作者扎卡里Z.的布朗博士和珍妮弗Alleva想感谢本固相合成技术和有益的讨论马修佛罗里达州帕克的初步发展。这项工作是支持国防威胁降低局（国防部DTRA）（HDTRA1-09-1-0009）和由Cephalon公司的支持霍斯特·威策尔奖学金奖

1。安装<ol><li>固相合成反应的设置是聚丙烯滤芯或玻璃反应釜，真空条件下通过聚丙烯管连接到一个封闭的过滤瓶，如在图1所示。可以通过磁力搅拌棒或通过反应器鼓泡氮混合反应。 </li><li>一个气歧管连接到配备干燥管和油起泡氩气瓶还建议，因为它允许下惰性气氛中的反应容器，并允许从密封容器中的试剂去除。 </li><li>所有操作均在通风橱和适当的个人防护装备（安全眼镜，白大褂和丁腈手套）是必需的。 </li></ol> 2。载入中第一双肽到树脂<ol><li>重达114毫克HMBA-AM树脂（0.88毫摩尔/克负荷，100μmol）到8 mL的反应釜和加入磁力搅拌棒。封顶日顶部ê船只用橡胶隔垫和氩气吹扫管至少5分钟。 </li><li>在此期间，重达117.3毫克图3（586.63克/摩尔，2EQ）和59.2毫克1化合物1 - （三甲苯2 -磺酰）-3-硝基-1，2,4 -三唑（MSNT，296.0克/摩尔，2EQ）到15毫升的一次性离心管，并溶解在2 mL无水二氯甲烷（DCM）。加入24μL1 - 甲基咪唑（NMI，80.81毫升/摩尔，3EQ）解决方案组合，直到完全溶解。 </li><li>转移激活解决方案，通过注射器的反应容器中，并允许下氩隔夜（10小时），搅拌。 </li><li>取出隔，并排出反应混合物。洗树脂与DCM（5倍）和二甲基甲酰胺（DMF）（5倍）。执行“甲基红试验”，在10.1节中描述的评估树脂负载程度。如果树脂甲基红试验过程中保持红色，则步骤应重复2.2和2.3。一个黄色的颜色，指示负甲基红试验，是首选;然而，因为任何剩余的羟基组将在下一步中，稍微积极的结果（浅橙色树脂的颜色）的上限是可以接受的。 </li></ol> 3。脱的第一双肽同时封顶树脂<ol><li>然后加入1毫升的DCM的反应容器中添加冰醋酸滴加1 mL 33％的溴化氢，超过30秒（冒泡），并允许搅拌15分钟。排水和洗树脂用DCM（5倍），然后重复处理一次。 </li><li>洗树脂与DCM（5X），然后DMF（5倍）。抵消树脂由5％V / V解决方案的N，N - 二异丙基（DIPEA）在DMF中，然后用DCM（5倍）和二甲基甲酰胺（5倍）再次清洗两次。执行“甲基红试验”和“醌测试”的10.1和10.2节中讨论。甲基红试验和醌测试的阳性结果应该是阴性。 </li></ol> 4。耦合叔/ TBU保护官能双阿米无酸<ol><li>三次洗涤，无水的DCM重新惰性气氛中的含树脂反应釜，然后附上隔和氩线。清除和清洗船只，加入1-2毫升无水的DCM和出租搅拌30秒，然后排空容器氩行起泡的，直到开始上升。这样做至少一次。 </li><li>准备一个0.15 M的解决方案功能的双氨基酸（3EQ）的1 - 羟基-7-azabenzotriazole（HOAt，136.11克/摩尔，18eq）2:1的DCM在2毫升和245毫克二甲基甲酰胺在火焰干测试管在氩气气氛。新增47μL的diisopropylcarbodiimide，（DIC时，156.6毫升/摩尔，3EQ），搅拌90分钟。 </li><li>加入666μL无水DMF 35微升DIPEA（174.19毫升/摩尔，2EQ）树脂，并允许搅拌5分钟。 </li><li>转让前活性双氨基酸的反应容器中的溶液通过注射器，并允许搅拌过夜。 </li><li>排出反应混合物，并用无水DCM的两次，而根据<html>氩气。 </li><li>促进关闭哌嗪二酮，增加0.25中号的HOAt（136.11克/摩尔，10eq）和DIC（156.6毫升/摩尔，10eq）在1:1 DCM的4毫升的解决方案：二甲基甲酰胺，并允许在氩气搅拌1个小时。 </li><li>取出隔，并排出反应混合物。洗树脂与DCM（5倍）和二甲基甲酰胺（5倍）。如果需要的话，执行第10.2节中所讨论的“醌测试”。 </li></ol> 5。中银/ TBU保护功能化的双氨基酸脱的<ol><li>加入2毫升95:5 trifluororacetic乙酸（TFA）的解决方案：triisopropylsilane（TIPS）的反应容器，并允许它搅拌1小时。排水和洗用DCM（5X）树脂约30秒，然后重复过程一次。 </li><li>洗树脂与DCM（5X），然后DMF（5倍）。抵消树脂由5％V / V解决方案，在DMF DIPEA然后洗DCM（5倍）和二甲基甲酰胺（5倍）再次清洗两次。如果需要的话，执行第10.2节中所讨论的“醌测试”。 </li></ol>乐“&gt; 6。重复步骤4和5所需的合成有针对性的双肽。 7。官能双肽Prolidine的完<ol><li>酰化日益增长的双肽prolidine年底可单独或一起通过哌嗪二酮。此外，为此可以离开保护，这将被切割后，给予该游离氨基酸。如果需要，执行“醌测试”，在10.2节讨论评估的耦合效率。 </li></ol> 8。