我们感谢劳伦凡沃森霍夫，Sunhee黄某和达里亚Mochly - 罗森有益的讨论。这项工作是支持的健康资助NIH RC4 TW008781-01 C-IDEA（SPARK）全国学院与NQ的资助者在研究设计，数据收集和分析，发布决定，或准备的手稿没有作用。

1.设备和试剂的制备<ol><li>准备设备<ol><li>执行使用正确的个人防护装备在通风橱内的所有步骤。 </li><li>化学合成上使用的是微波肽合成仪，带有Discover的附加模块配备有光纤温度探头，用于控制在一个聚四氟乙烯反应器中的微波功率传输（30毫升，用玻璃料）的固体支持物或在一次性聚丙烯肽盒（12毫升，用粗玻璃料）。 </li><li>为适当的混合，氮气供应连接到反应容器中，或者可替换地密封聚丙烯盒的两端，并将其放置在旋转振荡器上。 </li><li>地漏反应混合物或洗涤，通过废液捕获连接到室内真空。 </li><li>放置光纤探头进入反应容器。 </li></ol></li><li>准备试剂<ol><li>通过称量Rink酰胺AM树脂100-200目制备树脂（0.204毫克）中，添加5毫升1:N个1混合物，N-二甲基甲酰胺（DMF）/二氯甲烷（DCM）加入到反应容器/聚丙烯盒的树脂向下洗，振摇2-4小时溶胀正确，漏。 </li><li>制备0.2M的9-芴基（Fmoc） -氨基酸溶液通过溶解对应Fmoc-氨基酸的DMF和涡混合物直到氨基酸被溶解（ 表1）。 </li><li> （苯并三唑-1-基） - - N，N，N&#39;，N&#39;四甲基脲六氟磷酸盐（HBTU）在100毫升DMF中，并涡旋直到固体的混合物溶解（表中溶解18.96 克邻制备0.45M的活化剂溶液混合1）。 </li><li> 34.8 毫升 N，N-二异丙基乙胺（DIEA）结合65.2毫升1-甲基-2-吡咯烷酮（NMP）（见表1）制备的2M活化剂碱溶液混合。 </li><li>准备0.1M的去保护解决方案，混合溶解3.37克1-羟基苯水合物三唑（HOBt）在250ml的哌啶的DMF 20％体积/体积溶液和涡旋直到固体的混合物溶解（ 表1）。 </li></ol></li></ol> 2. Fmoc保护的氨基酸偶联<ol><li>氨基酸偶合<ol><li>添加氨基酸（2.5毫升）/活化剂（1毫升）/活化碱（0.5毫升）到反应容器/聚丙烯盒，并让反应继续进行300秒（25瓦，75℃， 表2）。排出溶液。 </li><li>洗用DMF将树脂。添加DMF中的树脂120秒（7毫升，0 W，RT）和排出溶液。重复五次。 </li></ol></li><li> Fmoc去保护<ol><li>加7 ml的DMF中20％哌啶的用0.1M的HOBt到反应容器/聚丙烯墨盒和温育30秒（45瓦，75℃， 表2）。 </li><li>沥干反应混合物。 </li><li>加7 ml的DMF中20％哌啶的用0.1M的HOBt到反应容器/聚丙烯盒孵育180秒（45瓦，75℃， 表2）。 </li><li>沥干反应混合物。 </li><li>洗用DMF将树脂。添加DMF中的树脂120秒（7毫升0瓦，室温）和漏溶液。重复五次。 注意:任选地，此处暂停程序，恢复在稍后的日期。 </li></ol></li><li>氨基酸偶联步骤之后，在4℃下用DCM和存储洗涤树脂至少数天（一段较长的时间存储的树脂在 - 20℃）。 <ol><li>移动从反应容器内的树脂在聚丙烯盒。 </li><li>洗用DCM树脂。添加DCM至树脂120秒（7毫升，0 W，室温）和漏溶液。重复三次。 </li><li>与顶盖和活塞封紧袋口聚丙烯滤芯。 </li><li>开始一个新的合成之前，使树脂溶胀3-4小时的DMF（7毫升）中。 </li></ol></li><li>监控综合<ol><li>使用凯撒（茚三酮）测试或氯苯醌测试，以迅速确定的进度合成。任选地，进行小规模的裂解反应，以确定合成的肽的纯度和质量。见第9条。 注:有关进一步的故障排除见表 3。 </li></ol></li><li>根据需要合成针对性肽重复步骤2.1和2.2: Arg(Pbf)-Asn(Trt)-Gly-Gln(Trt)-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Lys(Boc)-Lys(Mtt)-Gly-Gly-Tyr(But)-Gly-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf). </li></ol> 3.酸酐/酸偶合<ol><li>酐偶合<ol><li>洗用NMP的树脂。添加NMP中的树脂120秒（7毫升，0 W，室温）和漏溶液。重复三次。 </li><li>溶解10当量的NMP（5ml）中的相应的酸酐，加入1当量4-二甲氨基吡啶（DMAP）和10当量的DIEA到该溶液中（ 表1）。 </li><li>添加10:1:酸酐/ DMAP的混合物10 / DIEA至树脂并孵育300秒（25瓦，75℃， 表2）。排出溶液。 </li><li>洗用NMP的树脂。添加NMP中的树脂120秒（7毫升，0 W，室温）和漏溶液。重复三次。 </li></ol></li><li>酸偶合<ol><li>洗用DMF将树脂。添加DMF中的树脂120秒（7毫升，0 W，室温）和漏溶液。重复三次。 </li><li>溶解10当量的相应的二羧酸的DMF（5毫升）中。加入1当量的DMAP和10当量N，N&#39;-Diisopropylcarbodiimide（DIC）到该溶液中（ 表1）。 </li><li>混合30分钟的预活化该混合物。 </li><li>该混合物添加到树脂中，孵育300秒（25瓦，75℃，表2）和漏溶液。 </li><li>洗用DMF将树脂。添加DMF中的树脂120秒（7毫升，0 W，室温）和漏溶液。重复步骤三次。 </li></ol></li></ol> 4，N-二甲基三苯甲基（MTT）保护基团去保护离子注意:赖氨酸侧链保护用N-甲基三苯甲基（MTT）81，保护基，可以有选择地对酸不稳定的条件82,83下脱保护。去保护MTT振荡器上手动保护基没有微波能量。 <ol><li>树脂传递到聚丙烯筒配有盖塞和活塞。 </li><li>洗用DCM树脂。添加DCM至树脂120秒（7毫升，0 W，室温）和漏溶液。重复三次。 </li><li>加入15-25毫升的1％三氟乙酸（TFA），5％三异丙基硅烷（TIS），和94％DCM的混合物中的至每克树脂的聚丙烯墨盒。 