Name: synthesis of ph dependent pyrazole, imidazole, and isoindolone dipyrrinone fluorophores using a claisen-schmidt condensation approach
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Description: Watch this Scientific Journal Video about Synthesis of pH Dependent Pyrazole, Imidazole, and Isoindolone Dipyrrinone Fluorophores using a Claisen-Schmidt Condensation Approach at JoVE.com

Z.R.W.和N.B感谢NIH（2P20GM103440-14A1）的慷慨资助，以及荣杰大学和内华达大学，拉斯维加斯，他们协助获得 1H和 13CNMR。此外，我们要感谢国家安全委员会视觉媒体学生，阿诺德·普莱森西亚-弗洛雷斯，奥布里·雅各布斯和阿利斯泰尔·库珀在本手稿的电影摄影部分的拍摄和动画过程中的帮助。

1. 二苯酮模拟合成通用程序 16-25
<ol><li>在圆底烧瓶中溶解5.0 mL乙醇中的苯丙酮/异丙酮（1.00 mmol）和相应的乙醇/二甲醛（1.00 mmol）。</li>
	<li>将无瓶KOH（24.0 mmol，10M，2.40 mL）加入烧瓶中的一部分。</li>
	<li>搅拌并倒流混合物，直到反应完成由 TLC 确认（有关反应时间列表，请参阅 表 1）。使用了二氯甲烷中10%甲醇的TLC，并观察到这些类比在0.62到0.86范围内或周围具有Rf 值。在?...

<ol>
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克莱森-施密特凝结方法提供了一个相当强大的手段，通过相对操作简单的协议产生苯丙酮、二甲苯酮和二苯酮氟。虽然荧光二苯酮类比的合成是本研究的重点，但应当指出，类似的条件可以应用于访问其他双环甲基苯丙酮环系统，如二苯酮23、24、25和苯丙酮-富兰辅音26以及 3H-pyrazol-3-一-furan 辅音<sup cla...