脱的FMOC和双肽第四纪酰化<ol><li>添加2mL的解决方案的20％，在DMF哌啶和20分钟的反应不一。排水和洗树脂与二甲基甲酰胺（5倍），然后再次重复过程。 </li><li>洗树脂与DCM（5X），然后DMF（5倍）。 </li><li>准备在2毫升N-甲基吡咯烷酮（NMP）的一个氨基酸（3EQ）0.15 M的解决方案，与114毫克2 - （7 - 氮杂-1H-苯并三唑-1 - 基）-1,1,3,3 - 四甲基咪唑六氟磷酸盐<html> （哈图，380.2克/摩尔，3EQ）和104.5μLDIPEA（174.19毫升/摩尔，6eq）拌匀。加入反应釜，并允许搅拌6小时。 </li><li>洗树脂与DCM（5X），然后DMF（5倍）。 </li></ol> 9。从开往氨基酸和切割树脂树脂中银集团<ol><li>反应容器中加入2毫升1:1反式脂肪酸：DCM的解决方案，并允许搅拌30分钟。排水和洗树脂用DCM（5倍），然后重复处理一次。 </li><li>洗净沥干树脂与DCM（5倍），然后DMF（5X）30秒。 </li><li>加入2 mL 10％的无水DMF DIPEA的解决方案中，并允许搅拌24-48小时。 </li><li>预先称重的圆底烧瓶中收集到反应混合物。转移30μL此解决方案，以450μL，在LC-MS法小瓶四氢呋喃，并提交分析。与二甲基甲酰胺的额外等分洗净树脂和收集到的圆底烧瓶中，然后在真空中除去溶剂。 </li></ol>dflinebreak“&gt; 10。双肽的分离纯化<ol><li>原油溶解在二甲基亚砜（100-250μL）和转移到高效液相色谱小瓶插入少量双肽。地方半prepitive的高效液相色谱系统（惠普1100系列）插入到自动进样器配备的Xterra准备MS C18的5微米7.8x150 mm柱和100μL注射液循环。 </li><li>执行多个样品50水的5-95％乙腈梯度程序超过30分钟，同时在274 nm处监测微升0.1％甲酸注射。在预先称重的一次性离心管收集产品峰和冻结干燥用冻干机，应注意采取与第一次运行比较分析学习内容管理系统，通常是观察一个峰的保留时间略有转变。 </li></ol> 11。评估方法<ol><li>甲基红试验7：删除〜1毫克干树脂通过一次性吸管，冲洗成4毫升的反应容器。加入500μL无水DCM的20毫克甲基红，50μL，列印，N&#39;-diisopropylcarbodiimide双丙烯酰胺（DIC）和5 mg 4 - 二甲氨基吡啶（DMAP）的解决方案，并允许搅拌5-10分钟。排水和洗树脂与DCM的，直到滤液变成无色。积极的迹象是剩余的橙色或红色的树脂。 </li><li>醌测试12：换乘1毫克干树脂通过一次性吸管小瓶。 0.8毫米在DMF溶液醌和2％的乙醛在DMF溶液中加入3滴，让坐在室温5-10分钟。积极的迹象是变成蓝色/紫色的树脂。 </li><li>激活陷阱检测：活性化合物的合成过程中，可评估转移激活的解决方案，以液相色谱 - 质谱（LC-MS）的小瓶含50μL吡咯烷少量（5-10微升）。手工搅拌几秒钟（soluti应该变成黄色），然后稀释450μL四氢呋喃（THF），并提交LC-MS分析。 </li><li>分析LC-MS评估的最终产品和活性中间体可以使用惠普1200系列的LC-MS系统配备沃特世的Xterra的MS C18 3.5微米4.6毫米×150毫米列和5-95％的水乙腈梯度​​系统甲酸0.1％，超过30分钟。 </li></ol> 12。代表结果同时原油的例子（ 图4）和纯化（ 图5）提供学习内容管理系统的痕迹。纯化产量的约10％，预计使用上述方法。 <img alt="图1" src="/files/ftp_upload/4112/4112fig1.jpg" /> 图1。固相合成实验装置图。 <img alt="图2" src="/files/ftp_upload/4112/4112fig2.jpg" /> 图2。双氨基酸/双肽的有关术语。 <img alt="图3" src="/files/ftp_upload/4112/4112fig3.jpg" /> 图3。整体的综合计划。 <a href="http://www.jove.com/files/ftp_upload/4112/4112fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4a" src="/files/ftp_upload/4112/4112fig4a.jpg" /> 图4a。高效液相色谱法在274 nm的原油产品的跟踪。 <img alt="图4b" src="/files/ftp_upload/4112/4112fig4b.jpg" /> 图4b。MS粗品峰值的频谱。 <img alt="图5a" src="/files/ftp_upload/4112/4112fig5a.jpg" /> 图5a。高效液相色谱纯化，产品在274纳米微量元素。 <img alt="图5b" src="/files/ftp_upload/4112/4112fig5b.jpg" /> 纯化生产的质谱图5b。T峰。

