Name: facile protocol for the synthesis of self-assembling polyamine-based peptide amphiphiles (ppas) and related biomaterials
Uploaded: 2018-06-25T14:00:00
Description: Watch this Scientific Journal Video about Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials at JoVE.com

该项目由内布拉斯加州大学医学中心 (开办基金, MC) 资助;NIH-COBRE, 5P20GM103480 (t Bronich) 和美国化学学会, PRF 57434 号-DNI7 (MC S)。

1. PPAs 合成通用议定书
<ol><li>计算合成的尺度 (通常为毫摩尔)。此刻度基于目标 PPA 金额的质量。请记住, 随着氨基酸序列的增加, 许可证的反应效率逐渐降低。因此, 准确的反应效率是难以计算的。</li>
	<li>根据树脂的载荷计算出所用树脂的重量。装载在树脂的容器或分析协议被发现并且表达在毫摩尔/g。以下公式可用于计算树脂的重量:</li>
	<li>仔细权衡 2-chlorotrityl 氯树脂 (在我们的情况下, 负载?...

<ol>
	<li>Cui, H., Pashuck, E. T., Velichko, Y. S., Weigand, S. J., Cheetham, A. G., Newcomb, C. J., Stupp, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+and+x-ray-triggered+crystallization+at+long+range+in+self-assembling+filament+networks.">Spontaneous and x-ray-triggered crystallization at long range in self-assembling filament networks.</a> Science. 327, 555-559 (2010).</li><li>Pashuck, E. T., Cui, H., Stupp, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+supramolecular+rigidity+of+peptide+fibers+through+molecular+structure.">Tuning supramolecular rigidity of peptide fibers through molecular structure.</a> Journal of the American Chemical Society. 132, 6041-6046 (2010).</li><li>Stupp, S. I., Zha, R. H., Palmer, L. C., Cui, H., Bitton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+biomolecular+soft+matter.">Self-assembly of biomolecular soft matter.</a> Faraday Discussions. 166, 9-30 (2013).</li><li>Conda-Sheridan, M., Lee, S. S., Preslar, A. T., Stupp, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Esterase-activated+release+of+naproxen+from+supramolecular+nanofibres.">Esterase-activated release of naproxen from supramolecular nanofibres.</a> Chemical Communications. 50, 13757-13760 (2014).</li><li>Mata, A., Palmer, L., Tejeda-Montes, E., Stupp, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+biomolecules+for+nanoengineered+biomaterials+for+regenerative+medicine.">Design of biomolecules for nanoengineered biomaterials for regenerative medicine.</a> Nanotechnology in Regenerative Medicine. , 39-49 (2012).</li><li>Samad, M. B., Chhonker, Y. S., Contreras, J. I., McCarthy, A., McClanahan, M. M., Murry, D. J., Conda-Sheridan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+Polyamine-Based+Peptide+Amphiphiles+with+Tunable+Morphology+and+Physicochemical+Properties.">Developing Polyamine-Based Peptide Amphiphiles with Tunable Morphology and Physicochemical Properties.</a> Macromolecular bioscience. 17, (2017).</li><li>Nel, A. E., M&#228;dler, L., Velegol, D., Xia, T., Hoek, E. M., Somasundaran, P., Klaessig, F., Castranova, V., Thompson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+biophysicochemical+interactions+at+the+nano-bio+interface.">Understanding biophysicochemical interactions at the nano-bio interface.</a> Nature Materials. 8, 543 (2009).</li><li>Gujrati, M., Malamas, A., Shin, T., Jin, E., Sun, Y., Lu, Z. -. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+cationic+lipid-based+nanoparticles+facilitate+endosomal+escape+and+reduction-triggered+cytosolic+siRNA+release.">Multifunctional cationic lipid-based nanoparticles facilitate endosomal escape and reduction-triggered cytosolic siRNA release.</a> Molecular Pharmaceutics. 11, 2734-2744 (2014).</li><li>Zhu, Y., Li, J., Kanvinde, S., Lin, Z., Hazeldine, S., Singh, R. K., Oupicky&#769;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-immolative+polycations+as+gene+delivery+vectors+and+prodrugs+targeting+polyamine+metabolism+in+cancer.">Self-immolative polycations as gene delivery vectors and prodrugs targeting polyamine metabolism in cancer.</a> Molecular Pharmaceutics. 12, 332-341 (2014).</li><li>Planas-Portell, J., Gallart, M., Tiburcio, A. F., Altabella, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper-containing+amine+oxidases+contribute+to+terminal+polyamine+oxidation+in+peroxisomes+and+apoplast+of+Arabidopsis+thaliana.">Copper-containing amine oxidases contribute to terminal polyamine oxidation in peroxisomes and apoplast of Arabidopsis thaliana.</a> BMC Plant Biology. 13, 109 (2013).</li><li>Nash, I. A., Bycroft, B. W., Chan, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dde+-+A+selective+primary+amine+protecting+group:+A+facile+solid+phase+synthetic+approach+to+polyamine+conjugates.">Dde - A selective primary amine protecting group: A facile solid phase synthetic approach to polyamine conjugates.</a> Tetrahedron Letters. 37, 2625-2628 (1996).</li><li>Ralhan, K., KrishnaKumar, V. G., Gupta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piperazine+and+DBU:+a+safer+alternative+for+rapid+and+efficient+Fmoc+deprotection+in+solid+phase+peptide+synthesis.">Piperazine and DBU: a safer alternative for rapid and efficient Fmoc deprotection in solid phase peptide synthesis.</a> RSC Advances. 5, 104417-104425 (2015).</li><li>Casero, R. A., Marton, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+polyamine+metabolism+and+function+in+cancer+and+other+hyperproliferative+diseases.">Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases.</a> Nature Reviews Drug Discovery. 6, 373 (2007).</li><li>Wuts, P. G. M., Greene, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protection for the Amino Group. In Greene's Protective Groups in Organic Synthesis. , 696-926 (2006).</li><li>Palasek, S. A., Cox, Z. J., Collins, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+racemization+and+aspartimide+formation+in+microwave-enhanced+Fmoc+solid+phase+peptide+synthesis.">Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis.</a> Journal of Peptide Science. 13, 143-148 (2007).</li></ol>

