Name: synthetic methodology for asymmetric ferrocene derived bio-conjugate systems via solid phase resin-based methodology
Uploaded: 2015-03-12T14:00:00.000+00:00
Duration: 7 min 7 s
Description: Watch this Scientific Journal Video about Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology at JoVE.com

KG是由RA韦尔奇基金会授予P-1760支持，数学和科学教育（以KG）的TCU安卓研究所，TCU研究和创新活动格兰特（以KG）和TCU电监会格兰特（以JHS）。

1.合成生物素的Fc半胱氨酸（1） <ol><li>固相方法，以产生树脂结合的1。 <ol><li>放置生物素加载树脂（250毫克，0.145毫摩尔）转化为烧结注射器和通过制定二甲基甲酰胺（5毫升）中，并摇动在实验室摇动器在注射器20分钟使树脂溶胀。排出溶液和二甲基甲酰胺重复肿胀一次。 </li><li>通过加入4-6毫升20％哌啶的二甲基甲酰胺在注射器随后振荡10-15分钟，除去Fmoc保护基。与另一4-6毫升哌啶，重复脱保护过程。甲醇（1:1），3倍的甲醇:二氯甲烷（1:1）的序列的3倍二甲基甲酰胺，3倍二甲基甲酰胺洗涤树脂，3×二氯甲烷，约5 ml的每种。做的小珠的一小部分（〜10）茚三酮试验（+），以确认成功地由脱保护的蓝色的存在下在加热时。 </li><li>混合含有1&#39;-Fmoc-氨基二茂铁-1-羧酸溶液（203.3毫克，0.4350毫摩尔），1-羟基苯并三唑水合物（58.8毫克，0.413毫摩尔），二异丙基碳二亚胺（0.0673毫升，0.435毫摩尔），二异丙基乙基胺（0.0757毫升，0.435毫摩尔），以及一个4:二氯甲烷1:1混合物和二甲基甲酰胺。得出这样的成烧结注射器并轻轻摇动在实验室摇动器6小时。然后排出从注射器的溶液和洗涤如前所述。 </li><li>进行茚三酮试验 - 如上所述，确认耦合（）。茚三酮测试仍然可以在尽管从含有部分铁的附着得到的珠粒的橙色确认耦合有用。 </li><li>然后通过加入二甲基甲酰胺20％哌啶的除去Fmoc基团，并如上所述洗涤。茚三酮试验（+）应当用于确认去除Fmoc。 </li><li>制备的Fmoc-半胱氨酸（TRT）组成的溶液-OH（254.8毫克，0.4350毫摩尔），1-羟基苯并三唑水合物（58.8毫克，0.4125毫摩尔），二异丙基碳二亚胺（0.0673毫升，0.4350毫摩尔），二异丙基乙基胺（0.0757毫升，0.4350毫摩尔），并在4:二氯甲烷和二甲基甲酰胺的1:1混合物。加入该半胱氨酸耦合鸡尾酒烧结注射器，轻轻摇动6小时。洗，用前述的协议。 </li><li>确认使用茚三酮试验耦合（ - ），然后除去，用20％哌啶和洗涤将Fmoc组分。验证的游离末端胺使用茚三酮试验（+）。 </li></ol></li><li>从树脂裂解1。 <ol><li>使TFA溶液（9.45毫升），水（0.25毫升），1,2-乙二硫醇（0.25毫升），三异丙基硅烷（0.1毫升）中，将其添加到注射器并轻轻摇晃4小时。 </li><li>收集在Eppendorf管中所产生的红棕色溶液，蒸发缓慢用空气流为TFA。 </li><li>添加冷二乙醚（约15毫升）至Eppendorf管中，以沉淀1，这将形成温和搅拌。隔离通过离心的产物（1克，5分钟）。牛逼的乙醚洗涤液（〜60毫升数量）和离心机母鸡重复周期，得到1，为红色/褐色固体。 </li></ol></li></ol> 2.表征和分析1 <ol><li>确认该身份用1 H（16次扫描）和13 C NMR（512次扫描）在氘化甲醇（300微升）和ESI-MS分析，在图2所示的连接和组成相匹配。 期望的结果如下: 1 H NMR谱（CD 3 OD）δ/ ppm的:1.407-1.684（米，6H），2.245（T，2H），2.665-3.150（米，12H），4.015（吨，1H），4.104（D，2H ），4.274（Q，1H），4.426（D，2H），4.479（Q，1H），4.595（吨，1H）和13 C NMR谱（CD 3 OD）δ/ ppm的:24.644（CH 2），25.472 （CH 2），28.051（CH 2），28.300（CH 2），35.474（CH 2），38.698（CH 2），39.241（CH 2），39.717（CH 2），55.340 / 55.538（CP-环）， 60.286（C1H），61.964（CH），62.521 / 62.821（CP-环），66.038 / 66.170（CP-环），69.153 / 69.328（CP-环），71.468 / 71.593（CP-环），76.466（CH），171.770 （C = O），175.361（C = O）。 ESI-MS（M / Z）:实测值:639.00 [1 + NA] +，理论值:639.1 [1 + NA] +和HR-MS（M / Z）:实测值:617.2049 [1 + H] +，理论值: 617.1622 [1 + H] +。 </li><li>执行高效液相色谱法，元素分析来确认的孤立1的组合物。 使用C8进行HPLC色谱反相柱用100％甲醇以0.5ml / min的流速。注意:HPLC保留时间分别为:3.198-4.674分钟。 </li></ol>在金表面3.固定的1 <ol><li>切聚合物2支撑的黄金滑入了〜0.25平方。 </li><li>填在50ml的烧杯中的1（〜1毫米）DI水的溶液。 </li><li>金滑动添加到烧杯中，并覆盖用表玻璃。所有流载玻片孵育O / N在室温无搅拌。 </li><li>从溶液中除去金滑动，并允许它在空气中干燥。 </li><li>获得扫 ​​描使用扫描型电子显微镜（或等同物）来观察固定1电子显微镜图像。 </li></ol>