注意:TFA是一种强酸和腐蚀性，是非常刺激皮肤，眼睛和肺组织。 <ol><li>保持在罩在所有时间的TFA浓缩溶液。 </li><li>使用适当的个人防护设备（保护眼睛，一个白大褂和手套）和工作在一个通风良好的引擎盖。更换手套及时如果他们与TFA，并立即清理任何溢出接触。如果皮肤或眼睛进来与酸接触，立即用清水冲洗患处洗净额外的15分钟。 </li></ol></li><li>将聚丙烯盒在振荡器上振摇5分钟，在RT。 </li><li>通过施加真空排水从聚丙烯盒的溶液。 </li><li>重复步骤4.3-4.5，三次。 </li><li>洗用DCM树脂。添加DCM至树脂120秒（7毫升，0 W，室温）和漏溶液。重复五次。 </li></ol> 5.环化线性肽<ol><li>洗用DCM树脂。添加DCM至树脂120秒（7毫升，0 W，室温）和漏溶液。重复五次。 </li><li>在50ml聚丙烯管中，溶解5当量苯并三唑-1- LY-氧基-三- pyrrolidinophosphonium hexafluorphosphate（加入PyBOP）的二溴甲烷（DBM，5毫升）中，并添加10当量的DIEA到该溶液中（ 表1）。 </li><l我&gt;添加该混合物至树脂并孵育300秒（25瓦，75℃， 表2）。排出溶液。 </li><li>洗用DCM树脂。添加DCM至树脂120秒（7毫升，0 W，室温）和漏溶液。重复三次。 </li></ol> 6.切割的侧链基团和脱保护<ol><li>用DCM和乙醚洗涤树脂。 <ol><li>添加DCM至树脂120秒（7毫升，0 W，室温）和漏溶液。重复两次。 </li><li>加入乙醚至树脂120秒（7毫升，0 W，室温）和漏溶液。重复两次。 </li></ol></li><li>干燥在真空干燥器中的树脂在RT至少3小时以上氢氧化钾（KOH 1-10克）。 </li><li>称量干燥后的树脂和将其传送到一个​​聚丙烯盒。 </li><li>添加一个预冷却的三氟乙酸（TFA）裂解混合物以10ml（例如，90％TFA，2.5％水，2.5％TIS和5％苯酚），以每之一克树脂。 </li><li>摇动3小时在RT。 </li><li>通过将树脂过滤到50ml聚丙烯管收集为TFA裂解溶液。对于过滤，使用玻璃料是在12 ml的聚丙烯盒。 </li><li>加冷乙醚（35毫升）到管中。 </li><li>离心5分钟，在1207×g离心在4℃下。 </li><li>倾倒出乙醚层。 </li><li>重复步骤6.7-6.9，五次。 </li></ol> 7.干燥主干环肽<ol><li>保持沉淀的肽在同一试管中并使其干燥在通风橱中30分钟。 </li><li>溶解在1肽:水和乙腈（ACN）的1:1混合物。 </li><li>冷冻在液氮中的最终产物溶液。 </li><li>冻干最终产品。 </li></ol> 8.定性骨干环肽<ol><li>溶解产物在水（400微升）的小样本（1毫克）。 </li><li>注入溶解肽（10-200微升）进行反相高效液相chromatograpHY（RP-HPLC）系统，以测试该肽纯度34。 </li><li>检查用质谱基质辅助激光解吸电离（MS-MALDI）84的肽的质量。 <ol><li>拌1微升（100μM）的肽在以1:1（体积/体积）乙腈的混合物:水与1微升基质的（5毫克/毫升，α氰基-4-羟基肉桂酸）以1:1（ⅴ乙腈/ v）的混合物:水用TFA（0.1％）。 </li><li>斑点1微升所述MS-MALDI板上。 </li><li>干燥样品，并将它放在质谱仪。 </li></ol></li><li>称量肽并计算百分产率。 </li><li>储存在-20℃。 </li></ol> 9.监测综合<ol><li>凯撒（茚三酮）测试85 <ol><li>制备试剂溶液。 <ol><li>通过在25ml蒸馏水中溶解16.5毫克氰化钾（KCN）的制备A液。稀释1毫升上述溶液以49毫升吡啶。 </li><li>准备溶液B通过将1g茚三酮在20毫升乙醇中。 </li><li>准备溶液C通过在20毫升乙醇将40克苯酚。 </li></ol></li><li>使用Kaiser试验来检查完成氨基酸偶联的或保护基团的脱保护。 <ol><li>从树脂转移数珠至试管。 </li><li>加入三滴（约100微升）各溶液（A，B和C），混匀。 </li><li>加热试管加热块在110℃下进行5分钟的。 注:蓝彩色珠子（成果）表示不完全偶联反应或Fmoc保护基团的去保护。 </li></ol></li></ol></li><li>醌测试86 <ol><li>制备以下试剂新鲜每个测试。 <ol><li>制备2％四氯苯醌的DMF溶液，溶液A. </li><li>制备2％乙醛的DMF溶液，溶液B. </li></ol></li><li>执行氯苯醌测试，以检查完成氨基酸耦合或的t他脱保护的保护基团。 <ol><li>混合100微升溶液A与100微升溶液B在1.5ml管中。 </li><li>滴珠粒中并轻轻摇动5分钟。 注意:深棕色着色珠（阳性结果）表明Fmoc保护基或不完全的偶联反应的脱保护。 </li></ol></li></ol></li><li>小规模的裂解反应<ol><li>除去少量的树脂向一个3 ml的聚丙烯盒配备帽塞和活塞。 </li><li>治疗与2ml的混合物95％TFA，2.5％水和2.5％TIS。 </li><li>振摇在室温30分钟的混合物。 </li><li>删除使用聚丙烯盒的玻璃料通过过滤树脂并通过氮气流蒸发溶剂。 </li><li>溶于水的残余物，并使用HPLC和/或MS分析产物。 </li></ol></li></ol> 10. 杜氏利什曼原虫鞭毛体活力的文化含量<ol> &lt;LI&gt; 杜氏利什曼原虫 （ 杜氏利什曼原虫 ）的生长和处理条件<ol><li>文化L.原虫前鞭毛体在Eagle氏培养基（DMEM）的Dulbecco氏改性用4.5克/升葡萄糖，L-谷氨酰胺和丙酮酸钠在26℃。 </li><li>对待L.原虫前鞭毛体与在26℃的环肽（100μM）处理24小时。 </li></ol></li><li> 杜氏利什曼原虫 （ 杜氏利什曼原虫 ）的可行性分析<ol><li>评估寄生虫存活与20μl的AlamarBlue根据制造商的协议。 </li><li>确定的AlamarBlue减少通过测量荧光（在570 nm激发和590 nm发射）。较高的荧光值越大表明代谢活性，提高寄生虫生存能力。 </li></ol></li></ol>

