Name: constructing cyclic peptides using an on-tether sulfonium center
Uploaded: 2022-09-28T13:30:00
Description: Watch this Scientific Journal Video about Constructing Cyclic Peptides Using an On-Tether Sulfonium Center at JoVE.com

感谢国家重点研发计划（2021YFC2103900）的资金支持;中国自然科学基金（21778009和21977010）;广东省自然科学基金（2022A1515010996、2020A1515010521）：深圳市科技创新委员会（RCJC20200714114433053、JCYJ201805081522131455、JCYJ20200109140406047）;以及深港脑科学研究所-深圳基本科研机构资助（2019SHIBS0004）。作者感谢 化学科学， 皇家化学学会的期刊支持参考文献30和美国化学学会有机 化学杂志参考文献31。

1. 设备准备
注意：吗啉、N，N-二甲基甲酰胺 （DMF）、二氯甲烷 （DCM）、N，N-二异丙基乙胺 （DIPEA）、TFA、吗啉、哌啶、乙醚和甲醇有毒、易挥发和腐蚀性。这些试剂会通过吸入、摄入或皮肤接触对人体造成伤害。对于所有化学实验，请使用防护设备，包括一次性手套、实验外套和防护眼镜。
<ol><li>通过标准手动Fmoc固相肽合成（SPPS）29在Rink-?...

<ol>
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本文描述的合成方法提供了一种在肽序列中使用Cys和Met合成环肽的方法，其中基本线性肽通过常见的固相肽合成技术构建。对于Cys和Met之间环肽的双烷基化，整个合成路线可分为三个主要过程：Cys在树脂上的脱保护，接头的偶联以及Cys和Met在三氟乙酸裂解溶液中的环化。值得注意的是，Cys保护基团的去除被发现是随后闭环反应的关键步骤。因此，trt-Cys被去保护，直到溶液没有明显的黄色。进一步...