一些共生双循环系统，其中两个芳香环由单体桥连接，通过键旋转进行异构，当兴奋与光子（图1A）1，2，3，4，5。兴奋的同位素一般会放松到地面状态通过非辐射衰变过程6。如果对键旋转的能量屏障增加得足够大，则有可能限制或防止光同步。相反，光子激发导致兴奋的单一状态，经常通过荧光放松，而不是非辐射衰变（图1B）。抑制光异构化最常通过机械地通过共价链系住两个芳香环系统来限制粘结旋转，从而将分子锁定在特定的同位素状态。这种方法已被用于创建几个不同的荧光三环二苯酮和二甲基甲烷类比，如：3H，5H-二倍丙酮[1，2-c：2'，1'f]皮里米丁-3-一（1），xan异种生物（2）6，7， 皮罗洛林多利齐尼迪安类比（3）8，和博迪皮类比9 （4，图 2），其中聚丙烯和 / 或苯乙烯环系统系上甲基， 碳基， 或波龙二氟链接器。通常，1-4 拥有ΦF > 0.7 表明这些系统作为氟合金单位非常有效。
也可以通过通过环系统共同连接以外的其他方法限制光异构化。例如，绿色荧光蛋白（GFP）的酚类和二胺酮环（图2）受蛋白质环境限制旋转：与自由溶液10中的同一色素单元相比，限制性设置使量子产量增加三个数量级。据认为，GFP的蛋白质支架通过固态和静电效应11提供了旋转屏障。最近，我们的小组与内华达大学的奥多小组合作，里诺发现了另一种氟化物系统，其结构与基于二苯酮的xanthoglow系统（图2）12具有相似性。然而，这些二苯酮类比不同于血管内氢键中的xanthoglow系统，而不是共价键，阻止光分母化，并导致荧光双环系统。此外，皮拉佐尔、伊米达佐尔和同位素二苯酮类比可以在质子和脱质状态下结合氢气：脱质导致兴奋和发射波长的红色转移，可能是由于系统电子性质的变化。虽然氢结合被报道通过限制旋转13、14、15、16来增加量子产量，但我们不知道任何其他荧光系统，其中受限异位化在分子质子和脱质状态中都起到荧光作用。因此，这些pH依赖二苯酮氟酚在这方面是独一无二的。
在本视频中，我们专注于荧光二苯酮模拟系列的合成和化学特征。特别是，人们强调克莱森-施密特凝结方法，该方法用于构建一系列完整的荧光模拟。这种反应依赖于一种以基础为媒介的恶毒的离子的产生，这种离子攻击醛组，产生一种酒精，随后被消除。对于二苯酮模拟系列，皮罗林酮/异丙酮被转换为一个归因，以促进对附着在苯丙烯或二甲基苯甲酸酯环上的醛组的攻击（图3）：消除后，形成了一个完全结合的双循环系统，由甲氨酸桥连接。值得注意的是，整个系列的二苯酮类比可以由现成的商业材料构建，可以以单一的单锅反应序列生产，通常为中等至高产量（产量范围约为 50-95%）。由于大多数二苯酮类比在性质上是高度晶体的，在标准工作条件之外，很少需要纯化来生产分析纯样品。因此，这种氟化物系统只需几步即可从现成的商业材料中获取，并且可以在相对较短的时间内合成、纯化和准备进行分析或生物研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-ethyl-4-methyl-3-pyrrolin-2-one</td>
			<td>Combi-Blocks</td>
			<td>&#160;[766-36-9]</td>
			<td>Yellow solid reagent</td>
		</tr>
		<tr>
			<td>isoindolin-1-one</td>
			<td>ArkPharm</td>
			<td>&#160;[480-91-1]</td>
			<td>Off-white solid reagent</td>
		</tr>
		<tr>
			<td>5-bromoisoindolin-1-one</td>
			<td>Combi-Blocks</td>
			<td>&#160;[552330-86-6]</td>
			<td>Pink solid reagent</td>
		</tr>
		<tr>
			<td>2-formylimidazole</td>
			<td>Combi-Blocks</td>
			<td>&#160;[10111-08-7 ]</td>
			<td>Off-white solid reagent</td>
		</tr>
		<tr>
			<td>Imidazole-4-carbaldehyde</td>
			<td>ArkPharm</td>
			<td>&#160;[3034-50-2]</td>
			<td>Solid reagent</td>
		</tr>
		<tr>
			<td>1-H-pyrazole-4-carbaldehyde</td>
			<td>Oakwood Chemicals</td>
			<td>&#160;[35344-95-7]</td>
			<td>Solid reagent</td>
		</tr>
		<tr>
			<td>1-H-pyrazole-5-carbaldehyde</td>
			<td>Matrix Scientific</td>
			<td>&#160;[3920-50-1]</td>
			<td>Solid reagent</td>
		</tr>
		<tr>
			<td>Solid KOH Pellets</td>
			<td>BeanTown Chemicals</td>
			<td>[1310-58-3]</td>
			<td>White solid pellets</td>
		</tr>
		<tr>
			<td>Siliflash Silica Gel</td>
			<td>Scilicycle</td>
			<td>R12030B</td>
			<td>Fine white powder</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) (x10)</td>
			<td>Growcells</td>
			<td>MRGF-6235</td>
			<td>Colorless translucent liquid</td>
		</tr>
		<tr>
			<td>Beckman Coulter DU-800 UV/Vis Spectrophotometer and Software</td>
			<td>Beckman Coulter</td>
			<td>N/A</td>
			<td>Spectroscopy Instrument and Software</td>
		</tr>
		<tr>
			<td>Fluoromax-4 Spectrofluorometer</td>
			<td>Horiba Scientific</td>
			<td>N/A</td>
			<td>Spectroscopy Instrument</td>
		</tr>
		<tr>
			<td>FluorEssence Fluoremetry Software V3.5</td>
			<td>Horiba Scientific</td>
			<td>N/A</td>
			<td>Spectroscopy Software</td>
		</tr>
		<tr>
			<td>Finnpipette II Micropipette (sizes: 100-1,000, 20-200, and 0.5-10 &#181;L)</td>
			<td>Fischerbrand</td>
			<td>N/A</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Wilmad-LabGlass Rotary Evaporator (Model: WG-EV311-V-PLUS)</td>
			<td>SP Scienceware</td>
			<td>N/A</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>DuoSeal Vacuum Pump (Model Number: 1405)</td>
			<td>Welch</td>
			<td>N/A</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>GraphPad Prism 4</td>
			<td>GraphPad</td>
			<td>N/A</td>
			<td>Data Analysis Software</td>
		</tr>
		<tr>
			<td>SympHony pH Meter (Model: Sb70P)</td>
			<td>VWR</td>
			<td>N/A</td>
			<td>Equipment</td>
		</tr>
	</tbody>
</table>

synthesis of ph dependent pyrazole, imidazole, and isoindolone dipyrrinone fluorophores using a claisen-schmidt condensation approach