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从使用一个完全自动化的微波合成的利什曼原虫的蛋白质缺乏衍生骨干环肽的焦点库的合成描述。环肽的焦点库与保守的药效和各种接头的发展。加入各种接头如戊二酸酐，琥珀酸酐，己二酸，庚二酸，赖氨酸，鸟氨酸，和其他构件块的可用于增加品种的环肽的构象空间。有重点的环肽库的合成，使研究人员筛选最佳的构象空间。因为环肽的构象的变化取决于参数例如环的尺寸和位置，可?...

蛋白质-蛋白质相互作用（质子泵抑制剂）起到最生物过程的关键作用，从细胞内的信号转导至细胞死亡1。因此，针对生产者价格指数是具有根本的重要性，以基础研究和治疗应用。质子泵抑制剂可以通过特定的和稳定的抗体来调节，但抗体是昂贵的并且难以制造，并且具有生物利用度差。另外，质子泵抑制剂可通过小分子靶向。小分子更容易合成和廉价相比抗体;然而，它们是相对不灵活和更适合于细孔比大的蛋白质-蛋白质界面2,3。多样的研究表明，肽，它们比抗体简单和更便宜，比小分子更加灵活，可以结合蛋白接口和调节质子泵抑制剂4,5。全球治疗肽市场总值约1,500十亿美元在2013年增长10.5％禾LLY 6。此外，有50多个市售的肽，周围270肽在临床试验的不同阶段，和大约400肽在先进的临床前阶段7。尽管众多的肽被用作药物，肽仍然构成限制其广泛的应用，包括生物利用度差和稳定性，低效率在交叉细胞膜，和构象柔性8,9-几个挑战。一个替代方案克服这些缺点是应用不同的修改，例如本地（D-氨基酸和N-烷基化）和全局（环化）约束8,10-12。这些修改也自然出现。例如，环孢菌素A，免疫抑制剂的环肽天然，包含一个单一的D-氨基酸和经历N-烷基化修饰13,14。 修饰的天然氨基酸以诱导本地约束，如D-和N-烷基化，通常会影响所述肽9氏生物活性。然而，环化，其中感兴趣的序列可以保持不变，是更可能保留生物活性。环化是一个非常有吸引力的方式通过减少不同的构象之间的平衡，以限制肽构象的空间。它通常通过限制肽与活性构象，其介导只有一个函数增加生物活性和选择性。环化也通过保持肽在较少受到降解酶识别的构象提高肽的稳定性。事实上，环肽均表现相比相应的线性15-17具有改善的代谢稳定性，生物利用度，和选择性。 然而，环化可以是一把双刃剑，因为在某些情况下，限制可能阻止肽从达到的生物活性构象。为了克服这一障碍，集中库中的所有肽具有相同的主sequencË因此不断的药效可以合成。在库中的肽的不同在于，影响其结构参数，如环的大小和位置，以便随后筛选最生物活性的构象9,18。 肽可以既在溶液中并通过固相肽合成（SPPS）的方式，也就是现在的更普遍的肽合成方法，并将进一步讨论的合成。 SPPS是通过该化学转化通过接头连接在固体载体上进行，以制备多种合成化合物19的方法。 SPPS使组装肽由氨基酸连续耦合在从C末端，其连接到一个固体支持物以逐步的方式，以N-末端。的N-α-氨基酸侧链必须与保护是稳定在期间延伸肽使用的反应条件组，以确保在加入每ST一个氨基酸被掩蔽EP。在最后的步骤中，将肽从树脂释放，侧链保护基团伴随除去。而该肽被合成，所有的可溶性试剂可以通过过滤从肽 - 固体载体基质被移除，并在每个偶联步骤的末尾冲走。有了这样的系统，在高浓度的大过量的试剂可以驱动偶合反应至完成，所有的合成步骤可以在同一个容器中无材料 20的任何传输来执行。 虽然SPPS具有如生产不完全反应，副反应，不纯的试剂，以及困难监测反应21的一些局限，SPPS的优点已经使它的"金标准"的肽合成。这些优点包括选项掺入非天然氨基酸，自动化，容易纯化，最小化物理损失，以及使用过量的试剂，导致高收益。 SPPS已被证明是在困难的序列21,22，荧光修饰23，和肽文库24,25的合成极为有用。 SPPS也是其他聚链组件，例如寡核苷酸26,27，寡糖28,29，和肽核酸30,31非常有用的。有趣的是，在某些情况下，SPPS被证明是有利的，用于合成被在溶液32,33传统上制成的小分子。 SPPS可以用在小规模的研究和教学34,35以及大规模产业36-38。 这主要应用于SPPS方法为肽合成两种合成策略是氧羰基（BOC）和9-芴基（Fmoc）。引入SPPS原始策略是Boc时，这需要强酸条件从第r除去侧链保护基团和裂解肽ESIN。将Fmoc-基于肽合成，不过，使用中等强度的碱的条件，是对酸不稳定的Boc协议39较温和的替代方案。将Fmoc策略利用了设置在合成的最后步骤中除去而裂解从在酸性条件下的树脂肽正交叔丁基（TBU）侧链保护。 对于在固体载体上的肽合成的一般原则是在图1的初始氨基酸，通过在N-α-末端的临时保护基团掩盖，被装载到从C末端的树脂。甲半永久性保护基掩蔽侧链还用于在必要时（图1，步骤1）。