这里描述的协议可以用来合成 PPAs 作为 PAs 和相关的肽基分子 (如混合 peptoids)。虽然使用许可证合成肽是一个简单的过程, 但合成含有生物自导分子的肽尤其具有挑战性。多胺如胺、胺、diethyelenetriamine等, 可作为靶向分子定位于肿瘤细胞13。PPAs 可以自组装成具有不同形貌的纳米结构6。他们的正电荷还可以帮助更长的循环时间 (由于细胞膜逃逸) 和不同的代...

自组装肽 amphiphiles (PAs) 是一类生物材料, 通常由以下部分组成: (a) 亲水性头, (b) 链接器区域, (c) 疏水尾。文献中描述的大多数 PAs 具有亲水性的头, 由带电或极性氨基酸残留物1,2,3,4组成。PAs 已经在生物医学中发现了广泛的应用, 包括药物传递, 疾病诊断, 再生医学等5。在氨基酸序列的基础上, PAs 可以形成各种各样的纳米结构, 包括球形胶束和纳米丝。我们最近报告了一类混合多胺基肽 amphiphiles, 称为 PPAs6。这些生物材料的形貌、自组装动力学和代谢降解都与它们的溶出头组有关。此外, 苯丙醇纳米结构并没有对哺乳动物细胞 (MiaPaCa2 和 HeLa 细胞系) 在经过测试的浓度上表现出毒性。基于苯丙醇的 nanocarriers 是有吸引力的药物运载工具, 因为: (1) 多胺的摄取和新陈代谢已被证明在癌细胞中增加, (2) 阳离子纳米结构可以实现细胞膜逃逸7,8,这导致细胞内的循环和居住更高, (3) 与 PA 相比, 它们应该具有明显的代谢剖面;例如, 它们会更稳定地朝向人体内发现的蛋白酶 (尽管它们可能对其他酶 (如胺氧化酶)9,10敏感。此外, PPAs 已经发现有不同的形态, 物理化学性质, 纳米颗粒的硬度, 和组装动力学取决于不同的长度和电荷的单个 PPA 分子6。在这里, 我们描述了一个详细的协议, 以合成, 鉴定和纯化的 PPAs, 也可以用于制备 PAs 或类似的混合肽分子。
由于多胺在其受保护的形式中通常不具有商业用途, 而且由于保护多胺的主要和次级胺对共轭氨基酸和其他分子具有极其重要的意义, 我们概述了合成步骤, 以实现其保护。该协议的总体目标是提供一种简单的方法, 共轭多胺氨基酸。多胺缺乏羧酸基团;因此, 它们不能耦合到溜冰场酰胺或王树脂。相反, 建议合成协议的树脂, 如 2-chlorotrityl 氯。苯丙醇合成的主要挑战是初级和二胺功能基团的存在。为了我们的目的, 我们保护多胺中的所有次生胺, 同时保持基胺上的主要氨基基团自由地允许偶联反应。在固体相肽合成 (许可证) 原理的基础上, 对其进行了坚实的支持, 以促进每个耦合和脱步骤后的工作。下面的协议用于 PPAs 的手动和自动合成 (尽管在自动化系统中对某些步骤的验证将具有挑战性)。这些分子的合成也可以在自动合成器上进行, 或借助微波反应器 (自动或半自动) 进行。在图 1中总结了反应方案。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57908/57908fig1.jpg"> 图 1:(a) 合成 PPAs 的一般反应方案。(B) 可用于合成 PPAs 的代表性多胺。<a href="/files/ftp_upload/57908/57908fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table>
	<tbody>
		<tr>
			<td>2-Chlorotrityl chloride resin&#160;</td>
			<td>AappTec</td>
			<td>RTZ001</td>
			<td></td>
		</tr>
		<tr>
			<td>SynthwareTM synthesis vessel&#160;</td>
			<td>Aldrich</td>
			<td>SYNP120050M</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Acros</td>
			<td>AC406920250</td>
			<td>Fisher Sci. Catalogue #</td>
		</tr>
		<tr>
			<td>Wrist Shaker</td>
			<td>Boekel Scientific</td>
			<td>401000-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Kaiser test kit</td>
			<td>Sigma-Aldrich</td>
			<td>60017</td>
			<td></td>
		</tr>
		<tr>
			<td>2-[(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-amino]-ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>CDS004772</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous Methanol</td>
			<td>Acros</td>
			<td>AC610981000</td>
			<td>Fisher Sci. Catalogue #</td>
		</tr>
		<tr>
			<td>Chloranil test kit</td>
			<td>TCI</td>
			<td>TCC1771-KIT</td>
			<td>VWR Catalogue #</td>
		</tr>
		<tr>
			<td>Di-tert butyl di-carbonate&#160;</td>
			<td>Acros</td>
			<td>AC194670250</td>
			<td>Fisher Sci. Catalogue #</td>
		</tr>
		<tr>
			<td>Dimethylformamide</td>
			<td>Fisher Scientific</td>
			<td>BP1160-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrazine</td>
			<td>Acros</td>
			<td>AC296815000</td>
			<td>FIsher Sci. Catalogue #</td>
		</tr>
		<tr>
			<td>(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)</td>
			<td>p3biosystems</td>
			<td>31001</td>
			<td></td>
		</tr>
		<tr>
			<td>4-methyl piperidine&#160;</td>
			<td>Acros</td>
			<td>AC127515000</td>
			<td>FIsher Sci. Catalogue #</td>
		</tr>
		<tr>
			<td>Trifluoroacetic Acid</td>
			<td>AappTec</td>
			<td>CXZ035</td>
			<td></td>
		</tr>
		<tr>
			<td>Triisopropyl Silane</td>
			<td>Sigma-Aldrich</td>
			<td>233781</td>
			<td></td>
		</tr>
		<tr>
			<td>Ether</td>
			<td>Fisher Scientific</td>
			<td>E138-1</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-Cyano-4-hydroxycinnamic acid</td>
			<td>Sigma-Aldrich</td>
			<td>C8982</td>
			<td></td>
		</tr>
		<tr>
			<td>9-Aminoacridine</td>
			<td>Sigma-Aldrich</td>
			<td>92817</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Syringe Filters: PTFE Membrane</td>
			<td>Fisher Scientific</td>
			<td>09-730-21</td>
			<td></td>
		</tr>
	</tbody>
</table>