蛋白质-蛋白质相互作用（PPI）1在药物研发中起着关键作用。通过化学方法构建具有固定构象的稳定肽是开发PPIs模拟基序的最重要方法之一2。迄今为止，已经开发了几种靶向PPI的环肽用于临床3。大多数肽被限制在α螺旋构象中，以降低构象熵并改善代谢稳定性、靶标结合亲和力和细胞通透性4，5。在过去的20年中，Cys6，7，赖氨酸8，9，色氨酸10，精氨酸11和Met 12，13的侧链已入到非天然氨基酸中，以将肽固定成环状构象。这种环肽可以靶向独特的化学空间或特殊位点，从而触发共价反应以形成蛋白质-肽共价结合14，15，16，17。在Yu等人最近的一份报告中，氯乙酰胺被锚定在肽配体结构域上，确保具有出色蛋白质特异性的共价偶联反应18。此外，Walensky等人将丙烯酰胺和芳基磺酰氟（ArSO2F）等亲电弹头进一步掺入肽中19，形成稳定的肽共价抑制剂，提高肽抑制剂的抗肿瘤作用。因此，引入额外的官能团以共价修饰蛋白质-肽配体20是非常重要的。这些基团不仅与侧链上的蛋白质反应，而且还稳定肽21的二级结构。然而，由于复杂的合成路线和化学基团22，23的非特异性结合，由肽配体诱导的共价修饰蛋白的应用受到限制。因此，迫切需要合成环肽的有效策略。
受环肽2，24，25，26的多种策略的启发，该协议试图开发一种简单有效的稳定肽的方法。此外，我们注意到稳定肽的侧链基团在空间上接近肽配体时可以与目标蛋白共价反应。2013年，戴明小组通过开发一种生产选择性修饰肽蛋氨酸27的新方法，填补了化学修饰Met的缺乏。基于这一背景，Shi等人重点研究了侧链闭环形成锍盐中心的发展。当肽配体与靶蛋白结合时，锍盐基团与空间接近的Cys蛋白共价反应。近年来，Shi等人设计了一种稳定环肽28的新方法。环肽上的锍盐被具有巯基的还原剂还原，该还原剂可逆地还原为Met。然而，该反应效率低，对后续的生物学应用研究有害。在目前的研究中，设计了Met-Cys和丙炔基溴-Cys闭环反应，在环肽的侧链上保留单个锍盐。锍盐充当了一种新的弹头，在空间接近下与蛋白质Cys共价反应。简而言之，Cys和Met突变的肽通过分子内烷基化环化，导致系绳上锍中心的产生。在这个过程中，侧链桥的形成对环肽至关重要。总体而言，该协议描述了使用简单的反应条件和操作实现的详细基于锍的肽环化。目的是为进一步广泛的生物学应用开发一种潜在的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,3-bis(bromomethyl)-benzen</td>
			<td>Energy</td>
			<td>D0215</td>
			<td></td>
		</tr>
		<tr>
			<td>1,3-Dimethylbarbituric acid</td>
			<td>Energy</td>
			<td>A46873</td>
			<td></td>
		</tr>
		<tr>
			<td>1H NMR and HSQC</td>
			<td>Bruker</td>
			<td>&#160;AVANCE-III 400</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Hydroxybenzotriazole hydrate</td>
			<td>Energy</td>
			<td>E020543</td>
			<td></td>
		</tr>
		<tr>
			<td>2-(7-azabenzotriazol-1-yl)-N,N,N&#39;,N&#39;-tetramethyluronium hexafluorophosphate (HATU)</td>
			<td>Energy</td>
			<td>A1797</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptopyridine</td>
			<td>Energy</td>
			<td>Y31130</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Aminocaproic acid</td>
			<td>Energy</td>
			<td>A010678</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic anhydride</td>
			<td>Energy</td>
			<td>A01021454</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Aldrich</td>
			<td>9758</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium carbonate</td>
			<td>Energy</td>
			<td>12980</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Energy</td>
			<td>W330229</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Heating Cooling Drybath</td>
			<td>&#160;Thermo Scientific</td>
			<td>88880029</td>
			<td></td>
		</tr>
		<tr>
			<td>Diisopropylethylamine (DIPEA)</td>
			<td>Energy</td>
			<td>W320014</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl formamide (DMF)</td>
			<td>Energy</td>
			<td>B020051</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Energy</td>
			<td>A10027</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrospray Ionization Mass</td>
			<td>SHIMADZU2020</td>
			<td>&#160;LC-MS2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Ala-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R30101</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Arg(Pbf)-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R30201</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Cys(Trt)-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R30501</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Gln(Trt)-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R30601</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Glu(OtBu)-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R30701</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-His(Boc)-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R30902</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Ile-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R31001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Lys(Boc)-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R31201</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Met-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R31301</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Pro-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R31501</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Ser(tBu)-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R31601</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Thr(tBu)-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R31701</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Trp(Boc)-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R31801</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Tyr(tBu)-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R31901</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Val-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R32001</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Energy</td>
			<td>W810042</td>
			<td></td>
		</tr>
		<tr>
			<td>High Performance Liquid 
			Chromatography</td>
			<td>SHIMADZU</td>
			<td>LC-2030</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Aldrich</td>
			<td>9758</td>
			<td></td>
		</tr>
		<tr>
			<td>Morpholine</td>
			<td>Aldrich</td>
			<td>M109062</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#39;-Diisopropylcarbodiimide</td>
			<td>Energy</td>
			<td>B010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Ninhydrin Reagent</td>
			<td>Energy</td>
			<td>N7285</td>
			<td></td>
		</tr>
		<tr>
			<td>Propargyl bromide</td>
			<td>Energy</td>
			<td>W320293</td>
			<td></td>
		</tr>
		<tr>
			<td>Rink Amide MBHA resin</td>
			<td>Nanjing Peptide Biotech Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Solid Phase Extraction (SPE) Sample Collection Plates</td>
			<td>&#160;Thermo Scientific</td>
			<td>60300-403</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrakis(triphenylphosphine) palladium</td>
			<td>Energy</td>
			<td>T1350</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-way stopcocks</td>
			<td>Bio-Rad</td>
			<td>7328107</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Energy</td>
			<td>B010737</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>J&#38;K</td>
			<td>101398</td>
			<td></td>
		</tr>
		<tr>
			<td>Triisopropylsilane (TIS)</td>
			<td>Energy</td>
			<td>T1533</td>
			<td></td>
		</tr>
	</tbody>
</table>