甲氨酸桥结合的双环芳香化合物是一系列生物相关分子的常见成分，如猪皮林、二苯酮和药物。此外，这些系统的有限旋转通常会导致高度到中度荧光系统，如在 3H，5H-迪皮罗洛 [1，2-c：2'， 1'f] pyrimidin-3- 1， 黄磷、皮罗林多利齐尼迪奥内类比、博迪皮类比以及绿色荧光蛋白 （GFP） 的酚类和伊米达佐林环系统。本手稿描述了一种廉价且操作简单的方法，即执行克莱森-施密特凝结，以产生一系列荧光pH依赖性皮拉佐尔/伊米达佐尔/异丙酮二苯酮类比。虽然该方法说明了二苯酮类比的合成，但它可以转化为产生广泛的结合双环芳香化合物。本方法所使用的克莱森-施密特凝结反应的范围仅限于在基本条件下（核嗜好成分）和不可抗性醛（电嗜酸成分）下可凝结的核嗜好者和电嗜好者。此外，核嗜血和电亲反应物必须包含不会无意中与氢氧化物反应的功能组。尽管有这些限制，这种方法提供了完全新颖的系统，可以用作生物或分子探针。

克莱森-施密特凝结反应提供了使用协议部分描述的单锅程序（见第1步）访问二苯酮类比（16-25，图4）。模拟16-25全部由凝结丙酮9，溴化物酮10，或异丙酮11与1H-伊米达佐尔-2-碳水化合物（12），产生1 H-伊米达佐尔-5-卡博卡尔醛（13）， 1H- 皮拉佐尔-3-卡?...

Watch this Scientific Journal Video about Synthesis of pH Dependent Pyrazole, Imidazole, and Isoindolone Dipyrrinone Fluorophores using a Claisen-Schmidt Condensation Approach at JoVE.com

Synthesis of pH Dependent Pyrazole, Imidazole, and Isoindolone Dipyrrinone Fluorophores using a Claisen-Schmidt Condensation Approach

克莱森-施密特凝结反应是产生甲氨酸桥结合双环芳香化合物的重要方法。通过利用阿尔多尔反应的基础介导变种，可以通过一种通常便宜且操作简单的合成方法访问一系列荧光和/或生物相关分子。

使用克莱森-施密特凝结方法合成pH依赖皮拉佐尔、伊米达佐尔和伊森多龙二苯酮氟磷

Methine-bridged conjugated bicyclic aromatic compounds are common constituents of a range of biologically relevant molecules such as porphyrins, ...