facile protocol for the synthesis of self-assembling polyamine-based peptide amphiphiles (ppas) and related biomaterials

多胺基多肽 Amphiphiles (PPAs) 是一种新型的自组装的两亲性生物材料-与肽 Amphiphiles (PAs) 有关。传统的 PAs 有被充电的氨基酸作为溶出的小组 (赖氨酸, 精氨酸), 直接地连接到油脂片断或可能包含由中性氨基酸制成的链接器区域。调谐肽序列的 PAs 可以产生不同的形态。同样, PPAs 具有疏水性段和中性氨基酸, 但也含有多胺分子作为溶水 (亲水性) 基团。与 PAs 的情况一样, PPAs 也可以自组装成不同的形貌, 包括小棒, 扭曲纳米带, 和熔融纳米片, 当溶解在水中。然而, 在单个多胺分子上存在主要和次要胺, 在合成 PPAs 时会带来重大挑战。在本文中, 我们展示了一个简单的协议, 基于文献先例, 以实现一个简便的合成 PPAs 使用固相肽合成 (许可证)。该议定书可扩展到考绩制度和其他类似系统的综合。我们还说明了从树脂, 鉴定和纯化所需的步骤。

在合成纯化后, 在理化或生物评价之前, 建议 PPAs 的质量进行重新检查, 用解析 HPLC 法确定纯度。对于材料表征或生物评价, PPAs 需要纯度 > 95%。图 2显示了 HPLC 示踪 (顶端) 和 MALDI 光谱 (底部) 确认产品的存在。高效液相色谱分析系统将整合曲线下的区域 (联合自卫队), 联合自卫 > 95% 可与产品纯度相关。在紫外基高效液相色谱系统中, 期望看到一个单一?...

Watch this Scientific Journal Video about Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials at JoVE.com

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials

多胺基肽 amphiphiles (PPAs) 的合成是一个重要的挑战, 由于存在多种胺氮, 这需要明智地使用保护组来掩盖这些反应功能。本文介绍了一种简便的制备这些新型自组装分子的方法。

自组装多胺基多肽 Amphiphiles (PPAs) 及相关生物材料的简易合成协议

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). ...

Research

JoVE Journal

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Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

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Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

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We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

A Direct, Early Stage Guanidinylation Protocol for the Synthesis of Complex Aminoguanidine-containing Natural Products

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The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important ...

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Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

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Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...