<ol>
	<li>Atherton, E., Sheppard, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Solid Phase Peptide Synthesis: A Practical Approach. , (1989).</li><li>Brown, Z. Z., Alleva, J., Schafmeister, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-Phase+Synthesis+of+Functionalized+Bis-Peptides.">Solid-Phase Synthesis of Functionalized Bis-Peptides.</a> Biopolymers. 96, 578-585 (2010).</li><li>Schafmeister, C. E., Brown, Z. Z., Gupta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape-Programmable+Macromolecules.">Shape-Programmable Macromolecules.</a> Acc. Chem. Res. 41, 1387-1398 (2008).</li><li>Brown, Z. Z., Schafmeister, C. E. <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+Hexa-+and+Pentasubstituted+Diketopiperazines+from+Sterically+Hindered+Amino+Acids.">Synthesis of Hexa- and Pentasubstituted Diketopiperazines from Sterically Hindered Amino Acids.</a> Org. Let. 12, 1436-1439 (2010).</li><li>Nielson, J., Lyngso, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+Solid-Phase+Synthesis+of+Balanol+Analogues.">Combinatorial Solid-Phase Synthesis of Balanol Analogues.</a> Tet. Lett. 37, 8439-8442 (1996).</li><li>Blankemeyer-Menge, B., Nimtz, M., Frank, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Efficient+Method+for+Anchoring+Fmoc-Amino+Acids+to+Hydroxyl-Functionalized+Solid+Supports.">An Efficient Method for Anchoring Fmoc-Amino Acids to Hydroxyl-Functionalized Solid Supports.</a> Tet. Lett. 31, 1701-1704 (1990).</li><li>Komba, S., Sasaki, S., Machida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Colorimetric+Test+for+Detection+of+Hydroxyl+Groups+in+Solid-Phase+Synthesis.">A New Colorimetric Test for Detection of Hydroxyl Groups in Solid-Phase Synthesis.</a> Tet. Lett. 48, 2075-2078 (2007).</li><li>Demner, O., Dijkgraaf, I., Schottelius, M., Wester, H. J., Kessler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+Functional+Groups+into+Peptides+via+N-Alkylation.">Introduction of Functional Groups into Peptides via N-Alkylation.</a> Org. Lett. 10, 2015-2018 (2008).</li><li>Plas, S. E. V. a. n. d. e. r., Van Hoeck, E., Lynen, F., Sandra, P., Madder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+New+SPE+Material+for+EDCs:+Fully+Automated+Synthesis+of+a+Library+of+Tripodal+Receptors+Followed+by+Fast+Screening+by+Affinity+LC.">Toward a New SPE Material for EDCs: Fully Automated Synthesis of a Library of Tripodal Receptors Followed by Fast Screening by Affinity LC.</a> Eur. J. Org. Chem. 11, 1796-1805 (2009).</li><li>Kaiser, E., Colescot, R. L., Bossinger, C. D., Cook, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color+Test+for+Detection+of+Free+Terminal+Amino+Groups+in+Solid-Phase+Synthesis+of+Peptides.">Color Test for Detection of Free Terminal Amino Groups in Solid-Phase Synthesis of Peptides.</a> Anal. Biochem. 34, 595-598 (1970).</li><li>Chan, W. C., White, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fmoc Solid Phase Peptide Synthesis: A Practical Approach. , (2000).</li><li>Vojkovsky, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Secondary+Amines+on+Solid-Phase.">Detection of Secondary Amines on Solid-Phase.</a> Peptide Research. 71, 236-237 (1995).</li></ol>