靶肽的合成是从C末端组装到N末端 ​​的N-α-临时保护基的脱保护的重复循环（图1，步骤2）和耦合下一保护的氨基酸（图1 </str<html>翁&gt;，步骤3）。后最后一个氨基酸被加载（图1，步骤4），将肽从树脂载体切下并半永久性保护基团被除去（图1，步骤5）。 <img alt="图1" src="/files/ftp_upload/53589/53589fig1.jpg" /> 的固相肽合成图 1.常规 方案的 N-α-保护的氨基酸通过接头与树脂（步骤1）使用羧基锚定。所需的肽组装在从C末端到N末端通过从N-α（步骤2）和氨基酸耦合（步骤3）的临时保护基团（TPG）的脱保护的重复循环以线性方式。完成的合成（步骤4）后，将半永久性保护基（SPG）的过程中肽裂解（步骤5）去保护。获得="_空白"&gt;点击此处查看该图的放大版本。 完整的肽链的组装后，环化可以通过多种替代来实现:（A）的头-尾环化-这是一种方便的方式，但不限于，因为它仅提供一个用于环化（图2A）的选项，（B）的环化使用来自感兴趣序列中的氨基酸，包含生物活性官能团的-然而，使用这些氨基酸可通过，而不会干扰增加的氨基酸（或其他构件块）的影响的生物活性（图2B），和（C）环化生物活性序列。引入这些分子是普遍，因为它允许生产集中的库，而无需修改的兴趣（图2C）的序列。 <img alt="图2" src="/files/ftp_upload/53589/53589fig2.jpg" /> Figure 2. 可替代的肽环化法 （A）的头至尾环化，通过C末端和N末端 ​​之间的肽键。官能团之间（B）的环化，如半胱氨酸残基（1），或赖氨酸的侧链之间形成酰胺键之间的二硫键来天冬氨酸/谷氨酸（2），或侧链为N-或C-末端（3 -4）; （C）环化通过增加额外的氨基酸或氨基酸衍生物或小分子，例如前（R0）和（R7）的生物活性序列之后， <a href="https://www.jove.com/files/ftp_upload/53589/53589fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 微波辅助合成采用微波辐射加热反应，从而加速有机化工转化40,41。微波化学是基于试剂/能力溶剂吸收所微波能量并将其转换成热42。前的技 ​​术变得普遍，主要缺点必须克服，包括可控性和合成协议再现性和缺乏可用的系统为适当的温度和压力控制的43,44。做使用厨房微波合成几个短肽（7-10个氨基酸）与显著提高耦合效率和纯度45的微波辅助肽合成的第一份报告。此外，微波能量被证明减少链聚集，降低副反应，限制了外消旋化，并提高连接速度，这是所有艰难和漫长的序列46-53的关键。 目前在固体载体上使用微波照射于肽或相关化合物的合成中是广泛的，包括（A）中合成的有机溶剂54的水代替; （B）的合成肽的同常见的翻译后修饰，如糖 ​​肽55-58或59-61磷酸，其合成通常是困难的，因为位阻氨基酸衍生物的低耦合效率; （C）的合成与修饰肽主链，如azapeptides，其可以由替换的氨基酸残基的C（α）与氮原子62，或拟肽，其侧链连接到被形成的酰胺氮而非Cα原子63,64; （d）合成环肽65-71的;和（E）的合成组合库51,72中。在许多情况下，作者报告更高的效率和降低的合成时间使用微波辐射相比于常规协议。 用合理的设计73-75，我们开发均来自支架大号 eishmania的受体FO抗寄生虫肽- [R激活C-激酶（LACK）。拉克起着利什曼原虫属感染76的早期阶段发挥重要作用。寄生虫表达缺乏较低水平不缺乏参与必不可少的寄生虫信号传导过程和蛋白质的合成78寄生甚至免疫缺陷小鼠77。因此，缺乏的是一个关键的支架蛋白79和宝贵的药物靶点。重点放在中保守的寄生虫，但不是在宿主哺乳动物同系物的RACK在拉克序列，我们确定了一个8个氨基酸的肽（RNGQCQRK），在培养降低利什曼原虫属的生存能力。 在这里，我们描述了一种协议，用于从上述的LACK蛋白序列衍生骨架的环肽的合成。合成使用微波加热通过SPPS方法用Fmoc / TBU协议的坚实支持的肽。肽是通过酰胺键缀合到一个TAT 47-57（YGRKKRRQRRR）载体肽在SPPS的一部分。的各种货物到细胞中的TAT-基于运输已经使用了超过15年，并输送货物到亚细胞器已经证实80。四种不同的接头，琥珀酸和戊二酸酐以及己二酸和庚二酸，被用来进行环化，以​​产生二至五个碳原子的羧酸的接头。已完成的环化利用微波能量，和没有微波能量被手动完成的最终裂解和侧链去保护步骤。使用一个自动化微波合成器中的改进的产品纯度，增加了产品的产量，并降低了合成的持续时间。这是一般的协议可以被应用到利用肽理解重要的分子机制在体外和体内 ，并进一步发展潜力的药物用于人类疾病的其他研究。

