Name: functionalized spirocyclic heterocycle synthesis and cytotoxicity assay
Uploaded: 2021-02-09T13:30:00
Description: Watch this Scientific Journal Video about Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay at JoVE.com

这项工作由教师研究委员会向KSH（美国阿苏萨太平洋大学研究和赠款办公室）的赠款资助。A.N.G. 和 J.F.M. 是学术本科生研究经验 （SURE） 奖学金的获得者。S.K.M.和B.M.R.是STEM研究奖学金（美国阿苏萨太平洋大学科学研究中心）的获得者。我们感谢Matthew Berezuk博士和Philip Cox博士对生物测定的指导。

注意：本协议中使用的几种化学品和生物试剂具有毒性和致癌性。使用前，请查阅相关材料安全数据表 （MSDS）。在开始实验之前，使用适当的个人防护装备（职业安全与健康管理局批准的护目镜，适当的手套，实验室外套，全长裤和露趾鞋）。此外，在进行合成和处理有毒化学品和试剂（通风橱）时，应采取适当的安全措施。
1. 螺环杂环6和7的固相合成
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<ol>
	<li>Shangary, S., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+inhibitors+of+the+MDM2-p53+protein-protein+interaction+to+reactivate+p53+function:+a+novel+approach+for+cancer+therapy.">Small-molecule inhibitors of the MDM2-p53 protein-protein interaction to reactivate p53 function: a novel approach for cancer therapy.</a> Annual Review of Pharmacology and Toxicology. 49, 223-241 (2009).</li><li>Zhao, Y., Aguilar, A., Bernard, D., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+inhibitors+of+the+MDM2-p53+protein-protein+interaction+(MDM2+inhibitors)+in+clinical+trials+for+cancer+treatment.">Small-molecule inhibitors of the MDM2-p53 protein-protein interaction (MDM2 inhibitors) in clinical trials for cancer treatment.</a> Journal of Medicinal Chemistry. 58 (3), 1038-1052 (2015).</li><li>Paolo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+effective+virtual+screening+protocol+to+identify+promising+p53-MDM2+inhibitors.">An effective virtual screening protocol to identify promising p53-MDM2 inhibitors.</a> Journal of Chemical Information and Modeling. 56 (6), 1216-1227 (2016).</li><li>Shieh, S. Y., Ikeda, M., Taya, Y., Prives, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage-induced+phosphorylation+of+p53+alleviates+inhibition+by+MDM2.">DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2.</a> Cell. 91 (3), 325-334 (1997).</li><li>Hwang, B. J., Ford, J. M., Hanawalt, P. C., Chu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+p48+xeroderma+pigmentosum+gene+is+p53+dependent+and+is+involved+in+global+genomic+repair.">Expression of the p48 xeroderma pigmentosum gene is p53 dependent and is involved in global genomic repair.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (2), 424-428 (1999).</li><li>Oliner, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncoprotein+MDM2+conceals+the+activation+domain+of+tumor+suppressor+p53.">Oncoprotein MDM2 conceals the activation domain of tumor suppressor p53.</a> Nature. 362, 857-860 (1993).</li><li>Nag, S., Qin, J., Srivenugopal, K. S., Wang, M., Zhang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MDM2-p53+pathway+revisited.">The MDM2-p53 pathway revisited.</a> Journal of Biomedical Research. 27 (4), 254-271 (2013).</li><li>Bond, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+nucleotide+polymorphism+in+the+MDM2+promoter+attenuates+the+p53+tumor+suppressor+pathway+and+accelerates+tumor+formation+in+humans.">A single nucleotide polymorphism in the MDM2 promoter attenuates the p53 tumor suppressor pathway and accelerates tumor formation in humans.</a> Cell. 119 (5), 591-602 (2004).</li><li>Isobe, M., Emanuel, B. S., Givol, D., Oren, M., Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+gene+for+human+p53+tumor+antigen+to+band+17p13.">Localization of gene for human p53 tumor antigen to band 17p13.</a> Nature. 320 (6057), 84-85 (1986).</li><li>Gupta, A. K., Bharadwaj, M., Kumar, A., Mehrotra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiro-oxindoles+as+a+promising+class+of+small+molecules+inhibitors+of+p53-MDM2+interaction+useful+in+targeted+cancer+therapy.">Spiro-oxindoles as a promising class of small molecules inhibitors of p53-MDM2 interaction useful in targeted cancer therapy.</a> Topics in Current Chemistry. 375 (1), 1-25 (2017).</li><li>Griffin, S. A., Drisko, C. R., Huang, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricyclic+heterocycles+as+precursors+to+functionalized+spirocyclic+oximes.">Tricyclic heterocycles as precursors to functionalized spirocyclic oximes.</a> Tetrahedron Letters. 58, 4551-4553 (2017).</li><li>Drisko, C. R., Griffin, S. A., Huang, K. S. <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+[4.4]spirocyclic+oximes.">Solid-phase synthesis of [4.4]spirocyclic oximes.</a> Journal of Visualized Experiments. (144), e58508 (2019).</li><li>Lawrence, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linked+parallel+synthesis+and+MTT+bioassay+screening+of+substituted+chalcones.">Linked parallel synthesis and MTT bioassay screening of substituted chalcones.</a> Journal of Combinatorial Chemistry. 3 (5), 421-426 (2001).</li><li>. MTT assay protocol Available from: <a target="_blank" href="https://www.abcam.com/kits/mtt-assay-protocol">https://www.abcam.com/kits/mtt-assay-protocol</a> (2020)</li></ol>