The authors have nothing to disclose.

不对称二茂铁衍生物的合成是具有挑战性的溶液。例如，曾试图以产生1的溶液导致期望的产物（低于20％）的产量低。同样地，利用1&#39;-氨基二茂铁羧酸（没有的Fmoc）和树脂结合的生物素的反应导致与报道Baristic 等经聚合的产物一致不溶产物。和最小的产品。47这是通过二茂铁进一步复杂化和其衍生物是光敏感的，该氨基同源容易在溶液中二聚化。这些问题使广泛的反应和workups具有挑战性。然而，这反应性可以使用最初由梅里菲尔德开发用于合成肽的固相方法来规避。二茂铁-肽系统的合成已经获得关注由几组，并导致非对称的肽-二茂铁的系统的文库。46,48-52工作本文中所描述详细说明了第一非对称二茂铁含有生物素的二茂铁 - 半胱氨酸生物缀合物的合成。该化合物用作通过该其他小分子受体，可考虑检测生物相关的分子的模型。在这项工作中被用作固定化系绳到金表面。 合成的第一阶段是，得到固定化生物素芯上使用胺系接头的固态树脂。 N个 -Biotin- -N&#39;-Fmoc乙二胺树脂可商购可购买作为生物素NovaTag树脂和用作生物缀合物1的基础。使用哌啶的二甲基甲酰胺中的20％溶液和正（蓝色）茚三酮试验证实成功脱保护除去Fmoc-氨基保护基。所述树脂结合的生物素的游离胺，然后使用利用偶合鸡尾酒二异丙基乙基胺构成的锚二茂铁，二异丙基碳二亚胺和1-羟基苯并三唑水合物。40,46的各种二茂铁前体可用于这种目的，并且包括二茂铁羧酸和1&#39;-Fmoc-氨基二茂铁羧酸。使用前系统产量的单取代的生物共轭物仅具有修饰生物素附属物的环戊二烯基环中的一个。后者的二茂铁衍生物是由环戊二烯基环的与羧酸和Fmoc保护的氨基正交取代。这样的取代可以为二茂铁芯允许在环戊二烯基环的单独修饰，以产生生物共轭物，如1的扩展非对称变形。浅黄色珠粒转变为经二茂铁的生物素树脂芯的成功耦合一个明亮的橙色色调， 如图2。 在合成的下一阶段的各种连接子可以连接到树脂结合的二茂铁同类的游离胺。对于此系统的目的，半胱氨酸被用作它是含有氨基甲酸硫醇盐。硫醇盐部分允许附着生物共轭到金表面的，如下面所讨论。修饰半胱氨酸的树脂结合的系统用Fmoc-半胱氨酸（MMT）-OH的反应进行。除去Fmoc基团，在二甲基甲酰胺20％哌啶的产生1的树脂结合的形式。所述树脂结合的生物共轭物从固相载体除去使用三氟乙酸（TFA），水和三异丙基硅烷的溶液的生物有机金属体系的裂解。酸溶液，加入冷的乙醚蒸发掉，得到的生物共轭物1，为红橙色固体确定为是的TFA盐1证实了元素和NMR分析。纯度通过HPLC分析证实。 以显示原理的证明半胱氨酸组件提供一个附件系绳用于生物缀合物1，1沉积在金表面进行了探讨。该公知的Au-S亲和力允许对1上的聚合物支持的金表面的固定化容易的。在该实验中，金表面进行清洁，并轻轻研磨。幻灯片然后浸入1〜1mM的水溶液，并使其设置O / N。然后在Au表面用蒸馏水和用Kimwipe干燥。金载玻片然后使用SEM评价修饰。在图4所示的图像是代表1单层具有形成到金表面。在表面上的缺陷被假定是在1单层"洞"的结果，并且根据进一步的探索由我们的基团。 总体而言，该合成方法以产生二茂铁的生物缀合物可以在金表面被固定的报告。这项工作是新颖之处在于合成不对称二茂铁化合物是由副反应导致低收益率和纯度的数组挑战。二茂铁，生物共轭类似于1具有作为潜在的生物传感器，克服这些困难的合成是极为重要的。采用固相法类似于固相肽合成中，充分表征的生物共轭1，包含生物素-二茂铁-半胱氨酸已经产生。另外，扫描电镜是用来表明，该系统能够粘附到金表面由于半胱氨酸组分的硫醇盐部分。总体而言，本文所提供的简单的合成方法可以很容易地进行修改的肽序列和生物共轭系统的应用的阵列。