没有利益冲突的声明。

这里提供了一个双氨基酸建筑块，采用常见的固相多肽合成技术的功能之二肽合成方法合成方法。单体合成的“反-4-羟脯氨酸3这些”PRO4的积木是高度可扩展的，并已成功完成海因阶段在600毫摩尔（234克）规模（未出版）。一旦手单体，采用固相技术提供了比我们目前的解决方案相方法，通过消除反应的工作和中间纯化需要更快速的双肽合成方法。 在固相合成的主要挑战是合成的进展情况和解决问题的，因为没有中间隔离的诊断。这导致许多比色法测试的发展，包括识别，如果自由胺（凯泽测试10）或免费hydroxYLS（甲基红试验7）暴露树脂。不幸的是，常用的凯撒测试10是在我们的固相合成由于二级胺或附加到一个季碳胺几乎独占使用一般不适用。为在HMBA树脂评估的其他选项包括测试分裂使用，如肼11，定量Fmoc保护裂解紫外/可见1,11监测亲核，捕获和分析传入激活化合物。 在固相合成的另一个被忽视的问题是重复操作所需要的合成步骤的性质。这是头脑，强烈建议使用电子表格或清单时执行任何手动固相多肽合成。 使用比较常见的α-氨基酸的固相合成二肽的困难，包括由于立体欣潜力更加困难接头需要上树脂哌嗪二酮封drance，同时deprotections（BOC /的TBU; CBZ / TBU）。另一个困难在于实现从树脂相比更常规的手段时，使用这个“安全陷阱”的方法，定量释放。考虑到这些因素，这是非常可能的，这种方法的进一步优化，可以达到目前的努力是我们小组正在改善这里介绍的方法。

