该方法的经验和缺乏其他仪器，我们选择电泳作为纯化脱保护的寡聚体的首选替代物。然而，HPLC可以替代地用作有效的方法15 。通过质谱法，基质辅助激光解吸/电离 - 飞行时间（MALDI-TOF）进行纯化寡核苷酸的表征，使用我们组16报道的方法。 结构表征和其他<html> 通过 CD进行获得的双链体的稳定性。具体地说，我们利用CD来测定经修饰和未修饰的RNA寡核苷酸的热变性转变，遵循在约束下带的椭圆率的降低。 270个纳米，以及带的消失（与负椭圆率）与λ 最大 210nm处。提供杂交前后的光谱比较，以说明其差异并提供所采用的方法的验证。 CD的使用在核酸和氨基酸17中的结构基序的测定中被广泛接受，因此可用作确定各种结构和热力学参数的工具18 ;然而，没有多少例子用于评估热变性过渡的技术。一些情况包括测定含有G-四链体DNA的DNA的热稳定性屁股="外部参照"&gt; 19,20或双链体RNA和21的发夹。 本报告旨在为非专家读者或观众提供一系列工具，以便顺利开始此类研究。这将有助于加强和比较其他研究实验室的方法和技术，这些研究实验室参与了这个令人兴奋的科学分支。本报告的内容从各种来源中增加了该技术的现有协议，并丰富和便利了每一步视觉辅助的体验。

<table border="0" cellpadding="0" cellspacing="0" width="778">
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			<td height="20" style="height:20px;width:445px;">AbsolveTM</td>
			<td style="width:152px;">PerkinElmer</td>
			<td style="width:117px;">6NE9711</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile 99.9%, HPLC Grade</td>
			<td>Fisher Scientific</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile 99.9%, anhydrous for DNA sequencing</td>
			<td>Fisher BioReagents</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acrylamide, 99+%</td>
			<td>ACROS Organics</td>
			<td>164850025</td>
			<td></td>
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			<td height="20" style="height:20px;">Ammonium chloride 98+%</td>
			<td>Alfa Aesar</td>
			<td>12125-02-9</td>
			<td></td>
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			<td height="20" style="height:20px;">Ammonium citrate, dibasic 98%</td>
			<td>Sigma Aldrich</td>
			<td>3012-65-5</td>
			<td></td>
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			<td height="20" style="height:20px;">Ammonium Fluoride&#160; 98.0%, ACS grade</td>
			<td>Alfa Aesar</td>
			<td>12125-01-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium hydroxide 28-30% in water, ACS Plus</td>
			<td>Fisher Chemical</td>
			<td>1336-21-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium persulfate</td>
			<td>ACROS Organics</td>
			<td>1444</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Argon-ultra high purity</td>
			<td>&#160;Airgas</td>
			<td>7440-37-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bis-acrylamide Ultra pure</td>
			<td>VWR-Amresco</td>
			<td>172</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Boric Acid</td>
			<td>Fisher Scientific</td>
			<td>A73-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Diethyl pyrocarbonate, 97%</td>
			<td>ACROS Organics</td>
			<td>A0368487</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, anhydrous, histological grade</td>
			<td>Fisher Chemical</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediamine tetraacetic acid, disodium salt dehydrate 100.2%</td>
			<td>Fisher Chemical</td>
			<td>6381-92-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formamide&#160;</td>
			<td>Thermo Scientific</td>
			<td>75-12-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrochloric acid, 36.5-38.0%, Certified ACS Plus</td>
			<td>Fisher Chemical</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium chloride hexahydrate, 99%</td>
			<td>Fisher Scientific</td>
			<td>7786-30-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol, 99.9%,&#160; HPLC Grade</td>
			<td>Fisher Chemical</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methylamine 40% in water</td>
			<td>Sigma Aldrich</td>
			<td>74-89-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Methyl-2-pyrrolidinone, andhydrous, 99.5%</td>
			<td>Aldrich</td>
			<td>872-50-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-TOFTM 96 Well Insert (123 x 81 mm)&#160;</td>
			<td>MDS SCIEX</td>
			<td>1020157</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase Away</td>
			<td>Molecular BioProducts</td>
			<td>7005-11</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium acetate, anhydrous 99.2%, Certified ACS</td>
			<td>Fisher Chemical</td>
			<td>127-09-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride, 100.5%, Certified ACS</td>
			<td>Fisher Chemical</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium phosphate monobasic dihydrate 99.0%</td>
			<td>Sigma</td>
			<td>13472-35-0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2&#8217;,4&#8217; Triethylamine, 99+%</td>
			<td>Alfa Aesar</td>
			<td>121-44-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TEMED</td>
			<td>Amresco</td>
			<td>761</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triethylamine trihydrofluoride, 98%</td>
			<td>Aldrich</td>
			<td>73602-61-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trifluoroacetic acid, 99%</td>
			<td>Alfa Aesar</td>
			<td>76-05-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6&#8217;-Trihydroxyacetophenone monohydrate 98%</td>
			<td>Sigma Aldrich</td>
			<td>480-66-0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP154-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Urea</td>
			<td>Fisher Scientific</td>
			<td>U15-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagents for the RNA synthesis:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Deblocking mix, 3% trichloroacetic acid in dichloromethane</td>
			<td>Glen Research</td>
			<td>&#160;40-4140-57</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cap Mix A, THF/Pyridine/Acetic anhydride</td>
			<td>Glen Research</td>
			<td>40-4110-52</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cap Mix B, 10% 1-methylimidazole in THF</td>
			<td>Glen Research</td>
			<td>40-4120-52</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Activator, 0.25 M 5-ethylthio-1H-tetrazole in anhydrous acetonitrile</td>
			<td>Glen Research</td>
			<td>&#160;30-3140-52</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oxidizing Solution, 0.02 M iodine in THF/Pyridine/Water</td>
			<td>Glen Research</td>
			<td>40-4330-52</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U-RNA-CPG</td>
			<td>Glen Research</td>
			<td>20-3330-xx</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ac-G-RNA-CPG&#160;</td>
			<td>Glen Research</td>
			<td>20-3324-xx</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ac-G-CE Phosphoramidite&#160;</td>
			<td>Glen Research</td>
			<td>10-3025-xx</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U-CE Phosphoramidite</td>
			<td>Glen Research</td>
			<td>10-3030-xx</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ac-C-CE Phosphoramidite</td>
			<td>Glen Research</td>
			<td>10-3015-xx</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bz-A-CE Phosphoramidite&#160;</td>
			<td>Glen Research</td>
			<td>10-3003-xx</td>
			<td></td>
		</tr>
	</tbody>
</table>