<table><tbody><tr><td>&lt;strong&gt;REAGENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Solid support, Rink Amide AM resin ML</td><td>CBL</td><td>BR-1330</td><td>loading: 0.49 mmol/g</td></tr><tr><td>Fmoc-Ala-OH</td><td>Advanced Chemtech</td><td>FA2100</td><td></td></tr><tr><td>Fmoc-Arg(Pbf)-OH</td><td>Advanced Chemtech</td><td>FR2136</td><td></td></tr><tr><td>Fmoc-Asn(Trt)-OH</td><td>Advanced Chemtech</td><td>FN2152</td><td></td></tr><tr><td>Fmoc-Asp(OBut)-OH</td><td>Advanced Chemtech</td><td>FD2192</td><td></td></tr><tr><td>Fmoc-Cys(Trt)-OH</td><td>Advanced Chemtech</td><td>FC2214</td><td></td></tr><tr><td>Fmoc-Gln(Trt)-OH</td><td>Advanced Chemtech</td><td>FQ2251</td><td></td></tr><tr><td>Fmoc-Glu(OtBu)-OH</td><td>Advanced Chemtech</td><td>FE2237</td><td></td></tr><tr><td>Fmoc-Gly-OH</td><td>Advanced Chemtech</td><td>FG2275</td><td></td></tr><tr><td>Fmoc-His(Trt)-OH</td><td>Advanced Chemtech</td><td>FH2316</td><td></td></tr><tr><td>Fmoc-Ile-OH</td><td>Advanced Chemtech</td><td>FI2326</td><td></td></tr><tr><td>Fmoc-Leu-OH</td><td>Advanced Chemtech</td><td>FL2350</td><td></td></tr><tr><td>Fmoc-Lys(Boc)-OH</td><td>Advanced Chemtech</td><td>FK2390</td><td></td></tr><tr><td>Fmoc-Met-OH</td><td>Advanced Chemtech</td><td>FM2400</td><td></td></tr><tr><td>Fmoc-Phe-OH</td><td>Advanced Chemtech</td><td>FF2425</td><td></td></tr><tr><td>Fmoc-Pro-OH</td><td>Advanced Chemtech</td><td>FP2450</td><td></td></tr><tr><td>Fmoc-Ser-(tBu)-OH</td><td>Advanced Chemtech</td><td>FS2476</td><td></td></tr><tr><td>Fmoc-Thr(tBu)-OH</td><td>Advanced Chemtech</td><td>FT2518</td><td></td></tr><tr><td>Fmoc-Trp(Boc)-OH</td><td>Advanced Chemtech</td><td>FW2527</td><td></td></tr><tr><td>Fmoc-Tyr(But)-OH</td><td>Advanced Chemtech</td><td>FY2563</td><td></td></tr><tr><td>Fmoc-Val-OH</td><td>Advanced Chemtech</td><td>FV2575</td><td></td></tr><tr><td>1-Methyl-2-pyrrolidinone (NMP)</td><td>Sigma</td><td>328634</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Toxic/Highly flammable/Irritant.</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide (DMF)</td><td>Alfa Aesar</td><td>43465</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Toxic.&lt;br /&gt;
Use high quality DMF to eliminate side reactions such as Fmoc removal as a result of the dimethylamine traces from DMF decomposition.</td></tr><tr><td>Dichloromethane (DCM)</td><td>Sigma</td><td>D65100</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Harmful</td></tr><tr><td>Dibromomethane (DBM)</td><td>Sigma</td><td>D41868</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Harmful</td></tr><tr><td>Trifluoroacetic acid (TFA)</td><td>Sigma</td><td>T62200</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Corrosive/Toxic</td></tr><tr><td>Trifluoroacetic acid, HPLC grade (TFA)</td><td>Sigma</td><td>91707</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Corrosive/Toxic</td></tr><tr><td>Diethylether</td><td>Sigma</td><td>31690</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Highly flammable/Harmful</td></tr><tr><td>Triisopropylsilane (TIS)</td><td>Sigma</td><td>233781</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Irritant/Flammable</td></tr><tr><td>Water, HPLC grade</td><td>Sigma</td><td>270733</td><td></td></tr><tr><td>Acetonitroile, HPLC grade (ACN)</td><td>Fisher Scientific</td><td>A998-4</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Flammable/Irritant/Harmful</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Diisopropylethylamine (DIEA)</td><td>Sigma</td><td>3440</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Corrosive/Highly flammable</td></tr><tr><td>Piperidine</td><td>Sigma</td><td>W290807</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Toxic/Highly flammable</td></tr><tr><td>Pyridine</td><td>Sigma</td><td>270970</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Highly flammable/Harmful</td></tr><tr><td>Ethanol (EtOH)</td><td>Sigma</td><td>459844</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Highly flammable/Irritant</td></tr><tr><td>1-Hydroxybenzotriazole hydrate (HOBt)</td><td>Sigma</td><td>157260</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Highly flammable/Irritant/Harmful</td></tr><tr><td>&lt;em&gt;O&lt;/em&gt;-(Benzotriazol-1-yl)-&lt;em&gt;N,N,N&amp;prime;,N&amp;prime;&lt;/em&gt;- tetramethyluronium hexafluorophosphate (HBTU)</td><td>Sigma</td><td>12804</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Irritant/Harmful</td></tr><tr><td>Benzotriazole-1-ly-oxy-tris-pyrrolidinophosphonium hexafluorphosphate (PyBOP)</td><td>Advanced