<table border="1"><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> HMBA-AM树脂</td><td> NovaBiochem </td><td> 855018 </td><td></td></tr><tr><td> MSNT </td><td> NovaBiochem </td><td> 851011 </td><td></td></tr><tr><td> NMI中断</td><td> Sigma-Aldrich公司</td><td> 336092 </td><td>有毒，有腐蚀性</td></tr><tr><td> DCM的</td><td> Sigma-Aldrich公司</td><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&amp;N4=D65100%7CSIAL&amp;N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&amp;F=SPEC" target="_blank">D65100</a> </td><td>致癌</td></tr><tr><td>无水DCM的</td><td>阿库罗斯福冈</td><td> 34846 </td><td>致癌</td></tr><tr><td> 33％溴化氢醋酸</td><td> Sigma-Aldrich公司</td><td> 248630 </td><td>无毒，更正osive，烟雾时开放</td></tr><tr><td> DIPEA </td><td> Sigma-Aldrich公司</td><td> 387649 </td><td>易燃，有毒，有腐蚀性</td></tr><tr><td>二甲基甲酰胺</td><td> Fisher Scientific则</td><td> AC27960 </td><td>易燃，有毒</td></tr><tr><td>无水DMF </td><td>阿库罗斯福冈</td><td> 34843 </td><td>易燃，有毒</td></tr><tr><td> HOAt </td><td>金斯瑞</td><td> C01568 </td><td></td></tr><tr><td>迪爱生</td><td>阿库罗斯福冈</td><td> BP590 </td><td>易燃，有毒，有腐蚀性</td></tr><tr><td>反式脂肪酸</td><td> Sigma-Aldrich公司</td><td> T6508 </td><td>有毒，有腐蚀性</td></tr><tr><td>提示</td><td>阿库罗斯福冈</td><td> 21492 </td><td>易燃，有毒</td></tr><tr><td>哌啶</td><td> Sigma-Aldrich公司</td><td> 104094 </td><td>易燃，有毒，有腐蚀性</td></tr><tr><td>哈图</td><td>金斯瑞</td><td> C01566 </td><td&gt;有毒</td></tr><tr><td> NMP的</td><td>阿库罗斯福冈</td><td> 36438 </td><td>有毒的</td></tr><tr><td> DMAP的</td><td> NovaBiochem </td><td> 851055 </td><td>有毒的</td></tr><tr><td>甲基红</td><td> Sigma-Aldrich公司</td><td> 250198 </td><td></td></tr><tr><td>四氢呋喃</td><td> Sigma-Aldrich公司</td><td> 401757 </td><td>易燃，有毒，过氧化氢的形成</td></tr><tr><td>吡咯烷</td><td> Sigma-Aldrich公司</td><td> P73803 </td><td>易燃，有毒，有腐蚀性</td></tr><tr><td>二甲基亚砜</td><td>费舍尔</td><td> D1281 </td><td></td></tr><tr><td>的SPPS反应船只</td><td>恩典</td><td> 211108 </td><td></td></tr><tr><td>学习内容管理系统</td><td>安捷伦</td><td> 1200系列</td><td></td></tr><tr><td>半准备立法会</td><td>惠普</td><td> 1100系列</td><td></td></tr><tR&gt; <td>冻干机</td><td> labconco </td><td> 7934027 </td><td></td></tr><tr><td> rotovapor </td><td>步琪</td><td>的R-210系列</td><td></td></tr><tr><td>氩气</td><td>公司Airgas </td><td>受体PP300CT </td><td></td></tr></table>

solid phase synthesis of a functionalized bis-peptide using "safety catch" methodology

1962年，RB梅里菲尔德出版的第一个过程，作为一种新的路由使用的固相多肽合成，高效合成肽。很快就证明了这种技术的优势在其溶液相在时间和劳动的前身。关于固体支持性质的改善，保护群体的就业和耦合方法在过去的五十年只增加了梅里菲尔德的原系统的有效性。今天，使用中银为基础的保护和基地/亲核裂解树脂战略或基于FMOC-保护和酸性裂解树脂战略，率先由RC谢泼德，是最常用的用于合成多肽1。 梅里菲尔德的固体支持战略的鼓舞下，我们已经制定了一个组装官能双肽，这是本文所述的中银/叔丁基固相合成策略。使用固相合成比较牛逼O解决方案相方法不仅有利于梅里菲尔德1所述的时间和劳动，但也允许在双肽库的合成更大的方便。我们在这里展示的综合集成了最终的卵裂阶段，使用两个步骤“安全赶上”机制，从哌嗪二酮形成树脂官能双肽释放。 双肽刚性，螺梯双氨基酸，能在可预见的和可设计的单体单位和每个单体之间的连接的类型和立体控制的方式，定位功能的低聚物。每双氨基酸是1立体纯净，循环支架包含两个氨基酸（与α-氨基甲酸）3,4。我们的实验室目前正在调查横跨多种领域，包括催化，蛋白质相互作用和N的潜在功能双肽anomaterials。

Watch this Scientific Journal Video about Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology at JoVE.com

Solid Phase Synthesis of a Functionalized Bis-Peptide Using &quot;Safety Catch&quot; Methodology

高效固相多肽合成的官能双肽三聚体，利用“安全赶上”HMBA树脂裂解过程的描述。

一个官能二肽的固相合成使用“安全锁扣”方法论

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

solid-phase-synthesis-functionalized-bis-peptide-using-safety-catch

Research

JoVE Journal

Biology

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

24.4K 次观看.  Temple University. 高效固相多肽合成的官能双肽三聚体，利用“安全赶上”HMBA树脂裂解过程的描述。

视频: Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described. Triplicate runs of 48 parallel experiments are reported, varying concentration, solvent and the excess of some of the reagents. The isolation of the product was achieved by a simple filtration and wash procedure. The highest yield obtained was 66% from 0.4 M methanol with 1.2 eq. of imine. This is significantly above the 49% yield obtained from the initial reaction under equimolar concentration at 0.4 M in methanol. Methanol solutions with reagent concentrations of 0.4M or 0.2M gave superior yields while all solvent systems at 0.07M performed poorly. At 0.2M, methanol and ethanol/methanol (60/40) mixtures were statistically equally good while THF/methanol (60/40) was poor and acetonitrile/methanol (60/40) was intermediate. Good reproducibility of the precipitate yields was obtained in these replicate experiments, allowing for subtle interaction effects to be positively identified.