protocol for the solid-phase synthesis of oligomers of rna containing a 2'-o-thiophenylmethyl modification and characterization via circular dichroism

已经使用固相合成来获得特定于DNA或RNA的核酸的规范和修饰的聚合物，其已经成为各种领域和不同研究目的的应用的流行方法。本文所描述的过程集中于合成，纯化和RNA 5的十二聚体的表征" - [CUA CGG AAU CAU] -3"含位于C2&#39;-ø -位零个，一个或两个的修改。探针基于2-噻吩基甲基，通过标准有机合成并入RNA核苷酸，并通过其各自的亚磷酰胺引入相应的寡核苷酸。本报告通过四种典型的核碱基（尿苷（U），胞嘧啶（C），鸟苷（G），腺苷（A））以及在2&#39;- O-氨基酸修饰的2-噻吩基甲基官能化核苷酸，位置;然而，该方法适用于一个大的变量多年来一直发展起来的各种修改。在受控孔玻璃（CPG）载体上合成寡核苷酸，然后在标准条件下， 即氨和甲胺（AMA），然后是氟化氢/三乙胺/ N-甲基吡咯烷酮的混合物从树脂上裂解并脱保护。相应的寡核苷酸通过经由反相色谱（的Sep-Pak，C 18 -column），接着洗脱，脱盐，并进行隔离聚丙烯酰胺电泳（20％变性）纯化。通过紫外 - 可见（UV-vis）和圆二色性（CD）光度分析分别评估定量和结构参数。本报告旨在为有兴趣参与这一领域的初学者和专家研究人员提供资源和指导。随着新技术和方法的发展，预计将作为一项正在进行的工作。对thi内的方法和技术的描述s文件对应于使用亚磷酰胺化学的DNA / RNA合成仪（在2013年翻新并购买）。