Chemtech</td><td>RC8602</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Irritant</td></tr><tr><td>Ninhydrin</td><td>Sigma</td><td>454044</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Harmful</td></tr><tr><td>Phenol</td><td>Sigma</td><td>P3653</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Corrosive/Toxic</td></tr><tr><td>Potassium cyanide (KCN)</td><td>Sigma</td><td>11813</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Very Toxic</td></tr><tr><td>Potassium hydroxide (KOH)</td><td>Sigma</td><td>221473</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Toxic</td></tr><tr><td>&lt;em&gt;N,N&amp;rsquo;&lt;/em&gt;-Diisopropylcarbodiimide (DIC)</td><td>Sigma</td><td>38370</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Flammable/ Toxic</td></tr><tr><td>4-Dimethylaminopyridine (DMAP)</td><td>Sigma</td><td>522805</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Toxic/Irritant</td></tr><tr><td>Glutaric anhydride</td><td>Sigma</td><td>G3806</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Flammable/Irritant/Harmful</td></tr><tr><td>Succinic anhydride</td><td>Sigma</td><td>239690</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Irritant/Harmful</td></tr><tr><td>Adipic acid</td><td>Sigma</td><td>A26357</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Toxic/Irritant</td></tr><tr><td>Pimelic acid</td><td>Sigma</td><td>P45001</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Toxic/Irritant</td></tr><tr><td>Chloranil</td><td>Sigma</td><td>23290</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Toxic/Irritant</td></tr><tr><td>Acetaldehyde</td><td>Sigma</td><td>402788</td><td>&lt;strong&gt;Caution&lt;/strong&gt; Flammable/ Toxic</td></tr><tr><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>Allegra 6R centrifuge</td><td></td></tr><tr><td>Lyophilizer</td><td>Labconco</td><td>freezone 4.5</td><td></td></tr><tr><td>Vacuum pump</td><td>Franklin Electric</td><td>model 1101101416 with 3/4 HP</td><td>Alcatel pump with Franklin Motor&amp;nbsp;</td></tr><tr><td>Polypropylene cartridge 12 ml</td><td>Applied Separation</td><td>2419</td><td></td></tr><tr><td>Cap plug for 12 ml polypropylene cartridge</td><td>Applied Separation</td><td>8157</td><td></td></tr><tr><td>Polypropylene cartridge 3 ml</td><td>Applied Separation</td><td>2413</td><td></td></tr><tr><td>Cap plug for 3 ml polypropylene cartridge</td><td>Applied Separation</td><td>8054</td><td></td></tr><tr><td>Stop cocks PTFE</td><td>Applied Separation</td><td>2406</td><td></td></tr><tr><td>Tubes flat, 50 ml</td><td>VWR</td><td>21008-240</td><td></td></tr><tr><td>Extraction manifold, 20 pos, 16 x 100 mm tubes</td><td>Waters</td><td>WAT200609</td><td></td></tr><tr><td>Shaker, BD adams nutator mixer</td><td>Fisher scientific</td><td>22363152</td><td></td></tr><tr><td>Nalgene HDPE narrow mouth IP2 bottles, 125&amp;nbsp;ml</td><td>Fisher scientific</td><td>03-312-8</td><td></td></tr><tr><td>Erlenmeyer flask</td><td>Fisher Scientific</td><td>FB-501, 500 ml</td><td></td></tr><tr><td>Heating block</td><td>Thermolyne</td><td>1760 dri bath</td><td></td></tr><tr><td>Disposable borosilicate glass tubes with plain end</td><td>Fisher Scientific</td><td>14-961-25</td><td></td></tr><tr><td>Micropipettes and tips Finnpipette</td><td>Thermo</td><td>20&amp;ndash;200 and 100&amp;ndash;1,000 &amp;mu;l</td><td></td></tr><tr><td>HPLC vials - micro vl pp 400 &amp;micro;l PK100&amp;nbsp;&amp;nbsp;</td><td>VWR</td><td>69400-124</td><td></td></tr><tr><td>HPLC vial- Blue Snap-It Cap</td><td>VWR</td><td>66030-600</td><td></td></tr><tr><td>Analytical HPLC column</td><td>Peeke Scientific</td><td>U1-5C18Q-JJ</td><td>ultro 120 5 &amp;micro;m C18Q, 4.6 mm ID 150 mm</td></tr><tr><td>Prep HPLC column, XBridge&amp;nbsp;</td><td>Waters</td><td>OBD C18 5 &amp;micro;m column</td><td>19 mm &amp;times; 150 mm</td></tr><tr><td>Mass spectrometer</td><td>Applied Biosystems</td><td>Voyager DE-RP&amp;nbsp;</td><td></td></tr><tr><td>Nitrogen cylinder</td><td></td><td></td><td></td></tr><tr><td>Desiccator</td><td></td><td></td><td></td></tr><tr><td>Analytical RP-HPLC system Shimadzu LC-20</td><td></td><td></td><td>equipped with: CBM-20A system controller, SPD-20A detector, CTO-20A column oven, 2 x LC-20AD solvent delivery unit, SIL-20AC autosampler, DGU-20A5 degasser (Shimadzu, MD, USA).</td></tr><tr><td>Preparative RP-HPLC system Shimadzu LC-20</td><td></td><td></td><td>equipped with: CBM-20A system controller, SPD-20A detector, CTO-20A column oven, 2 x LC-6AD solvent delivery unit and FRC-10A fraction collector (Shimadzu, MD, USA).</td></tr></tbody></table>