constructing cyclic peptides using an on-tether sulfonium center

近年来，环肽因其优异的生物活性而在药物发现领域受到越来越多的关注，因此，它们现在被临床使用。因此，寻求合成环肽的有效策略以促进其在药物发现领域的应用至关重要。本文报告了使用树脂上或分子内（分子间）联烷基化高效合成环肽的详细方案。使用该协议，利用固相肽合成，半胱氨酸（Cys）和蛋氨酸（Met）同时偶联在树脂上，从而合成线性肽。此外，使用可调系绳和系绳上的锍中心通过Met和Cys之间的联烷基化合成环肽。整个合成路线可分为三个主要过程：Cys在树脂上的脱保护，接头的偶联以及Cys与Met在三氟乙酸（TFA）裂解溶液中的环化。此外，受锍中心反应性的启发，炔丙基连接到Met上以触发硫醇-炔基加成并形成环肽。之后，将粗肽干燥并溶解在乙腈中，分离，然后通过高效液相色谱（HPLC）纯化。采用液相色谱-质谱联用（LC-MS）确认了环肽的分子量，并利用HPLC进一步确认了环肽与还原剂组合的稳定性。此外，通过1 H核磁共振（1H NMR）波谱分析了环肽中的化学位移。总体而言，该协议旨在建立合成环肽的有效策略。

所有线性肽均在Rink-amide MBHA树脂上通过标准手工Fmoc固相合成合成。如图5A所述构建环状六肽（Ac（环I）-wmaaac-nh2）模型。值得注意的是，通过Met烷基化产生了一个新的系绳手性中心，反相HPLC确认了环肽的两种差向异构体（Ia，Ib）。此外，使用反相HPLC的积分测定差向异构体的转化率和比例。由六肽Ac-WMAAAC-NH2产生的环AC-（环I）-WMAAAC-NH2肽1-Ia和1-Ib表现?...

Watch this Scientific Journal Video about Constructing Cyclic Peptides Using an On-Tether Sulfonium Center at JoVE.com

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

该协议介绍了通过半胱氨酸和蛋氨酸之间的双烷基化以及由炔丙基锍中心触发的简单硫醇 - 炔反应 合成 环肽。

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 ...