螺环化合物的合成基于该实验室先前进行的研究，但进行了一些修改（图1）11，12。通过红外光谱监测每个反应步骤的进度。迈克尔将REM接头1与糠胺一起加入得到聚合物结合的2（IR 1722 cm-1 → 1731 cm-1）。从上一份报告中，ISOC of 2产生了三环杂环化合物<...

已知小分子抑制E3泛素连接酶小鼠双分钟2同系物（MDM2）与p53的相互作用可恢复p53介导的诱导肿瘤细胞凋亡1，2，3。MDM2是p53通路的负调节因子，通常在癌细胞4，5，6，7，8，9中过表达。最近的晶体学和生化研究表明，含有螺旋环骨架的小分子可以有效抑制MDM2-p53相互作用10。螺旋环框架（图1，蓝色阴影）被认为是一个特权基序，因为这种刚性正交环系统的衍生化导致了新型治疗药物的发现。使用传统的有机合成技术时，访问这种有趣的架构提出了挑战。尽管已经研究了螺旋环分子在生物系统中的治疗效果，但这些分子的合成仍然是一个繁琐的过程。不需要的副产品、使用恶劣的条件和危险的过渡金属通常是有问题的。
螺旋环基序在药物开发中的潜在用途导致开发一种方案，利用固相合成来生成具有该基序的分子库以及其他可互换的官能团11，12。步骤之间产物和反应物的分离可以通过简单地使用连接到树脂珠和固相过滤容器的REM接头来实现。这将减少步骤并可能提高产量。这种合成方法可以产生大量潜在的候选药物。然而，这些分子在生物系统中的有效性需要进一步研究。
为了确定这些螺环化合物的细胞毒性，采用了MTT测定13，14 。该方法测量细胞活力，可用于间接确定细胞毒性。将不同浓度的抑制剂添加到96孔板中的培养细胞中，并通过比色分析测量线粒体脱氢酶将黄色MTT还原为紫色甲臜化合物的程度的活细胞比例（图2）。该活性通常报告为IC50 值 - 相对于未处理的对照，细胞生长抑制50%的浓度。本文描述了MTT测定的方案以及这些新型螺环分子的初步结果。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CELLS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>COS-7 cells (ATCC CRL-1651)</td>
			<td>ATCC</td>
			<td>CRL-1651</td>
			<td>African green monkey kidney cells</td>
		</tr>
		<tr>
			<td>CHEMICALS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-Bromooctane</td>
			<td>Sigma-Aldrich</td>
			<td>152951</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Allylbromide</td>
			<td>Sigma-Aldrich</td>
			<td>337528</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Benzylbromide</td>
			<td>Sigma-Aldrich</td>
			<td>B17905</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Cisplatin</td>
			<td>Cayman Chemical</td>
			<td>13119</td>
			<td>Cytotoxicity control</td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Dimethylformamide (DMF)</td>
			<td>Sigma-Aldrich</td>
			<td>227056</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, with L-glutamine</td>
			<td>Genesee Scientific</td>
			<td>25-500</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>FBS (Fetal bovine serum)</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>Furfurylamine</td>
			<td>Acros Organics</td>
			<td>119800050</td>
			<td>reagent&#160;</td>
		</tr>
		<tr>
			<td>Iodomethane</td>
			<td>Sigma-Aldrich</td>
			<td>289566</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)</td>