development of a backbone cyclic peptide library as potential antiparasitic therapeutics using microwave irradiation

蛋白质 - 蛋白质相互作用（质子泵抑制剂）是密切参与几乎所有的生物过程，并与许多人类疾病。因此，有一个重大努力的目标在基础研究和在制药工业中质子泵抑制剂。蛋白质 - 蛋白质接口通常是大而平的，而且往往缺乏口袋，小分子靶向此类网站的发现复杂化。使用抗体靶向的替代方法有由于不良的口服生物利用度，低格，透气性好，生产效率低下的限制。 使用的肽靶向PPI接口具有几个优点。肽具有更高的构象柔性，提高的选择性，并且通常便宜。然而，肽具有其自身的局限性，包括稳定性差，效率低下穿越细胞膜。为了克服这种局限性，肽的环化可以被执行。环化已被证明提高肽选择性，代谢稳定性，和生物利用率。然而，预测环肽的生物活性的构象是不平凡的。为了克服这一难题，人们有吸引力的方法是筛选一个焦点库到屏幕，其中所有主链的环肽具有相同的一级序列，但是不同之处在于影响其构象的参数，如环的大小和位置。 我们描述了一个详细的协议用于合成靶向特定寄生虫质子泵抑制剂骨干环肽的文库。采用合理的设计方法，我们开发了从支架L蛋白eishmania受体激活C-激酶（LACK）的肽。我们假设，即是保守的寄生虫，但不是在哺乳动物宿主同源的拉克序列，可以表示相互作用位点的蛋白质，是对于寄生虫&#39;能力至关重要。合成的环肽是使用微波照射以减少反应时间和增加效率。开发骨干环肽具有不同的戒指尺寸库便于系统的屏幕为大多数生物活性构象。此方法提供合成环肽的一般，快速和容易的方式。

这里，我们描述的聚焦小库骨干环肽的特异性靶向的利什曼原虫寄生虫的重要质子泵抑制剂和作为抗寄生虫药（综述约靶向质子泵抑制剂作为抗寄生虫药87的肽）的发展。通过新颖骨干环肽的合成，药效是保守的伸出尺寸的支架。这里提出的焦点库的力量是改变肽支架尺寸，同时允许构象自由通过环化受限程度的能力。做骨干环肽的整个合成使用自动微波合成器在固体载体上，之后...

Watch this Scientific Journal Video about Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation at JoVE.com

Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation

A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.

一个主干环肽库作为潜在的抗寄生虫治疗微波辐射的发展

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is ...

development-backbone-cyclic-peptide-library-as-potential

Research

JoVE Journal

Chemistry

11.8K 次观看.  Stanford University School of Medicine. A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.

视频: Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

对于拟肽与他们抗利什曼原虫活性混合赖氨酸型/精氨酸型单体及评价合成的有效方法

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this ...

科研

生物化学

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

保利的微波辅助功能化（乙二醇）和On-树脂链聚合反应，并形成水凝胶的肽使用

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

化学

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 ...

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

免疫与感染

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

产生和回收的β-细胞的细胞球体从逐步增长的PEG-肽水凝胶

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

生物工程

Microwave-Assisted Preparation of 1-Aryl-1H-pyrazole-5-amines

A synthetic process for the preparation of a variety of 1-aryl-1H-pyrazole-5-amines was developed. The microwave-mediated nature of this method makes it efficient in both time and resources and utilizes water as the solvent. 3-Aminocrotononitrile or an appropriate &#945;-cyanoketone is combined with an aryl hydrazine and dissolved in 1 M HCl. The mixture is then heated in a microwave reactor at 150 &#176;C, typically for 10-15 min. The product can be readily obtained by basifying the solution with 10% NaOH and isolating the desired compound with a simple vacuum filtration. The use of water as a solvent in this reaction lends to its ease and utility in production, and this method is easily reproducible with a variety of functional groups. Typical isolated yields range from 70-90%, and reactions can be performed on the milligram to gram scale with little to no change in observed yields. Some of the applications of these molecules and their derivatives include pesticides, anti-malarials, and chemotherapeutics, among many others.