生物传感器是采用生物分子识别技术作为用于选择性分析平台和被用于它们的特异性，速度和低成本的小型设备。电化学生物传感器用于生物分子的检测是在这个领域的前沿，由于其简单性，成本效益和高灵敏度。这些传感器1,3的一般解剖是装备有识别分子的电极的特定的感兴趣的生物标志物。该生物标记物的由识别分子结合导致的电位或电流的局部变化可以通过简单的测量来检测。迄今为止，识别部分的范围可以从酶，抗体，4-8，9-12整个细胞，13-16受体，17-20肽21-23和DNA 24，并 ​​主要集中在大，生物分子25-28研究在这个舞台上的努力主要集中在免疫传感器磨片重的免疫球蛋白是固定有氧化还原活性核（如二茂铁）和用于检测的目标抗体。这些研究被排除在临床应用中，由于精度差，消耗的时间从使用抗原/抗体引起的并发症而产生1,3成长的注意力都集中在小分子检测的生物医学（小于1公斤/摩尔） ，食品和除了国家安全的环境利益。29生物传感器设备的最有名的例子是自测血糖监测仪，其具有耦合到一个口袋大小的安培计丝网印刷酶电极。这些系统通常利用在充电量通过葡萄糖氧化反应产生的总量的测定在一段时间的库仑法。适销对路的设备必须是便携，坚固耐用，手持尽量使用浅显的广大民众。 氧化还原标签，如二茂铁的necessaRY以提供电化学检测生物标志物或小分子的溶液中，因为大多数的生物标志物是无内在电化学活性。30-38二茂是有机金属分子，是金标准电化学，这使得它对于整合到电化学生物传感器的极佳选择。二茂铁基的氧化还原活性种已经获取了相当大的注意，由于它们的尺寸小，稳定性好，方便合成存取，易于化学修饰，相对亲脂性，并且易于氧化还原调谐的。3,30-42小分子基于所述二茂铁芯具有被广泛地用作金属离子和小分子的检测。32-38,43系统靶向较大的品种，如生物分子已经利用大的抗体或免疫球蛋白，以已被嵌入到一个电化学表面二茂铁衍生物的附件。1,3,39 ，44在每一种情况下，该电势和电流的Intensi的铁三 / 二铁氧化还原对TY被改变时分子偶联，从而产生一个新的分光手柄，指示所述分析物分子的存在。这种变化源于该环戊二烯基环的pI系统和铁d轨道之间产生的大量重叠。如果PI-系统被修改， 即衍生或反应，则该轨道相互作用将反过来，变化。这将影响到铁芯，并且可以观察到在的铁III /铁二夫妇的电位的移位。40,45,46这些性质使得用作定量剂在电化学免疫测定或生物传感器这种系统的吸引力。 为了产生特异于生物传感器的能力含二茂铁的系统是最佳​​的修改一个Cp环与生物受体特异于靶分子，并利用其它的Cp环作为分子系绳到电化学读数或ELEC处接上（ 图1）。这些不对称二茂铁衍生物的合成是由副反应并在分子间交联形成的二聚和聚合物质的形成了挑战。47然而，偶联化学制备的酰胺键是最直接的途径，以提供二茂铁的涉及生物组分，例如简单的衍生物肽和它们的代谢物。因此，首先在20世纪50年代开发的Merrifield用于肽合成的固相技术可以应用到含二茂铁的有机金属化合物。通过使用正交取代的1&#39;-Fmoc-氨基二茂铁-1-羧酸分子，二茂铁系统，它可以包含一个受体部分（生物素），电化学读数（二茂铁），并固定化 - 连接体组分（半胱氨酸）的具有已建成和在此详述。此生物缀合物的合成进行了讨论，以及证据固定在金表面上。这项工作represenTS生物素，二茂铁和氨基酸用于固定在金表面的组成的系统的第一表示。