克莱森-施密特凝结反应是一种古老的反应，最早由克莱森和施密特于1881年同时报道。它涉及在基座辅助下将以蓝色显示的酮或醛加入芳香醛中，以红色显示。最初，加入该归国物会导致酒精的形成，如括号所示。然而，随后的脱水最终产生一种衰减。由于芳烃不含α氢，因此不能形成一种单体。因此，通常弱碱基，如氢氧化物，可用于生成该网。多年来，克莱森-施密特凝结剂被用来产生许多化合物。然而，它已被广泛用于加入黄锥和黄烷酮的芳香环系统，这些系统以红色和蓝色显示。蓝色部分表示来自外壳和红色是芳香部分。根据替代模式，小木屋和黄酮是一系列生物活性分子的基本核心，这些分子具有一系列活性，如抗菌、抗真菌、抗炎和抗肿瘤。从克莱森-施密特凝结反应产生的另一类广泛的分子是甲氨酸桥接化合物，我们在这项研究中举了一个例子。我们的实验室对色素胆汁的荧光成分感兴趣，胆汁素是天然产生的下限降解产物。胆红素及其许多成分的合成围绕克莱森-施密特型凝结，可以在蓝色肠剂和红色芳香醛成分显示的结构中可视化。通常，胆汁素的直接成分，如二苯酮，是非荧光的。然而，如果一个人用甲基或碳基组将两个氮群连接起来，产生的分子就会变得高度荧光，例如在xanthoglows的情况下。通常，二苯酮吸收紫外线或蓝光，从而产生 Z 到 E 等位素化过程。与未桥接的二苯酮一样，桥接二苯酮中的 N 也吸收紫外线或蓝光，但不同的是，它们通过荧光在兴奋状态下放松。我们最近发现了一系列二苯酮衍生物，它们实际上荧光，没有连接两个氮群的共价桥。相反，氢键似乎阻碍了Z到E等位化进程，导致荧光模式。此外，还意外地发现，这些分子可以在基本介质中脱质，导致脱质状态下的吸收辐射光谱出现红移。因此，这些分子可能具有作为比例pH探针的价值。使用荧光二苯酮衍生物，在传统的克莱森-施密特凝结反应中产生轻微的适应。该协议偏离了传统的克莱森-施密特凝结反应，因为从皮罗林酮或异丙酮中提取的恶毒的浓缩物是核嗜血源。恶毒的文书增加了皮拉佐尔或硫化物，以产生一个小图书馆的二苯酚类比。用于创建此库的程序在视频中进行了说明。然而，类似的步骤可以用来执行传统的克莱森-施密特反应。虽然克莱森-施密特反应已经而且仍然是一种广泛使用的合成反应，但这是该方法的第一个视频计数，我们对此有所了解。要准备通过阿尔多尔冷凝合成荧光二甲苯酮模拟物，权衡所选核嗜血杆菌和电嗜电物的等价物。然后将其添加到包含磁性搅拌棒的 25 毫升圆形底部烧瓶中。使用分级气缸测量五毫升乙醇。然后将乙醇添加到圆形底部烧瓶中。使用分级气缸测量先前准备的 10 摩尔氢氧化钾的 2.4 毫升。然后将氢氧化钾添加到烧瓶中。要将烧瓶设置为反流，请将足够的真空润滑脂涂抹在反应凝结器的地面玻璃接头上，以防止抓住地面玻璃接头。将冷凝器连接到冷水供应，然后将冷凝器的润滑接头连接到圆形底部烧瓶上。然后将烧瓶放在油浴或铝制加热块上，通过热耦合器和热板搅拌器保持恒温。加热以反流温度，同时允许反应搅拌。反应混合物应在一、三、六、十二和二十四小时内通过薄层色谱仪进行监测，以测量反应速率，并检查起始材料是否完全消耗。让烧瓶冷却到室温，然后使用旋转蒸发器蒸发乙醇溶剂。将烧瓶放在冰浴中，让烧瓶在五分钟内与冰浴温度平衡。通过在单个部分中加入1.7毫升醋酸，中和烧瓶中剩余的氢氧化钾。如果晶体形成后中和，然后按照真空过滤净化程序。如果没有观察到晶体形成，则遵循闪光柱色谱净化程序。将一张圆形滤纸放在漏斗顶部，用除离子水轻轻弄湿纸张，将其粘附在漏斗上。为了准备晶体的真空过滤，使用安装的橡胶适配器将赫希或布赫纳漏斗放入侧臂烧瓶中。为了避免过滤纸堵塞，可能会阻碍过滤，我们使用了比类似比例过滤过程更大的赫希或布赫纳漏斗。将圆底烧瓶中的内容倒入滤纸上，让混合物过滤。在过滤过程中使用10毫升冰冷的除离子水冲洗晶体。过滤后，将晶体转移到25毫升圆的底部烧瓶中。将轻薄的真空润滑脂涂抹在高功率真空线玻璃适配器上，然后将适配器连接到圆形底部烧瓶。用凯克夹子固定玻璃接头。要准备高功率真空来干燥任何剩余溶剂的晶体，请用干冰和调子的混合物充分冷却玻璃真空陷阱。将大功率真空管连接到连接到圆形底部烧瓶的玻璃适配器。打开大功率真空泵，让晶体干燥至少一小时。一旦晶体在真空下充分干燥，关闭真空泵并释放真空密封，以去除圆形底部烧瓶。称一下干晶体，报告反应百分比。要通过闪光柱色谱法为净化做好准备，请将未形成合成过程晶体的酸处理混合物添加到分离漏斗中。使用分级气缸测量 10 毫升二氯甲烷，并用它来稀释分离漏斗中的酸处理混合物。关闭并轻轻摇动分离漏斗，同时确保经常通风。在此之后，在分离漏斗中应可见两个独立的层。使用额外的五毫升二氯甲烷提取水层。再完成此步骤两次。将所有有机成分混合，加入足够的硫酸钠，以干燥有机成分。将干燥的有机馏分转移到圆形底部烧瓶中，然后使用旋转蒸发器去除二氯甲烷。再稀释剩余的二氯甲烷五毫升。使用大约 75 克硅凝胶准备一个列，并用它来对样品进行闪光柱色谱，使用二氯甲烷中的 10% 甲醇作为弹性物。使用旋转蒸发器从收集的分数中蒸发。按照真空过滤净化程序的先前描述，准备大功率真空泵和玻璃溶剂陷阱，使收集的固体在高真空下干燥至少一小时。一旦晶体在真空下充分干燥，称重干燥的晶体以报告反应百分比。为了确认库中每个二苯酮模拟器的结构，组合使用了多种光谱方法，包括核磁共振光谱、红外光谱和高分辨率质谱法。紫外线-维斯和荧光光谱学用于荧光二苯酮模拟的光物理特征。利用克莱森-施密特凝结反应，我们能够合成一个包含10种化合物的小库，包括一种不能参与分子间氢键的控制化合物。二苯酮模拟的产量从大约40%到几乎定量不等，并列在每个分子下面。量子产量最高的化合物，无论是质子形式还是去质形式，都来自2-原体，并显示在粉红色的盒子中。控制化合物，不荧光，是在一个青色盒。标准 365 纳米长波长灯下的二苯酮衍生物为观测到的荧光提供了光。人们可以直观地观察脱质产生的红移荧光。通过小瓶的颜色，它们从蓝色过渡到青色。有关二苯酮衍生物的光物理和其他物理特性的更多定量数据，我们引导观众在手稿的书面部分显示两个。总的来说，克莱森-施密特凝结反应提供了一系列与甲辛相关的双环芳香化合物。但是，存在一些限制。反应取决于使用可溶性核嗜血杆菌和不可溶性醛电嗜血杆菌，如芳烃，以进行成功的凝结。如果不能满足这一基本要求，尝试执行此反应可能导致无法将环系统和/或竞争侧产品的生成连接在一起。另一个考虑是，基本条件用于产生封闭的核嗜好者，这种核嗜好可能与容易与氢氧化物反应的功能组产生不兼容性。在这种情况下，可以用DBU、三聚氰胺、烟斗素、胡尼格碱和碳酸钠代替氮基或碳酸氢化合物。我们只是选择使用氢氧化钾，因为它的可用性和相对费用。尽管有这些限制，但协议中概述的方法可以通过程序简单且具有成本效益的单步反应，为许多系统提供芳香环耦合的手段。在我们合成的二苯酮模拟中，克莱森-施密特凝结已使迄今为止描述的pH依赖氟化物的最容易获得的途径之一。然而，未来的二苯酮模拟物设计将采用概述的程序，以产生荧光化合物，具有更强的分子间氢粘合能力和更低的pKa值。预期这些增强的 pH 依赖型探针将具有更高的量子产量，同时能够可视化 pH 波动，从而实现更广泛的细胞内事件。