描述了在C2&#39;- O-位上含有0,1或2个2-噻吩基甲基修饰的RNA十二烷基化物的合成以及其相应的纯化和表征。此外，还包括通过CD进行的结构分析的详细描述。 通过固相合成获得四条RNA（包括具有互补序列的链），随后每个寡核苷酸的纯度在300-700nmol之间。通过脱盐〜150 pmol的每个样品进行质谱，然后沉积在平板上用于MALDI-TOF分析（ 图5 ）。通过每种溶液的紫外光度分析进行定量，而CD用于鉴定双链结构的形成并记录其相应的T m。通过紫外 - 可见光谱法，无论比较单链样品还是双链体结构，经典和修饰的寡核苷酸之间没有观察到明显的差异。然而，在测量其CD光谱时观察到微小的变化（ 图6 ）。此外，三个双相结构的T m测量显示了不同的值，其表示通过在其中一条链上引入2&#39;-O-硫代苯甲基修饰而引起的不稳定性。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/56189/56189fig1.jpg" /> 图1 :获得RNA链的程序。使用CPG作为固体支持物和5-乙基硫代四唑作为活化剂的固相循环:（ i ）去离子化; （ ii ）耦合; （ iii ）氧化; （ iv ）上限和新循环;产生对应物丁基寡核苷酸（ v ）负载在CPG树脂上并含有所有保护基团;并随后在碱和氟化物存在下脱保护（ vi ），得到用于进一步分析的最终RNA寡核苷酸。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56189/56189fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/56189/56189fig2.jpg" /> 图2 :亚磷酰胺和保护基团的结构。在A，G和C的环外胺基上含有甲硅烷基的C2&#39;- O-基团和碱基不稳定基团的亚磷酰胺的化学结构。本研究使用O- TBBMS组。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56189/56189fig2large.jpg" target="_blank">请点击这里查看更大的版本</a>这个数字的n。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/56189/56189fig3.jpg" /> 图3 :相应寡核苷酸的逐步合成。循环如图所示编辑，所用代码和流量的关键字显示在右侧。逐步合成从提供的软件中粘贴。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56189/56189fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/56189/56189fig4.jpg" /> 图4 :获得的产量和个体耦合跟踪的表示。所示实施例已经从补体的合成改编以显示典型的寡核苷酸合成是。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56189/56189fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/56189/56189fig5.jpg" /> 图5 :寡核苷酸的MS（ON）1-3。 ON 1的MALDI-TOF - 3（从上到下）。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56189/56189fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" class="xfigimg" src="/files/ftp_upload/56189/56189fig6.jpg" /> 图6 :单链RNA和双链RNA的光谱，规范和修饰。含有零（ 1 ，A），一（ 2 ，B）或两（（ <strong&gt; 3，C）（蓝色）和之后（红色）杂交后的修饰。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56189/56189fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="表格1" src="/files/ftp_upload/56189/56189table1.jpg" /> 表1:亚 磷酰胺溶液计算。

Watch this Scientific Journal Video about Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism at JoVE

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

本文提供了在C2&#39;- O位上修饰的RNA的十二聚体的固相合成，纯化和表征的详细程序。 UV-vis和圆二色性光度分析用于量化和表征结构方面， 即单链或双链。

用于固相合成含有2&#39;-<em&gt; 0</em通过圆二色性修饰和表征

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

protocol-for-solid-phase-synthesis-oligomers-rna-containing-2-o

Nanyang Technological University

Research

JoVE Journal

Chemistry

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

18.8K 次观看.  University of Colorado Denver. 本文提供了在C2'- O位上修饰的RNA的十二聚体的固相合成，纯化和表征的详细程序。 UV-vis和圆二色性光度分析用于量化和表征结构方面， 即单链或双链。 