			<td>EMD Millipore</td>
			<td>Calbiochem 475989-1GM</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Phosphate-buffered Saline (PBS)</td>
			<td>Genesee Scientific</td>
			<td>25-507</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>REM Resin</td>
			<td>Nova Biochem</td>
			<td>8551010005</td>
			<td>Polymer support; 0.500 mmol/g loading</td>
		</tr>
		<tr>
			<td>trans-&#946;-nitrostyrene</td>
			<td>Sigma-Aldrich</td>
			<td>N26806</td>
			<td>Nitro-olefin reagent</td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Sigma-Aldrich</td>
			<td>244511</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Triethylamine (TEA)</td>
			<td>Sigma-Aldrich</td>
			<td>T0886</td>
			<td>Reagent for beta-elimination</td>
		</tr>
		<tr>
			<td>Trimethylsilyl chloride (TMSCl)</td>
			<td>Sigma-Aldrich</td>
			<td>386529</td>
			<td>Reagent; CAUTION - highly volatile; creates HCl gas</td>
		</tr>
		<tr>
			<td>GLASSWARE/INSTRUMENTATION</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL solid-phase reaction vessel</td>
			<td>Chemglass</td>
			<td>CG-1861-02</td>
			<td>Glassware with filter</td>
		</tr>
		<tr>
			<td>96 Well plate reader</td>
			<td>Promega (Turner Biosystems)</td>
			<td>9310-011</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>AVANCE III NMR Spectrometer</td>
			<td>Bruker</td>
			<td>N/A</td>
			<td>Instrument; 300 MHz; Solvents: CDCl3 and CD3OH</td>
		</tr>
		<tr>
			<td>Thermo Scientific Nicole iS5</td>
			<td>Thermo Scientific</td>
			<td>IQLAADGAAGFAHDMAZA</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Wrist-Action Shaker</td>
			<td>Burrell Scientific</td>
			<td>757950819</td>
			<td>Instrument</td>
		</tr>
	</tbody>
</table>

functionalized spirocyclic heterocycle synthesis and cytotoxicity assay

最近文献报道了螺环杂环是癌症治疗的潜在药物。这些新型正交环系统的合成具有挑战性。最近发表了一种合成这些化合物的有效方法，该方法描述了四步的固相合成，而不是先前报道的五个步骤。这种较短合成的优点是消除了有毒试剂的使用。发现低负载再生迈克尔（REM）接头基树脂在合成中至关重要，因为高负载版本阻止了添加含有大块苯基和芳香族侧链的试剂。比色3-（4′，5′-二甲基噻唑-2′-基）-2，5-二苯基四唑溴化物（MTT）测定用于体外检查这些新型螺旋环分子微摩尔浓度的细胞毒性。MTT很容易在商业上获得，并产生相对快速，可靠的结果，使该测定成为这些螺环杂环的理想选择。测试了正交环结构以及糠胺（合成方法中的前体，包含类似的5元环基序）。