Research

Education

JoVE Core

JoVE Journal

Organic Chemistry

Chemistry

Unit 1

&alpha;-Carbon Chemistry: Enols, Enolates, and Enamines

SCIENTISTS IN ACTION

Preparation and Use of Samarium Diiodide (SmI2) in Organic Synthesis: The Mechanistic Role of HMPA and Ni(II) Salts in the Samarium Barbier Reaction

Preparation and Use of Samarium Diiodide SmI2 in Organic Synthesis: The Mechanistic Role of HMPA and NiII Salts in the Samarium Barbier Reaction

二碘化钐的制备和使用（SMI<sub&gt; 2</sub&gt;）在有机合成中：HMPA和镍（II）盐的钐巴尔比耶反应的机理作用

Although initially considered an esoteric reagent, SmI2 has become a common tool for synthetic organic chemists. SmI2 is generated through the addition of ...

科研

化学

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

合成，细胞和交货<em&gt;在体内</em&gt;的树状为基础的pH值传感器应用

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals

硒化镉/硫化镉棒和纳米四脚合成种子

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded ...

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

BMP-2的共价结合利用表面上的自组装单层膜法

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Qualitative Identification of Carboxylic Acids, Boronic Acids, and Amines Using Cruciform Fluorophores

使用十字型荧光基团的羧酸，硼酸，胺的定性鉴别

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with ...

Synthesis and Characterization of Functionalized Metal-organic Frameworks

功能化的合成与表征金属 - 有机骨架

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

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

合成与盘状磷酸盐锆剥落获取胶体液晶

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

The Synthesis of [Sn10(Si(SiMe3)3)4]2- Using a Metastable Sn(I) Halide Solution Synthesized via a Co-condensation Technique

The Synthesis of [Sn10SiSiMe334]2- Using a Metastable SnI Halide Solution Synthesized via a Co-condensation Technique

的合成[锡<sub&gt; 10</sub&gt;（SI（SIME<sub&gt; 3</sub&gt;）<sub&gt; 3</sub&gt;）<sub&gt; 4</sub&gt;]<sup&gt; 2</sup<sup&gt; -</sup&gt;使用亚稳锡（I）卤溶液通过共缩聚合成技术

The number of well-characterized metalloid tin clusters, synthesized by applying the disproportionation of a metastable Sn(I) halide in the presence of a ...

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

[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 of Esters Via a Greener Steglich Esterification in Acetonitrile

乙腈绿色 Steglich 酯化合成酯的研究

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the ...