<table><tbody><tr><td>Biotin Novatag Resin</td><td>NovaBiochem</td><td>8550510001</td><td></td></tr><tr><td>TORVIQ 10 ml Luer Lock Fritted Syringe</td><td>Fisher</td><td>NC9299151</td><td></td></tr><tr><td>piperidine</td><td>Acros</td><td>P/3520/PB05</td><td></td></tr><tr><td>ninhydrin test</td><td>Sigma-Aldrich</td><td>60017-1ea</td><td></td></tr><tr><td>1&amp;rsquo;-Fmoc-amino-ferrocene-1-carboxylic acid</td><td>Omm Scientific</td><td>Special Order</td><td></td></tr><tr><td>&lt;em&gt;N,N&amp;prime;&lt;/em&gt;-Diisopropylcarbodiimide</td><td>Sigma-Aldrich</td><td>D125407-5G</td><td></td></tr><tr><td>Fmoc-Cys(Trt)-OH</td><td>Novabiochem</td><td>8520080025</td><td></td></tr><tr><td>trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>T5408</td><td></td></tr><tr><td>1,2-ethanedithiol</td><td>Sigma-Aldrich</td><td>2930</td><td></td></tr><tr><td>triisopropyl silane</td><td>Sigma-Aldrich</td><td>233781</td><td></td></tr><tr><td>Eppendorf tubes (20 ml)</td><td>any source</td><td></td><td></td></tr><tr><td>methanol</td><td>any source</td><td></td><td>dry with molecular sieves prior to use &amp;amp; store in 100 ml media bottle for easy usage</td></tr><tr><td>dichloromethane</td><td>any source</td><td></td><td>dry with molecular sieves prior to use &amp;amp; store in 100 ml media bottle for easy usage</td></tr><tr><td>dimethylformamide</td><td>any source</td><td></td><td>dry with molecular sieves prior to use &amp;amp; store in 100 ml media bottle for easy usage</td></tr><tr><td>centrifuge</td><td>any source</td><td></td><td></td></tr></tbody></table>

synthetic methodology for asymmetric ferrocene derived bio-conjugate systems via solid phase resin-based methodology

早期检测是治疗成功的关键大多数疾病的，并且是许多类型的癌症的诊断和治疗尤其迫切。所使用的最常见的技术是成像方式如磁共振成像（MRI），正电子发射断层（PET）和计算机辅助断层扫描（CT），并且最佳为了解该疾病的物理结构，但只能每四到执行一次由于使用的成像剂和总成本的六个星期。考虑到这一点，对"点的关怀"技术，如生物传感器，其评估疾病和/或治疗在临床医生的办公室疗效的阶段，做到及时，将彻底改变治疗方案的发展。1作为一种手段来探索基于二茂铁的生物传感器用于生物相关分子2的检测，方法被开发以产生本文所述的二茂铁-生物素的生物共轭物。本报告将重点放在可在金表面被固定生物素二茂铁半胱氨酸系统。