视频: Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

分裂和池合成及肽叔酰胺图书馆表征

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

科研

化学

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

功能化的合成与表征金属 - 有机骨架

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

合成方法通过固相树脂为基础的方法不对称二茂铁产生的生物共轭系统

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the hydroxylmethylene phosphine precursor with ammonia in methanol under a nitrogen atmosphere. The N-triphosPh ligand precipitates from the solution after approximately 1 hr of reflux and can be isolated analytically pure via simple cannula filtration procedure under nitrogen. Reaction of the N-triphosPh ligand with [Ru3(CO)12] under reflux affords a deep red solution that show evolution of CO gas on ligand complexation. Orange crystals of the complex [Ru(CO)2{N(CH2PPh2)3}-&#954;3P] (2) were isolated on cooling to RT. The 31P{1H} NMR spectrum showed a characteristic single peak at lower frequency compared to the free ligand. Reaction of a toluene solution of complex 2 with oxygen resulted in the instantaneous precipitation of the carbonate complex [Ru(CO3)(CO){N(CH2PPh2)3}-&#954;3P] (3) as an air stable orange solid. Subsequent hydrogenation of 3 under 15 bar of hydrogen in a high-pressure reactor gave the dihydride complex [RuH2(CO){N(CH2PPh2)3}-&#954;3P] (4), which was fully characterized by X-ray crystallography and NMR spectroscopy. Complexes 3 and 4 are potentially useful catalyst precursors for a range of hydrogenation reactions, including biomass-derived products such as levulinic acid (LA). Complex 4 was found to cleanly react with LA in the presence of the proton source additive NH4PF6 to give [Ru(CO){N(CH2PPh2)3}-&#954;3P{CH3CO(CH2)2CO2H}-&#954;2O](PF6) (6).

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Ruthenium phosphine complexes are widely used for homogeneous catalytic reactions such as hydrogenations. The synthesis of a series of novel tridentate ruthenium complexes bearing the N-triphos ligand N(CH2PPh2)3 is reported. Additionally, the stoichiometric reaction of a dihydride Ru&#8211;N-triphos complex with levulinic acid is described.

一系列钌的合成，表征和反应<em&gt;Ñ</em&gt; -triphos<sup&gt;博士</sup&gt;配合

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the ...

A Direct, Early Stage Guanidinylation Protocol for the Synthesis of Complex Aminoguanidine-containing Natural Products

The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important structural element found in many complex natural products and pharmaceuticals. Owing to the continual discovery of new guanidine-containing natural products and designed small molecules, rapid and efficient guanidinylation methods are of keen interest to synthetic and medicinal organic chemists. Because the nucleophilicity and basicity of guanidines can affect subsequent chemical transformations, traditional, indirect guanidinylation is typically pursued. Indirect methods commonly employ multiple protection steps involving a latent amine precursor, such as an azide, phthalimide, or carbamate. By circumventing these circuitous methods and employing a direct guanidinylation reaction early in the synthetic sequence, it was possible to forge the linear terminal guanidine containing backbone of clavatadine A to realize a short and streamlined synthesis of this potent factor XIa inhibitor. In practice, guanidine hydrochloride is elaborated with a carefully constructed protecting array that is optimized to survive the synthetic steps to come. In the preparation of clavatadine A, direct guanidinylation of a commercially available diamine eliminated two unnecessary steps from its synthesis. Coupled with the wide variety of known guanidine protecting groups, direct guanidinylation evinces a succinct and efficient practicality inherent to methods that find a home in a synthetic chemist&#39;s toolbox.

Here, we present a protocol for direct, early stage guanidinylation that enables rapid total synthesis of aminoguanidine-containing small organic molecules. An advanced synthetic intermediate used in the synthesis of a blood coagulation factor XIa inhibitor was prepared using this protocol.

一个直接的，早期胍基协议的复杂含氨基胍，天然产物合成

The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important ...

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of trifluoromethoxylated aromatic compounds remains a formidable challenge in organic synthesis. Conventional approaches often suffer from poor substrate scope, or require use of highly toxic, difficult-to-handle, and/or thermally labile reagents. Herein, we report a user-friendly protocol for the synthesis of methyl 4-acetamido-3-(trifluoromethoxy)benzoate using 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (Togni reagent II). Treating methyl 4-(N-hydroxyacetamido)benzoate (1a) with Togni reagent II in the presence of a catalytic amount of cesium carbonate (Cs2CO3) in chloroform at RT afforded methyl 4-(N-(trifluoromethoxy)acetamido)benzoate (2a). This intermediate was then converted to the final product methyl 4-acetamido-3-(trifluoromethoxy)benzoate (3a) in nitromethane at 120 &#176;C. This procedure is general and can be applied to the synthesis of a broad spectrum of ortho-trifluoromethoxylated aniline derivatives, which could serve as useful synthetic building blocks for the discovery and development of new pharmaceuticals, agrochemicals, and functional materials.

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

An operationally simple procedure for the synthesis of ortho-trifluoromethoxylated aniline derivatives via a two-step sequence of O-trifluoromethylation of N-aryl-N-hydroxyacetamide followed by thermally induced intramolecular OCF3-migration is reported.

协议的合成<em&gt;邻</em&gt; -trifluoromethoxylated苯胺类化合物

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

超分子胶体的合成与表征

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.

A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.

微流控干纺及再生丝素纤维的表征

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.