使用改良方案合成螺环肟6和7（图1）。迈克尔将糠胺添加到REM接头1b中得到聚合物结合树脂2。通过红外（IR）光谱通过检测1722cm-1处α，β-不饱和酯的消失来监测反应的进展（图3）。螺环结合树脂4由2通过瞬态中间体3形成。甲醇水解4得到3-[（3E）-（2S，4R）-2-苯基-3-羟基亚氨基4-羟甲基吡咯烷-1-基]-丙酸甲酯7，同时烷基化后β?...

Watch this Scientific Journal Video about Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay at JoVE.com

Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

在这里，我们描述了使用3-（4′，5′-二甲基噻唑-2′-基）-2，5-二苯基四唑溴化物（MTT）的生物测定，以测试先前合成的螺环肟。

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 ...

该协议利用可回收和改良的方法生产螺环化合物。MTT测定有助于回答有关这些新分子的细胞毒性的问题。该技术使用再生起始材料生产新型可修饰分子。MTT测定提供相对快速且易于分析的生物学结果 如前所述，首先进行螺环杂环六和七的固相合成。使用无菌PBS作为稀释剂制备20毫升每毫升5毫克的MTT溶液。过滤并将溶液储存在零下20摄氏度。然后，在DMEM中制备MTT溶液的一对一稀释液。在DMSO中测试化合物的1.5毫升微量离心管中制备每毫升储备溶液一毫升。将它们存放在零下 20 摄氏度。通过稀释原液浓度，每剂量制备200微升测试化合物的工作溶液。在无血清培养基中为1至1000。在组织培养种子中，COS将7个细胞在完全培养基中放入平底组织培养物上，以每孔200微升4， 000个细胞的浓度处理96孔板。使用多通道移液器。将细胞在含有5%二氧化碳的气氛中在37摄氏度下孵育24小时。第二天，使用连接到真空泵的玻璃牧场移液管从孔中吸出上清液。使用先前制备的工作溶液将细胞与测试化合物一式三份给药。然后将细胞放回培养箱。24小时后，从孔中吸出上清液，并向每个孔中加入200微升MTT溶液。将板孵育四小时。从孔中轻轻吸出上清液，而不会干扰紫色甲氮烷晶体。向每个孔中加入200微升DMSO以溶解晶体。然后将板在室温下孵育15分钟。使用96孔板读数器测量每个孔在590或600纳米处的吸光度。使用没有细胞的孔作为背景并平均吸光度值。使用改进的方案合成螺环 gox 接缝 6 和 7。通过使用红外光谱检测αβ不饱和以斯帖的消失来监测反应的进展。MTT测定用于确定细胞活力。已知顺铂在高浓度下诱导细胞死亡，作为阳性对照。正如预期的那样，顺铂的浓度越高，细胞活力就越低。然后使用MTT测定法测试螺环化合物六和七以及糠胺。糠胺和螺环型六线的细胞毒性相似。但螺环类化合物7的毒性明显大于糠胺和6。尝试此方案时，请记住，使用大块酰基卤化物进行的N-烷基化被发现会降低β消除速率，从而降低产品产量。

Research

JoVE Journal

Biology

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

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

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Synthesis and Calibration of Phosphorescent Nanoprobes 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 ...

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate [18F]SFB

微波辅助合成一锅 N -琥珀酰亚胺- 4 - [ 18 F] fluorobenzoate（[ 18 F] SFB）

Biomolecules, including peptides,1-9 proteins,10,11 and antibodies and their engineered fragments,12-14 are gaining importance as both potential ...

Intracellular Refolding Assay

细胞内复性检测

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

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

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 ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

快速合成及其与绿色荧光蛋白为基础的记者化学激活的转录因子筛选

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Heme Synthesis Levels in Mammalian Cells

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...