1，树脂结合形式示于图2。二茂铁组分的共价连接产生了一种橙色色调的树脂珠粒是持久连续洗涤和表示含有复杂的固定化铁如由相对于铁的吸收树脂珠粒的PEG部分。的1对无树脂形式是彩色的，以使树脂珠粒相同。以下除去从树脂珠的化合物，纯度和产率（68％），从所述方法得到的是远远优于典型的解决方案的方法。产品的元素分析表明，1被分离，为TFA盐:计算值（实测值）对C 26 H 36 FEN 6 O 4 S 2 4TFA值:C，38.07（38.90）; H，3.76（4.20）; N，7.83（7.70）。从一个典型的反应所得到的产率（105.1毫克，68％）是基于元素分析的结果。在氘代甲醇中省NMR分析ided 1 H NMR谱（CD 3 OD）δ/ ppm的:1.407-1.684（米，6H），2.245（T，2H），2.665-3.150（米，12H），4.015（吨，1H），4.104（D， 2H），4.274（Q，1H），4.426（D，2H），4.479（Q，1H），4.595（吨，1H）和13 C NMR谱（CD 3 OD）δ/ ppm的:24.644（CH 2）， 25.472（CH 2），28.051（CH 2），28.300（CH 2），35.474（CH 2），38.698（CH 2），39.241（CH 2），39.717（CH 2），55.340 / 55.538（CP-环） ，60.286（CH），61.964（CH），62.521 / 62.821（CP-环），66.038 / 66.170（CP-环），69.153 / 69.328（CP-环），71.468 / 71.593（CP-环），76.466（CH ），171.770（C = O），175.361（C = O）。 HPLC保留时间分别为:3.198-4.674分钟。在HPLC分析中观察到的多个峰被确认为1的TFA盐，使用上述元素分析如所述。质谱相关的结构示于图2:ESI-MS（M / Z）:实测值:639.00 [1 + NA] +理论值:639.1 [1 + NA] +和HR-MS（M / Z）:实测值:617.2049 [1 + H] +，理论值:617.1622 [1 + H] +。电子吸收光谱，获得在水和表现出λ 最大 （ε，M-1 -1）268（4,779.8），434（324.26）。 用于培养中生物共轭1的溶液的小金滑动的方法表示在图3中的示意图，一个薄的金层背面与聚合物材料加入到1的溶液，并使其孵育O / N。金载玻片然后用DI水洗涤，并使其干燥。与此同时，一个金载玻片共孵育在去离子水中，并以相同的方式洗涤。两个样品，在图4所示的SEM图像，表明金滑动共孵育1的表面进行改性。这表明1硫醇盐相互作用提供附着于金表面的锚。 <img alt="图1" src="/files/ftp_upload/52399/52399fig1.jpg" /> 图1.基础的生物传感器的。电化学生物传感器的具体实例，以直接检测靶在溶液中。 <img alt="图2" src="/files/ftp_upload/52399/52399fig2.jpg" /> 用于生产1.用于孵育在生物共轭1的溶液的小金滑动的方法图2的合成方法在图3中所表示的示意图，一个薄的金层背面与聚合物材料中加入的溶液1，让其孵化O / N。金载玻片然后用DI水洗涤，并使其干燥。与此同时，一个金载玻片共孵育在去离子水中，并以相同的方式洗涤。两个样品的SEM图像在图4中示出，表明金滑动共孵育1表面进行改性。这表明，1所述硫醇盐的相互作用提供附接的锚固到金表面。 <img alt="图3" src="/files/ftp_upload/52399/52399fig3.jpg" /> 图3.示意图表示1上的金滑动的固定。该方法包括在水中溶解的生物共轭并添加金滑动。孵育O / N后跟滑动的洗涤。分析成功的固定化是通过使用图4中所示的扫描电子显微镜。 <img alt="图4" src="/files/ftp_upload/52399/52399fig4.jpg" /> 金铺在一个塑料聚合物薄膜图4的SEM图像。（A）中孵育SANS 1和（B）FOllowing孵育1在水中的O / N，并用水漂洗。

Watch this Scientific Journal Video about Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology at JoVE.com

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

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. ...

synthetic-methodology-for-asymmetric-ferrocene-derived-bio-conjugate

Nanyang Technological University

Research

JoVE Journal

Chemistry

9.6K 次观看.  Texas Christian University. 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.