Microwave-Assisted Preparation of 1-Aryl-1H-pyrazole-5-amines

1-Aryl-1H-pyrazole-5-amines are prepared from aryl hydrazines combined with either 3-aminocrotononitrile or an &#945;-cyanoketone in a 1 M HCl solution using a microwave reactor. Most reactions are done in 10-15 minutes and pure product can be obtained via vacuum filtration with typical isolated yields of 70-90%.

微波辅助制备1-Aryl-1 H-pyrazole-5胺

A synthetic process for the preparation of a variety of 1-aryl-1H-pyrazole-5-amines was developed. The microwave-mediated nature of this method makes it ...

Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper tumor cells, leading to tumor recurrence. To conquer the limited penetration of biological barriers, cell-penetrating peptides (CPPs) have been discovered with excellent membrane translocation ability and have emerged as useful molecular transporters for delivering various cargoes into cells. However, conventional linear CPPs generally show compromised proteolytic stability, which limits their permeability across biological barriers. Thus, the development of novel molecular transporters that can penetrate biological barriers and exhibit enhanced proteolytic stability is highly desired to promote drug delivery efficiency in biomedical applications. We have previously synthesized a panel of short cyclic CPPs with aromatic crosslinks, which exhibited superior permeability in cancer cells and tissues compared to their linear counterparts. Here, a concise protocol is described for the synthesis of the fluorescently labeled cyclic polyarginine R8 peptide and its linear counterpart, as well as key steps for investigating their cell permeability.

This protocol describes the synthesis of cyclic cell-penetrating peptides with aromatic cross-links and the evaluation of their permeability across biological barriers.

构建环状细胞穿透肽以增强生物屏障的穿透力

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper ...

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as a consequence, they are now used clinically. It is, therefore, critical to seek effective strategies for synthesizing cyclic peptides to promote their application in the field of drug discovery. This paper reports a detailed protocol for the efficient synthesis of cyclic peptides using on-resin or intramolecular (intermolecular) bisalkylation. Using this protocol, linear peptides were synthesized by taking advantage of solid-phase peptide synthesis with cysteine (Cys) and methionine (Met) coupled simultaneously on the resin. Further, cyclic peptides were synthesized via bisalkylation between Met and Cys using a tunable tether and an on-tether sulfonium center. The whole synthetic route can be divided into three major processes: the deprotection of Cys on the resin, the coupling of the linker, and the cyclization between Cys and Met in a trifluoroacetic acid (TFA) cleavage solution. Furthermore, inspired by the reactivity of the sulfonium center, a propargyl group was attached to the Met to trigger thiol-yne addition and form a cyclic peptide. After that, the crude peptides were dried and dissolved in acetonitrile, separated, and then purified by high-performance liquid chromatography (HPLC). The molecular weight of the cyclic peptide was confirmed by liquid chromatography-mass spectrometry (LC-MS), and the stability of the cyclic peptide combination with the reductant was further confirmed using HPLC. In addition, the chemical shift in the cyclic peptide was analyzed by 1H nuclear magnetic resonance (1H NMR) spectra. Overall, this protocol aimed to establish an effective strategy for synthesizing cyclic peptides.

This protocol presents the synthesis of cyclic peptides via bisalkylation between cysteine and methionine and the facile thiol-yne reaction triggered by the propargyl sulfonium center.

使用系绳上的锍中心构建环肽

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as ...

Versatile Home-Built Microwave-Assisted System for Peptide Synthesis

An Inexpensive Adaptation of a Commercial Microwave Reactor for Solid Phase Peptide Synthesis

A home-built apparatus to perform solid phase peptide synthesis (SPPS), assisted by microwave irradiation and heating, is presented. In contrast to conventional SPPS reaction vessels, which drain solvent and byproducts via a frit located at the bottom of the vessel, the presented apparatus employs a gas dispersion tube under vacuum to remove solvent, byproducts, and excess reagents. The same gas dispersion tube supplies nitrogen gas agitation of the SPPS beads during the reaction steps of coupling and deprotection. Microwave heating is beneficial for SPPS couplings of sterically hindered residues, such as alpha-aminoisobutyric acid (Aib), an alpha,alpha-dialkylated amino acid residue. This home-built apparatus has been used to prepare, via manual Fmoc SPPS methods, heptameric and octameric peptides dominated by the Aib residue, which is notoriously difficult to couple under standard room temperature conditions and reagents. Further, typical commercial microwave SPPS reactors are dedicated exclusively to SPPS synthesis rendering them inaccessible to non-SPPS users. In contrast, the presented apparatus preserves the versatility of the microwave reactor for conventional microwave acceleration of chemical reactions, as the apparatus is trivially removed from the commercial microwave reactor.

A simple, inexpensive, and novel apparatus for performing solid phase peptide syntheses in a commercial microwave reactor is presented.

A home-built apparatus to perform solid phase peptide synthesis (SPPS), assisted by microwave irradiation and heating, is presented. In contrast to ...

<meta charset="utf8"></head><body>使用双报告基因的 脓肿分枝 杆菌高通量筛选测定

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

<meta charset="utf8"></head><body>作者聚焦:用于高效发现 脓肿分枝杆菌 治疗的可扩展药物筛选方案

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

分枝杆菌高通量药物筛选的分析开发

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...