The Ugi reaction has proved to be a convenient way to quickly create diverse libraries of compounds. It involves the reaction of an amine, an aldehyde, a carboxylic acid and an isonitrile typically in methanol at room temperature. In this video, we utilize a 48-slot Mettler-Toledo MiniBlock equipped with filtration tubes and a Mettler-Toledo MiniMapper automated liquid handler was used to deliver the reagents and solvent. The parameters of interest were the concentration, the solvent composition and the excess of some of the reagents.

卯木反应的优化使用平行合成和自动化液体处理

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described.  Triplicate runs ...

科研

生物学

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

氧成像在生物系统中的磷光纳米探针的合成和校准

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

实心板为基础的饮食限制在线虫</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (&alpha;Ig) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The &alpha;Ig control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly.

Using Luminex Corporation&#x2019;s xMAP microsphere technology, we have developed the Multiplexed Fluorometric ImmunoAssay (MFIA) for serosurveillance of various laboratory animal species. The MFIA is a suspension microarray where antigen, tissue control or immunoglobulins are covalently linked to color-coded polystyrene microspheres. The MFIA testing method as well as various troubleshooting topics is addressed.

复用荧光免疫检测方法和故障排除

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

液相亲和捕获试验用磁珠研究蛋白质相互作用：脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

固体支持基于膜的电

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

通过使用光稳定菁染料波长移的DNA杂交探针的合成

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, Rhizoctonia bataticola. In the literature, comprehensive and detailed step-by-step protocols on disease assays are sparse. This article provides complete details on the steps involved in setting up a blotting paper technique for quickly screening genotypes for resistance to DRR. The blotting paper technique is easy and less expensive. Another method, based on the sick pot approach, is a mimic of natural infection and can be applied to study the interacting components&#8212;plant, pathogen, and environment&#8212;involved in the disease triangle.
Moreover, in nature, DRR occurs mostly in rainfed chickpea cultivation areas, where soil moisture recedes as crop growth advances. Drought stress is known to predispose chickpea plants to DRR disease. Pathomorphological and molecular understanding of plant-pathogen interaction under drought stress can pave the way for the identification of elite DRR-resistant varieties from the chickpea germplasm pool. This article provides a stepwise methodology for the preparation of a sick pot and subsequent disease assay. Overall, the information presented herein will help researchers prepare R. bataticola fungal inoculum, maintain this pathogen, set up the blotting paper technique, prepare sick culture and sick pot, and assess pathogen infection in chickpea plants.

This study presents methodologies to study the pathomorphological and molecular mechanisms underlying chickpea&#8211;Rhizoctonia bataticola interaction. The blotting paper method is useful to rapidly study chickpea genotype responses, while the sick pot-based method can be used to simultaneously impose drought and R. bataticola infection and screen for tolerant genotypes.

鹰嘴豆的干根罗特病测定：详细方法

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, ...

Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring systems is challenging. An efficient methodology to synthesize these compounds was recently published that described the solid phase synthesis in four steps rather than the previously reported five steps. The advantage of this shorter synthesis is the elimination of the use of toxic reagents. Low-loading Regenerating Michael (REM) linker-based resin was found to be crucial in the synthesis as high-loading versions prevented the addition of reagents containing bulky phenyl and aromatic side chains. The colorimetric 3-(4&#8242;,5&#8242;-dimethylthiazol-2&#8242;-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was used to examine the cytotoxicity of micromolar concentrations of these novel spirocyclic molecules in vitro. MTT is readily available commercially and produces relatively fast, reliable results, making this assay ideal for these spirocyclic heterocycles. Orthogonal ring structures as well as furfurylamine (a precursor in the synthesis method containing a similar 5-member ring motif) were tested.

Here, we describe a bioassay using 3-(4&#8242;,5&#8242;-dimethylthiazol-2&#8242;-yl)-2,5- diphenyltetrazolium bromide (MTT) to test previously synthesized spirocyclic oximes.

功能化螺环杂环合成和细胞毒性测定

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring ...

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

从血浆和实体组织中分离和分析可追溯和功能化的细胞外囊泡

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...