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

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 ...

科研

化学

Synthesis of a Water-soluble Metal&#8211;Organic Complex Array

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as Ru(II), Pt(II), and Rh(III). The compound, named as a water-soluble metal-organic complex array (WSMOCA), is obtained through 1) the conventional solution-chemistry-based preparation of the corresponding metal complex monomers having a 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acid moiety and 2) their sequential coupling together with other water-soluble organic building units on the surface-functionalized polymeric resin by following the procedures originally developed for the solid-phase synthesis of polypeptides, with proper modifications. Traces of reactions determined by mass spectrometric analysis at the representative coupling steps in stage 2 confirm the selective construction of a predetermined sequence of metal centers along with the peptide backbone. The WSMOCA cleaved from the resin at the end of stage 2 has a certain level of solubility in aqueous media dependent on the pH value and/or salt content, which is useful for the purification of the compound.

A potential general method for the synthesis of water-soluble multimetallic peptidic arrays containing a predetermined sequence of metal centers is presented.

一个水溶性金属 - 有机络合物阵列合成

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as ...

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

利用光致硫醇-烯/yne Hydrothiolation 构造硫醚类/乙烯基硫化物栓系螺旋肽

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of solid-phase synthesis, regenerating Michael (REM) linker strategies, and 1,3-dipolar cycloaddition, a library of structurally similar heterocycles, both with and without a spirocyclic center, can be constructed. The main advantages of the solid-support synthesis are as follows: first, each reaction step can be driven to completion using a large excess of reagents resulting in high yields; next, the use of commercially available starting materials and reagents keep the costs low; finally, the reaction steps are easy to purify via simple filtration. The REM linker strategy is attractive because of its recyclability and traceless nature. Once a reaction scheme is completed, the linker can be reused multiple times. In a typical solid-phase synthesis, the product contains either a part of or the whole linker, which can prove undesirable. The REM linker is &#34;traceless&#34; and the point of attachment between the product and the polymer is indistinguishable. The high diastereoselectivity of the intramolecular 1,3-dipolar cycloaddition is well documented. Limited by the insolubility of the solid support, the reaction progression can only be monitored by a change in the functional groups (if any) via infrared (IR) spectroscopy. Thus, the structural identification of intermediates cannot be characterized by conventional nuclear magnetic resonance (NMR) spectroscopy. Other limitations to this method stem from the compatibilities of the polymer/linker to the desired chemical reaction scheme. Herein we report a protocol that allows for the convenient production of spirocyclic heterocycles that, with simple modifications, can be automated with high-throughput techniques.

Here we present a protocol to demonstrate an efficient method for the synthesis of spirocyclic heterocycles. The five-step process utilizes solid-phase synthesis and regenerating Michael linker strategies. Generally difficult to synthesize, we present a customizable method for the synthesis of spirocyclic molecules otherwise inaccessible to other modern approaches.

[4.4] 刺环氧基的固相合成

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

硫醇反应试剂的合成与生物共轭作用--用于原位选择性修饰免疫共轭的生成

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

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 ...

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

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides1.
Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides2, which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield1, but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation.
Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an &alpha;-amine)3,4. Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.

The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described.

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 ...

生物学

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 ...

Solid Phase Synthesis

Source: Vy M. Dong and Diane Le, Department of Chemistry, University of California, Irvine, CA
Merrifield&#39;s solid-phase synthesis is a Nobel Prize winning invention where a reactant molecule is bound on a solid support and undergoes successive chemical reactions to form a desired compound. When the molecules are bound to a solid support, excess reagents and byproducts can be removed by washing away the impurities, while the target compound remains bound to the resin. Specifically, we will showcase an example of solid-phase peptide synthesis (SPPS) to demonstrate this concept.

Solid Phase Synthesis: Principles, Peptide Synthesis

固相合成

Source: Vy M. Dong and Diane Le, Department of Chemistry, University of California, Irvine, CA
Merrifield&#39;s solid-phase synthesis is a Nobel Prize ...