在该协议中，构建了锍盐，可以充当与蛋白质半胱氨酸在特殊接近处反应的新手。该技术的优点是该反应快速高效，并且不含Met催化剂，并开发了一种简单有效的稳定肽的方法。首先，在500毫升烧杯或烧瓶中制备20%哌啶或50%吗啉和DMF的N-末端9-芴基-甲基氧羰基脱保护溶液。然后，向色谱柱中加入10毫升脱保护溶液，并向溶液中注入氮气30分钟或两次，持续5分钟。通过真空泵排出溶液，并依次用二氯甲烷和N，N-二甲基甲酰胺洗涤树脂五次。要检测树脂为深黄色溶液，在少量树脂中加入一毫升N，N-二甲基甲酰胺，并向玻璃管中加入200微升5%茚三酮。在130摄氏度下加热三分钟，以评估树脂颜色的变化。对于肽的偶联，在聚丙烯管中制备手稿中所述的混合物溶液，并将其溶解在三毫升N，N-二甲基甲酰胺中。然后，向溶液中加入173微升N，N-二异丙基乙胺或154微升N，N'二异丙基碳化二亚胺以使氨基酸失活。接下来，将混合物加入装有树脂的色谱柱中，并用氮气鼓泡两个小时。偶联后，在少量树脂中加入1毫升N，N-二甲基甲酰胺，向玻璃管中加入200微升5%茚三酮。在130摄氏度下加热三分钟，同时树脂变为无色，这证实了在脱保护步骤之前没有游离氨基。排干溶液后，如前所述洗涤树脂，并向色谱柱中加入10毫升脱保护溶液。然后，用氮气搅拌 30 分钟或两次，持续 5 分钟。再次，添加新鲜的溶液。向带有树脂的色谱柱中加入10毫升无水甲醇进行脱水，并用氮气干燥以备下次使用。称取100毫克树脂到柱中，并在偶联步骤之前用二氯甲烷和N，N-二甲基甲酰胺洗涤树脂。通过在100毫升烧杯或烧瓶中加入三氟乙酸，三异丙基硅烷和二氯甲烷混合物来制备用于去除三苯甲基-L-半胱氨酸保护基的溶液，以除去保护基。向色谱柱中加入5至10毫升制备的溶液以除去保护组10分钟。用氮气鼓泡重复六次，直到黄色完全消失。通过真空泵排出溶液后，如前所述清洗树脂。然后，通过在50毫升烧杯或烧瓶中加入二卤接头和N，N-二异丙基乙胺与N，N-二甲基甲酰胺，制备与脱保护半胱氨酸反应的溶液。向色谱柱中加入5至10毫升的反应溶液，与受保护的半胱氨酸反应至少3小时，氮气鼓泡。同样，通过真空泵排出溶液并按顺序洗涤树脂，如前所述。为了制备肽环化溶液，将三氟乙酸混合物加入通风橱中的20毫升烧杯或烧瓶中进行肽环化。然后，将5至10毫升的混合物溶液加入聚丙烯管中，并在三氟乙酸混合物下将树脂切割三小时。在脱水和洗涤树脂之前演示的偶联步骤之前，制备1%甲酸水溶液和1毫摩尔丙炔基溴，并加入到蛋氨酸肽溶液中。接下来，在室温下摇动蛋氨酸和炔丙基溴的偶联反应12小时。反应后，将产物溶解在聚丙烯管和乙腈中，并通过0.22微米的过滤膜过滤。然后，立即通过反相HPLC纯化溶液，并将其冷冻干燥成粉末以备下次使用。采用HPLC和LCMS光谱对半胱氨酸和蛋氨酸双烷基化合成的环状肽进行了表征， 结果表明差向异构体具有不同的保留时间和相同的分子量.差向异构体及其偶联产物的HPLC迹线证明了环肽与其产物之间的时间依赖性转化。采用LCMS法测定硫醇-炔型反应合成的环肽分子量，并用HPLC分离纯化肽。通过质子核磁共振和异核单量子相干光谱进一步表征了肽，揭示了其化学位移。获得了环状肽与二硫苏糖醇和氧化氘之间时间依赖性转化的质子核磁共振波谱，以探索肽因锍环打开而具有的稳定性。结果表明，24 h后未形成添加物或开环产物。该程序建立了合成环肽的有效策略。该反应表示，通过形成稳定环化肽的确认，选择性也得到了增强，表明环肽是有前途的药物催化剂。

Research

JoVE Journal

Chemistry

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

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

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 ...

科研

化学

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

生物分子有序结构由短肽的自组装形成

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

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

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 ...

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

环状聚合物的合成及其在熔融状态扩散运动的表征在单分子水平

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

洞察氨基酸和多肽与无机材料的使用相互作用的单分子力谱

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera

利用 iccd 摄像机对无氧环境下的三重收集有机化合物进行光物理表征

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables ...

Using Cyclic Voltammetry, UV-Vis-NIR, and EPR Spectroelectrochemistry to Analyze Organic Compounds

利用循环伏安法、紫外-近红外光谱和 EPR 光谱电化学对有机化合物进行分析

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance ...

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

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

12-Dithiolane 改性自组装肽的合成与表征

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment

药物药代动力学和毒理学评估的肠道/肝脏微生理系统

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests ...