这项研究得到了康奈尔大学的支持。这项工作利用康奈尔材料研究中心的共享设施, 这是支持通过 NSF MRSEC 计划 (赠款 DMR-1120296)。S.R.N. 承认, 支持由 NSF 研究生研究奖学金 (赠款 DGE-1650441) 和 Dr. 戴夫 Holowka 协助钙实验。在这一材料中发表的任何意见、发现、结论或建议都是作者的观点, 不一定反映国家科学基金会的观点。

注意: 在这种合成中使用浓酸和碱。在执行反应时使用所有适当的安全措施, 包括使用工程控制 (油烟罩) 和个人防护用品 (PPE), 包括安全眼镜、手套、实验室大衣、全长长裤和闭脚趾鞋. 
 1. 准备 [(哦 2 ) (NH 3 ) 4 Ru (& #181;-O) 汝 (nh 3 ) 4 (OH 2 )] cl 5 <ol> <li> 合成 [ru (nh 3 ) 5 cl] cl 2 12 <ol> <li> 溶解1.00 克 RuCl 3 和 #183; n h 2 O (按重量 40% Ru, 4.1 摩尔) 在 5 mL h 2 o 将深褐色溶液冷却到0和 #176; C 在冰浴中。以滴的方式添加11毫升 (0.23 摩尔) 的80% 水合肼溶液。最初的反应将与演化气体一起剧烈, 导致棕色溶液。使所得到的溶液在室温下搅拌16小时;最终的解决方案将是深红色的. 
		注意: 肼具有剧毒和致癌性。此外, 这种试剂的无水形式是爆炸性的。与往常一样, 使用适当的 PPE 和通风罩时处理。不要将这些解决方案集中于干燥. </li>
		<li> 对此解决方案, 添加约5-10 毫升的浓缩 HCl, 以调整 pH 值为2。在这一点上, 解决方案将是黄褐色的颜色. </li>
		<li> 将此解决方案加热到105和 #176; 搅拌1-2 小时黄色固体会沉淀出溶液。当没有更多的沉淀明显的形式, 消除热量. </li>
		<li> 允许反应混合物冷却到室温, 然后放置在0和 #176; C 冰浴10分钟. 通过真空过滤, 用5毫升乙醇和乙醚洗涤, 收集黄色固体. </li>
		<li> 完全溶解在15-25 毫升热水中的原油产品。将浓缩的 HCl 溶液放在冰浴中冷却10毫升。将黄色溶液过滤到冷却的 HCl 溶液中, 以诱发一个淡黄色纯固体的沉淀。过滤这沉淀物和洗涤以5毫升每 0.5 M HCl, 乙醇并且乙醚. </li>
		<li> 用红外光谱表征化合物。通过在3226厘米 -1 、1604 cm -1 、1297 cm -1 和 801 cm -1 的拉伸频率标识来验证纯度。2069 cm -1 的常见次要杂质被分配给 [Ru (NH 3 ) 5 N 2 ] Cl 3 。
	</ol> </li> <li> 合成 [(哦 2 ) (NH 3 ) 4 Ru (和 #181;-O) 汝 (nh 3 ) 4 (OH 2 )] Cl 5 <ol> 溶解100毫克 (0.34 摩尔) [ru (nh <li> 3 ) 5 cl]Cl 2 在50毫升的1米 NH 4 OH 在200毫升重壁圆形底压力容器。用塞子将烧瓶松散地盖上, 将反应混合物加热75和 #176; C 为 6 h. 从热中取出, 室温下搅拌4天, 产生深绿色溶液. 
		谨慎!加热密封容器会导致压力积聚。确保使用适当的压力安全玻璃器皿。对于这个反应, 密封容器的目的是尽量减少气体 NH 的损失 3 。因此, 将塞子松散地放置, 以释放多余的压力. </li>
	</ol> </li> <li> 通过阳离子交换色谱 <ol> 在25毫升烧杯中进行纯化, 悬浮 5 g 阳离子交换树脂 (例如, 阳离子交换树脂 50WX2 200-400 目 (H <li> + 窗体) 在10毫升 0.1 M HCl 中. 
		<li> 将此浆料装入一个10毫升的柱子 (10 毫米直径, 15 厘米高) 贴附50毫升溶剂油藏。用大约20-30 毫升的0.1 米 HCl 清洗树脂, 直到洗无色. </li>
		<li> 返回到步骤1.2.1 中隔离的绿色反应解决方案。在此解决方案中, 添加浓缩 HCl 以调整 pH 值至 2, 此时溶液颜色会变为褐色. </li>
		<li> 将此酸化溶液加载到1.3.2 中的阳离子交换树脂柱上, 轻轻移在树脂上。让洗完全耗尽, 并继续加载解决方案。重复此过程, 直到添加了整个解决方案。树脂的顶端将是深褐色/黑色。树脂的体积会略有减少. </li>
		<li> 使用玻璃珠覆盖树脂的顶部。在添加新的解决方案时, 这些将防止树脂受到干扰. </li>
		<li> 洗为20毫升的1米 HCl 的列. </li>
		<li> 洗增加 HCl 浓度1.5 米 (和 #8776; 50 毫升) 的列。一个黄色的解决方案将开始从列中脱落。将 HCl 浓度提高到2米, 然后继续洗脱, 直到洗无色或呈淡绿色黄色。这个过程将需要150-200 毫升的总容量. </li>
		<li> 将 HCl 浓度提高到2.5 米 (20-50 毫升)。在试管中收集洗作为分数。增加到 3 M HCl。该产品将洗从该专栏作为一个绿色棕色的解决方案。红棕色的分数也可能会从柱子上脱落。由于这些分数是氧化钌红色杂质, 不池与绿色棕色的分数. </li>
	</ol> </li> <li> 的特性和验证 [(OH 2 ) (NH 3 ) 4 汝 (和 #181;-O) 汝 (NH 3 ) 4 (OH 2 )] "Cl 5 <ol> <li> 测试所有分数步1.3。8紫外可见光谱学。为了完成这项任务, 添加100和 #181; 将给定分数的 L 升为2毫升的3米 NH3, 并通过紫外可见光谱进行分析。含有纯产品的分数将有一个大的吸光度带在 360 nm 和较低的强烈吸光度在 600 nm。480或 533 nm 的吸光度分别指示氧化钌红和钌红色杂质. </li>
		<li> 包含纯产品的池馏分, 并通过旋转蒸发将溶液蒸发干燥。该产品将作为一个绿色棕色固体隔离。产量通常在5-15 毫克 (10-20% 收益率) 的顺序。单晶体, 适用于 X 射线衍射, 可以获得的水蒸气扩散的乙醇水溶液的化合物. </li>
		<li> 为了验证纯度, 在 pH 值为7.4 的磷酸盐缓冲盐水 (PBS) 溶液中通过紫外-可见光谱分析化合物。纯度可以通过采取 360 nm 和 600 nm 峰的强度比值来评估。这个比率是31为一个纯净的化合物。对于不纯化合物, 比例将会更小. </li>
		<li> 用红外光谱法对 solid-state 样品进行分析。诊断带是在 3234 cm -1 , 3151 cm -1 , 1618 cm -1 , 1313 cm -1 , 和 815 cm -1 。常见的杂质带见于 1762 cm -1 和 1400 cm -1 , NH 4 Cl. 钌红的特征可以通过带在 1404 cm -1 、1300 cm -1 、1037 cm -1 和 800 cm -1 中标识. </li>
	</ol> </li> <li> 荧光光谱法评价线粒体钙的摄取抑制 警告!以下过程使用哺乳动物细胞。工作应在适当的层流罩, 是认证的生物安全等级 2 (BSL2) 研究. 
	#8203; 注意: [(哦 2 ) (NH 3 ) 4 Ru (#181;-O) 汝 (NH 3 ) 4 (OH 2 )] Cl 5 将被称为 [Ru] 在本节 <ol> <li> 中使缓冲含葡萄糖的盐水溶液 (BGSS) 作为化验介质。BGSS 是一个解决方案, 包括110毫米氯化钾, 1 mm 2 PO 4 , 1 毫米氯化镁 2 , 20 毫米 4-(2-羟基乙基)-1-piperazineethanesulfonic 酸 (HEPES), 5 毫米琥珀酸钠, 30 和 #181; M 乙二醇双 (和 #946; 乙氨基醚)- n, n, n 和 #39;, n 和 #39; -乙酸酸 (EGTA)。除了 EGTA, 将 pH 值调整到7.4。添加 EGTA 并调整 pH 值至7.4。50毫升的测定培养基中添加0.5 毫升的1毫克/毫升葡萄糖. </li>
		<li> 500 cm 中的培养 HeLa 细胞 2 Dulbecco 和 #39 的改良鹰培养基 (DMEM) 和10% 胎牛血清 (FBS) 在湿式恒温箱中, 5% CO 2 37 和 #176; c. 通过播种增强 100 mm Petri 皿中生长的 hela 细胞他们在 500 cm 2 培养皿。大盘中的总介质量为115毫升。每个大盘子将产生大约1800万细胞, 足够的两个荧光光谱实验。
		<ol> <li> 将单元格增长到 90-95% 70-100。取出培养基, 用15毫升 pH 7.4 PBS 冲洗细胞。在 PBS 中加入15毫升的1毫米乙酸酸 (EDTA), 孵育10分钟以分离细胞。将电池转移到14毫升圆底猎鹰管 </li> </ol> </li> <li> 使用台盼蓝和例的计数单元, 并用倒置显微镜计算每1.8 毫升容量达到750万细胞所需的细胞总数和培养基数量 中等。将细胞离心10分钟, 在5310和 #215; 醒酒上清液, 加入 BGSS 的计算量。重细胞轻轻。
		<ol> <li> 用于此检测, 准备40毫米 digitonin 在二甲基亚砜 (亚砜), 1 毫米钙 Green-5N 在 h 2 o 和10毫米 CaCl 2 在 h 2 o 中的库存解决方案. [汝] 库存解决方案, 在纯净水中, 可以从1-3 毫米不等。
			#8203; 注意: Green-5N 钙是光敏的。存储在黑暗中, 尽量减少曝光. </li>
		</ol> </li> <li> 设置 fluorimeter 以在 506 nm 处激发, 并在 532 nm 上读取试管控制在37和 #176 上的发射器; c. 用搅拌棒或轮子制作丙烯酸试管, 1.8 mL 细胞悬浮1.5.2 以上, 1.8 和 #181; L digitonin 解决方案, 3。6#181;l 钙 Green-5N (溶液), 9 和 #181; l [汝] (1 毫米的股票溶液, 5 和 #181; M 最终浓度)。在 fluorimeter 孵育细胞15分钟。
		<ol> <li> 将数据读取为激发/发射比而不是原始吸收。这种做法最大限度地减少了与光源强度波动相关的误差. </li>
			<li> 在没有 [Ru] 的情况下执行第一个样本分析, 以测量在单元格的响应中添加 CaCl 2 的效果. </li>
			<li> 使用1.5.4 中描述的设置开始分析 fluorimeter。等待约2分钟建立一个稳定的排放基线, 然后添加1.8 和 #181; L CaCl 2 (10 和 #181; M 最后的浓度)。在加入 CaCl 2 后, 发射强度会立即增加, 然后随着钙离子进入线粒体而在几分钟内衰变。等到衰变结束 (#8776; 5 分钟)。添加额外的钙 boluses, 以确定不处理的细胞线粒体钙吸收反应 [Ru]. </li>
		</ol> </li> <li> 在包含5和 #181 的另一个试管中; M [Ru], 重复上述1.5.4.3 中所述的实验。在抑制剂的存在下, 排放强度会增加, 但不会衰减。这种观察意味着阻断线粒体钙的摄取. </li>
	</ol>
	</li>
</ol>

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作者没有透露

线粒体钙吸收抑制剂 [(OH2) (nh3)4汝 (µ O) ru (nh3)4(OH2)] cl5可以从 [Ru (NH3)5cl] cl2中合成众所周知的钌 (III)起始材料, 如本程序所述。合成的 [Ru (NH3)5cl] cl2很容易实现, 但难度很小。在水合肼中搅拌 RuCl3 16 小时后, 溶液的 pH 值应调整为2与 HCl 的数值。pH 值下降是实现所需产品的关键。如果需要, 可以修改此合成, 以形成 [Ru (NH3)5br] br2 , 回流在评论而不是 HCl 中。
我们已经观察到在线粒体钙吸收抑制剂合成过程中, 减少不受欢迎的副产品形成的一些策略。值得注意的是, 保持烧瓶上限是这一反应成功的关键;如果烧瓶是开放的, 许多其他副产品, 包括钌红 (RuRed), 是形成了可观的数量。据推测, 气态氨是从打开的反应容器中消失的, 这一事件危及到所需产品的形成。烧瓶的大小和水氨的体积也是重要的参数, 不应与本议定书中所描述的那样显著地改变, 因为我们注意到, 这种反应的效率在较小的烧瓶中降低了。这种方法与我们先前披露的其他两种方法相比, 并没有显著增加这一化合物的产量。但是, 它确实包含较少的步骤, 从而导致较少的产品形成, 如 RuRed。低收率可能是由于存在少量的未 [Ru (NH3)5cl] cl2以及在该列顶部保留的未知高电荷杂质。虽然我们没有探索许多额外的反应条件, 它是可能的改善, 可以实现通过改变反应时间和反映物浓度, 以获得更高的产量。
[(哦2) (nh3)4ru (µ O) 钌 (nh3)4(oh2)] Cl5是本协议中最繁琐、最困难的部分。一个很好的填料柱将大大有助于分离;将树脂作为浆料装载是一种有效的包装方法。应注意的是, 随着盐酸浓度增加的过程中的柱洗脱, 树脂将收缩大小。为了成功地获得纯净的材料, 酸浓度增加的速率不应偏离规定的程序。最终产品将是一个绿色的棕色固体;一个基本的解决方案将是深绿色的, 但酸性的解决方案将是棕色的。如果观察到明亮的粉红色-红色溶液, 钌红色污染物存在。RuRed 的数量可以通过使用消光系数 (6.2万 M-1·cm-1在 533 nm) 来确定。这种纯化工艺对于强阳离子交换树脂柱比较普遍。
线粒体钙摄取抑制可以使用化 HeLa 细胞、荧光钙传感器钙 Green-5N 和 spectrofluorimeter。13,14此检测需要大量的单元格, 因此必须在非常大的 500 cm2 Petri 盘中放大区域性。这些大的文化烧瓶防止了额外的挑战, 减少微生物污染, 因为裸露的表面积很大。工作应在层流柜中进行, 以保持不育。荧光光谱法监测化 HeLa 细胞中钙 Green-5N 的荧光反应。添加一个外部的钙丸到试管触发立即增加荧光, 引起的钙离子与传感器的互动。在没有抑制剂的几分钟过程中, 由于线粒体中钙离子的摄取而导致的强度衰减, 这是一种不能被染料访问的细胞器官。当这种检测在抑制剂的存在下进行时, 添加一团钙离子会增加排放强度。然而, 由于线粒体钙摄取的衰减, 不存在, 验证线粒体钙的摄取抑制特性的化合物。这一过程可以作为钙吸收抑制剂的一般屏幕使用。此外, 此程序可应用于其他真核细胞的利益。这种方法依赖于外膜的性, 因此不提供关于一个复杂的细胞吸收能力的信息。
总之, 该协议描述了一种新型的钌线粒体钙吸收抑制剂的合成和纯化。该化合物对研究线粒体钙的生物学及其在哺乳动物细胞生理中的作用具有重要的价值。相对简单的检测线粒体钙吸收的方法也用于筛选和研究新的抑制剂。

线粒体钙是对正常细胞功能, 包括能量生产和细胞凋亡至关重要的一些过程的关键调节器。1,2,3线粒体钙 uniporter (MCU), 一种离子转运蛋白, 驻留在线粒体膜上, 调节钙离子流入线粒体。4,5,6单片机的化学抑制剂是继续努力研究这种转运蛋白和线粒体钙的功能和细胞作用的宝贵工具。化合物 [(小贩2) (nh3)4ru (µ) 汝 (nh3)4(o2CH)3 +, Ru360, 是唯一已知的单片机的选择性抑制剂之一, 其报告的 Kd值为24µM.7 ,8,9,10此复合物是在钌红色 (RuRed) 的商业配方中发现的常见杂质, triruthenium µ-羰基桥接 hexacation 的公式 [(NH3)5ru (µ) 汝 (nh3)4(µ) ru (nh3)5)6 +, 这也被用作钙吸收抑制剂。虽然 Ru360 是商用的, 但成本很高。此外, Ru360 的合成和分离受到困难的纯化程序和模糊的表征方法的挑战。
我们最近报告的替代程序访问一个 Ru360 模拟, [(OH2) (nh3)4汝 (µ) ru (新罕布什尔州3)4(oh2)] Cl5。11此复合抑制具有高亲和力的 MCU, 类似于 Ru360。在本协议中, 我们将描述这一化合物的最有效的合成, 它从 [Ru (NH3)5cl] cl2开始。使用强酸性阳离子交换树脂提纯产品, 并对此程序的常见缺陷进行了详细介绍。我们还提出了表征和评估化合物纯度的方法, 并描述了一个简单的方法来测试它的有效性, 阻断线粒体钙摄取。

<table style="border-collapse:collapse;width:663pt" width="884">
	<colgroup>
		<col style="width:182pt" width="243" />
		<col style="width:227pt" width="302" />
		<col style="width:129pt" width="172" />
		<col style="width:125pt" width="167" />
	</colgroup>
	<tbody>
		<tr height="23" style="height:17.25pt">
			<td class="xl69" height="23" style="width:182pt;height:17.25pt" width="243">Ruthenium Trichloride hydrate</td>
			<td class="xl69" style="width:227pt" width="302">Pressure Chemical</td>
			<td class="xl69" style="width:129pt" width="172">3750</td>
			<td class="xl70" style="width:125pt" width="167"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:182pt;height:15.75pt" width="243">Concentrated hydrochloric acid</td>
			<td class="xl69" style="width:227pt" width="302">J.T. Baker</td>
			<td class="xl69" style="width:129pt" width="172">9535</td>
			<td class="xl70"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl69" height="22" style="width:182pt;height:16.5pt" width="243">Concentrated ammonium hydroxide</td>
			<td class="xl71" style="width:227pt" width="302">Mallinckrodt Chemical Works</td>
			<td class="xl71" style="width:129pt" width="172">A669C-2 1</td>
			<td class="xl70"></td>
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		<tr height="24" style="height:18.0pt">
			<td class="xl69" height="24" style="width:182pt;height:18.0pt" width="243">Dowex 50 WX2 200-400 Mesh</td>
			<td class="xl69" style="width:227pt" width="302">Alfa Aesar</td>
			<td class="xl69" style="width:129pt" width="172">13945</td>
			<td class="xl70"></td>
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		<tr height="25" style="height:18.75pt">
			<td class="xl69" height="25" style="width:182pt;height:18.75pt" width="243">Calcium Green 5N</td>
			<td class="xl69" style="width:227pt" width="302">Invitrogen</td>
			<td class="xl69" style="width:129pt" width="172">C3737</td>
			<td class="xl70"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:182pt;height:15.75pt" width="243">Digitonin</td>
			<td class="xl69" style="width:227pt" width="302">Aldrich</td>
			<td class="xl71" style="width:129pt" width="172">260746</td>
			<td class="xl70"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:182pt;height:15.75pt" width="243">DMSO</td>
			<td class="xl69" style="width:227pt" width="302">Aldrich</td>
			<td class="xl69" style="width:129pt" width="172">471267</td>
			<td class="xl70"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:182pt;height:15.75pt" width="243">EGTA</td>
			<td class="xl69" style="width:227pt" width="302">Aldrich</td>
			<td class="xl69" style="width:129pt" width="172">E3889</td>
			<td class="xl70"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:182pt;height:15.75pt" width="243">KCl</td>
			<td class="xl69" style="width:227pt" width="302">USB</td>
			<td class="xl69" style="width:129pt" width="172">20598</td>
			<td class="xl70"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:182pt;height:15.75pt" width="243">KH2PO4</td>
			<td class="xl69" style="width:227pt" width="302">Aldrich</td>
			<td class="xl69" style="width:129pt" width="172">P3786</td>
			<td class="xl70"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:182pt;height:15.75pt" width="243">MgCl2</td>
			<td class="xl77" style="width:227pt" width="302">Fisher Scientific</td>
			<td class="xl69" style="width:129pt" width="172">M33-500</td>
			<td class="xl70"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:182pt;height:15.75pt" width="243">HEPES</td>
			<td class="xl69" style="width:227pt" width="302">Fluka</td>
			<td class="xl69" style="width:129pt" width="172">54466</td>
			<td class="xl70"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl69" height="22" style="width:182pt;height:16.5pt" width="243">Sodium Succinate</td>
			<td class="xl69" style="width:227pt" width="302">Alfa Aesar</td>
			<td class="xl69" style="width:129pt" width="172">33386</td>
			<td class="xl70"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl69" height="22" style="width:182pt;height:16.5pt" width="243">EDTA</td>
			<td class="xl69" style="width:227pt" width="302">J.T. Baker</td>
			<td class="xl69" style="width:129pt" width="172">8993-01</td>
			<td class="xl70"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:182pt;height:15.75pt" width="243">Glucose</td>
			<td class="xl69" style="width:227pt" width="302">Aldrich</td>
			<td class="xl69" style="width:129pt" width="172">G5000</td>
			<td class="xl70"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:182pt;height:15.75pt" width="243">200 Round bottom flask</td>
			<td class="xl69" style="width:227pt" width="302">ChemGlass</td>
			<td class="xl75">CG-1506-14</td>
			<td class="xl74" style="width:125pt" width="167"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:182pt;height:15.75pt" width="243">Glass stopper</td>
			<td class="xl65" style="width:227pt" width="302">ChemGlass</td>
			<td class="xl65" style="width:129pt" width="172">CG-3000-05</td>
			<td class="xl68"></td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl65" height="42" style="width:182pt;height:31.5pt" width="243">10 mm x 15 cm glass column with reservoirs</td>
			<td class="xl77" style="width:227pt" width="302">Custom - similar to Chemglass columns</td>
			<td class="xl77" style="width:129pt" width="172">Similar to CG-1203-20</td>
			<td class="xl68"></td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl65" height="20" style="width:182pt;height:15.0pt" width="243">DMEM</td>
			<td class="xl77" style="width:227pt" width="302">Corning</td>
			<td class="xl77" style="width:129pt" width="172">10-017-CV</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:182pt;height:15.75pt" width="243">FBS</td>
			<td class="xl77" style="width:227pt" width="302">Gibco</td>
			<td class="xl69" style="width:129pt" width="172">10437028</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:182pt;height:15.75pt" width="243">PBS</td>
			<td class="xl77" style="width:227pt" width="302">Corning</td>
			<td class="xl69" style="width:129pt" width="172">21-040-CV</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:182pt;height:15.75pt" width="243">Round bottom Falcon tubes</td>
			<td class="xl77" style="width:227pt" width="302">Fisher Scientific</td>
			<td class="xl69" style="width:129pt" width="172">14-959-11B&#160;</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:182pt;height:15.75pt" width="243">500 cm2 petri dishes</td>
			<td class="xl65" style="width:227pt" width="302">Corning</td>
			<td class="xl76" style="width:129pt" width="172">431110</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:182pt;height:15.75pt" width="243">Trypan blue</td>
			<td class="xl65" style="width:227pt" width="302">ThermoFisher Scientific</td>
			<td class="xl76" style="width:129pt" width="172">15250061</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:182pt;height:15.75pt" width="243">Hemacytometer</td>
			<td class="xl65" style="width:227pt" width="302">Aldrich</td>
			<td class="xl76" style="width:129pt" width="172">Z359629</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:182pt;height:15.75pt" width="243">Acrylic Cuvettes</td>
			<td class="xl65" style="width:227pt" width="302">VWR&#160;</td>
			<td class="xl76" style="width:129pt" width="172">58017-875</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl72" height="21" style="width:182pt;height:15.75pt" width="243">UV-Vis spectrometer</td>
			<td class="xl68">Agilent Model Cary 8454&#160;</td>
			<td class="xl65" style="width:129pt" width="172"></td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl72" height="21" style="width:182pt;height:15.75pt" width="243">Spectrofluorimeter</td>
			<td class="xl68">SLM Model 8100C</td>
			<td class="xl65" style="width:129pt" width="172"></td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl72" height="21" style="width:182pt;height:15.75pt" width="243">IR spectrometer</td>
			<td class="xl68">Bruker Hyprion FTIR with ATR attachment</td>
			<td class="xl65" style="width:129pt" width="172"></td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:182pt;height:15.75pt" width="243">Centrifuge</td>
			<td class="xl65" style="width:227pt" width="302">ALC Model PM140R</td>
			<td class="xl65" style="width:129pt" width="172"></td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:182pt;height:15.75pt" width="243">Inverted light microscope</td>
			<td class="xl65" style="width:227pt" width="302">VWR&#160;</td>
			<td class="xl65" style="width:129pt" width="172">89404-462</td>
			<td class="xl68"></td>
		</tr>
	</tbody>
</table>

synthesis and evaluation of a ruthenium-based mitochondrial calcium uptake inhibitor

我们详细介绍了线粒体钙吸收抑制剂的合成和纯化, [(oh2) (nh3)4汝 (µ) ru (新罕布什尔州3)4(OH2)5 +。这种化合物的优化合成开始从 [Ru (NH3)5cl] cl2在 1 M NH4OH 在一个封闭的容器, 产生一个绿色的解决方案。纯化是用阳离子交换色谱法完成的。这种化合物的特点和验证是纯紫外-可见光和红外光谱。荧光光谱法对化 HeLa 细胞线粒体钙摄取抑制特性进行了评价。

该方法描述了线粒体钙吸收抑制剂的合成 [(OH2) (nh3)4汝 (µ O) ru (nh3)4(OH2)] cl5从 [Ru (NH3)5cl] cl2开始, 一众所周知的钌 (III) 起动材料。[Ru (NH3)5Cl]Cl2的特征是红外光谱, 具有 3200 cm-1、1608 cm-1、1298 cm-1和 798 cm-1 (图 1) 的振动模式。小杂质在 2069 cm-1可以归因于 [Ru (NH3)5N2] Cl3。这 Ru (III) 种的反应与 1 M nh4OH 提供 [(oh2) (nh3)4ru (µ) 汝 (NH3)4(OH2)] Cl5。这一反应的进展证明了从黄色到绿色的溶液颜色的戏剧性变化。最后的反应溶液是深绿色的颜色。这种溶液的酸化与浓盐酸导致颜色改变为褐色;这种中和的副产品是氯化铵, 如果不进行护理, 可能污染最终产品。用强酸性网格 (H+型) 树脂对化合物进行阳离子交换色谱纯化。该树脂是平衡的第一个与 0.10 M HCl, 在该列上加载了 [(oh2) (nh3)4µ (nh3)4(哦2)] Cl5的解决方案。氯化铵副产品 elutes 与 1 M HCl 洗涤。含钌化合物在 HCl 浓度较高时洗。一系列黄色分数, 其中包含未 [Ru (NH3)5cl] cl2起始材料, 当 HCl 浓度为 1.5 m 时, 从列中脱落。所需的产品 elutes 在 2.5-3.0 M HCl 之间, 所产生的分数在颜色上呈绿色, 呈绿褐色。
在汇集产品的组分之前, 他们的纯净应该被核实。由于所需的化合物在其紫外可见光谱中表现出一定程度的 pH 依赖性, 我们建议在高碱性 3 M NH3溶液中加入小等分, 以确保所有分数的光谱特征都是相同的。纯分数只应显示在 360 nm 的强烈峰值和 600 nm 的弱峰值 (图 2)。峰顶在480和533毫微米附近表明钌褐色和红色的存在分别, 和一个重要峰顶在260毫微米, 以肩膀在290毫微米, 意味存在 [Ru (NH3)5cl] cl2起始材料 (图3).旋转蒸发的纯馏分提供所需的化合物作为一个绿色棕色固体。
该分离化合物可以进一步具有紫外可见和红外光谱的特点。紫外可见光谱 (图 2) 显示了上面所述的 360 nm 和 600 nm 吸收带。600 nm 波段的消光系数为850米-1cm-1 , 360 nm 带是 27000 m-1cm-1。与 600 nm 波段相比, 360 nm 波段的强度的比值应为 31, 在 pH 7.4 PBS 中, 这一指标可以有效地用来测量化合物的纯度。IR 频谱应显示在图 4中。例如, ru 的拉伸频率为850厘米-1的诊断。采用红外光谱法测定钌-钌拉伸, 确保最终产物中不存在氯化铵。氯化铵, 一种常见的杂质, 有振动模式在 1762 cm-1 (非常微弱) 和 1400 cm-1 (强), 可以很容易地从红外光谱 (图 5) 中辨别出来。钌红色是反应的一个共同的副产品, 可以在红外光谱中识别, 其延伸的范围为1404厘米-1、1300 cm-1、1037 cm-1和 800 cm-1, 但会出现与所需产品的某些重叠 (图 6)。
观察 digitonin-化 HeLa 细胞的钙吸收反应 (图 7)。星号表示添加了 CaCl2丸。在 [(哦2) (nh3)4ru (µ O) 钌 (nh3)4(oh2)) 中, Cl5在添加钙时观察到发射强度的增加, 但由于线粒体钙没有衰变摄取被观察。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56527/56527fig1.jpg"> 
图 1: [Ru (NH3)5cl] cl2的红外光谱[ru (nh3)5cl] cl2与非常小的 [ru (nh3)5N2] cl3杂质的红外光谱。红色箭头指示在2069厘米-1处的杂质。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56527/56527fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56527/56527fig2.jpg"> 
图 2: 具有代表性的纯材料紫外可见光谱.[(oh2) (nh3)4汝 (µ) 汝 (nh3)4(oh2)Cl5在 pH 值 7.4 PBS 中采取的紫外可见吸收光谱和近红外线 (内陷)。360 nm 的主要吸光度的消光系数为2.7万米-1 cm-1。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56527/56527fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56527/56527fig3.jpg"> 
图 3: 粗反应混合物的紫外可见光谱.[(oh2) (nh3)4汝 (µ) 汝 (nh3)4(oh2)Cl5在 pH 值 7.4 PBS 中提纯之前, 对原油反应混合物的紫外-可见光吸收光谱。红色箭头表示常见杂质。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56527/56527fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56527/56527fig4.jpg"> 
图 4: 具有代表性的纯材料红外光谱.红外光谱 [(oh2) (nh3)4汝 (µ O) ru (nh3)4(哦2)] Cl5。ru 为850厘米-1, 其他拉伸来自 NH3振荡器。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56527/56527fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56527/56527fig5.jpg" alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56527/56527fig5.jpg"></img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56527/56527fig5.jpg">/> 
图 5: 含 NH 的红外光谱4Cl 杂质.红外光谱的 [(oh2) (nh3)4汝 (µ O) ru (nh3)4(哦2)] Cl5与氯化铵杂质。红色箭头指示氯化铵在 1400 cm-1。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56527/56527fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56527/56527fig6.jpg"> 
图 6: 商用钌红的红外光谱.红外光谱 [(oh2) (nh3)4汝 (µ) ru (新罕布什尔州3)4(oh2)] Cl5 (黑色痕迹) 和商业钌红色 (红色痕迹)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56527/56527fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56527/56527fig7.jpg"> 
图 7: 具有代表性的钙吸收结果.由于 digitonin 化 HeLa 细胞中添加钙而引起的荧光增加, Green-5N 钙和 [(哦2) (nh3)4ru (µ) 汝 (nh3)4(oh2)] Cl5在 BGSS。空白包含没有 [(哦2) (nh3)4ru (µ O) 汝 (nh3)4(OH2)] Cl5和由于 Ca2 +吸收到线粒体中的荧光减少, 可见。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56527/56527fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor at JoVE.com

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

钌线粒体钙吸收抑制剂的合成与评价

提出了一种用于线粒体钙吸收的钌抑制剂的合成、纯化和表征的协议。本文介绍了一种评价其在化哺乳动物细胞中的有效性的方法。

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of ...

synthesis-evaluation-ruthenium-based-mitochondrial-calcium-uptake

Nanyang Technological University

Research

JoVE Journal

Chemistry

7.8K 次观看.  Cornell University. 提出了一种用于线粒体钙吸收的钌抑制剂的合成、纯化和表征的协议。本文介绍了一种评价其在化哺乳动物细胞中的有效性的方法。

视频: Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

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

Although initially considered an esoteric reagent, SmI2 has become a common tool for synthetic organic chemists. SmI2 is generated through the addition of molecular iodine to samarium metal in THF.1,2-3 It is a mild and selective single electron reductant and its versatility is a result of its ability to initiate a wide range of reductions including C-C bond-forming and cascade or sequential reactions. SmI2 can reduce a variety of functional groups including sulfoxides and sulfones, phosphine oxides, epoxides, alkyl and aryl halides, carbonyls, and conjugated double bonds.2-12 One of the fascinating features of SmI-2-mediated reactions is the ability to manipulate the outcome of reactions through the selective use of cosolvents or additives. In most instances, additives are essential in controlling the rate of reduction and the chemo- or stereoselectivity of reactions.13-14 Additives commonly utilized to fine tune the reactivity of SmI2 can be classified into three major groups: (1) Lewis bases (HMPA, other electron-donor ligands, chelating ethers, etc.), (2) proton sources (alcohols, water etc.), and (3) inorganic additives (Ni(acac)2, FeCl3, etc).3
Understanding the mechanism of SmI2 reactions and the role of the additives enables utilization of the full potential of the reagent in organic synthesis. The Sm-Barbier reaction is chosen to illustrate the synthetic importance and mechanistic role of two common additives: HMPA and Ni(II) in this reaction. The Sm-Barbier reaction is similar to the traditional Grignard reaction with the only difference being that the alkyl halide, carbonyl, and Sm reductant are mixed simultaneously in one pot.1,15 Examples of Sm-mediated Barbier reactions with a range of coupling partners have been reported,1,3,7,10,12 and have been utilized in key steps of the synthesis of large natural products.16,17 Previous studies on the effect of additives on SmI2 reactions have shown that HMPA enhances the reduction potential of SmI2 by coordinating to the samarium metal center, producing a more powerful,13-14,18 sterically encumbered reductant19-21 and in some cases playing an integral role in post electron-transfer steps facilitating subsequent bond-forming events.22 In the Sm-Barbier reaction, HMPA has been shown to additionally activate the alkyl halide by forming a complex in a pre-equilibrium step.23
Ni(II) salts are a catalytic additive used frequently in Sm-mediated transformations.24-27 Though critical for success, the mechanistic role of Ni(II) was not known in these reactions. Recently it has been shown that SmI2 reduces Ni(II) to Ni(0), and the reaction is then carried out through organometallic Ni(0) chemistry.28
These mechanistic studies highlight that although the same Barbier product is obtained, the use of different additives in the SmI2 reaction drastically alters the mechanistic pathway of the reaction. The protocol for running these SmI2-initiated reactions is described.

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

A straightforward procedure for the preparation of samarium diiodide (SmI2) in THF is described. The role of two main additives namely hexamethylphosphoramide (HMPA) and Ni(acac)2 in Sm mediated reactions is demonstrated in the Sm-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

Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded synthesis strategy is used in which spherical seeds of CdSe are prepared first using a hot-injection technique. By controlling the crystal structure of the seed to be either wurtzite or zinc-blende, the subsequent hot-injection growth of CdS off of the seed results in either a rod-shaped or tetrapod-shaped nanocrystal, respectively. The phase and morphology of the synthesized nanocrystals are confirmed using X-ray diffraction and transmission electron microscopy, demonstrating that the nanocrystals are phase-pure and have a consistent morphology. The extinction coefficient and quantum yield of the synthesized nanocrystals are calculated using UV-Vis absorption spectroscopy and photoluminescence spectroscopy. The rods and tetrapods exhibit extinction coefficients and quantum yields that are higher than that of the bare seeds. This synthesis demonstrates the precise arrangement of materials that can be achieved at the nanoscale by using a seeded synthetic approach.

A protocol for the seeded synthesis of rod-shaped and tetrapod-shaped multicomponent nanostructures consisting of CdS and CdSe is presented.

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 ...

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

抗癌金属配合物的合成与细胞毒性评价MTT法

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 &#176;C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 &#176;C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.
Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.

A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.

合成和Iodoaziridines纯化涉及定量选择最优固定相的色谱

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. ...

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

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

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

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 ...

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

不同二硫 Connectivities µ-芋螺毒素 PIIIA 异构体的合成与结构测定

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

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 of Esters Via a Greener Steglich Esterification in Acetonitrile

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the reaction is commonly performed using chlorinated or amide solvent systems, which are hazardous to human health and the environment. Our methodology utilizes acetonitrile as a greener and less hazardous solvent system. This protocol exhibits rates and yields that are comparable to traditional solvent systems and employs an extraction and wash sequence that eliminates the need for the purification of the ester product via column chromatography. This general method can be used to couple a variety of carboxylic acids with 1&#176; and 2&#176; aliphatic alcohols, benzylic and allylic alcohols, and phenols to obtain pure esters in high yields. The goal of the protocol detailed here is to provide a greener alternative to a common esterification reaction, which could serve useful for ester synthesis in both academic and industrial applications.

A modified Steglich esterification reaction was used to synthesize a small library of ester derivatives with primary and secondary alcohols. The methodology uses a non-halogenated and greener solvent, acetonitrile, and enables product isolation in high yields without the need for chromatographic purification.

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 ...

Synthesis Method for Cellulose Nanofiber Biotemplated Palladium Composite Aerogels

Here, a method to synthesize cellulose nanofiber biotemplated palladium composite aerogels is presented. Noble metal aerogel synthesis methods often result in fragile aerogels with poor shape control. The use of carboxymethylated cellulose nanofibers (CNFs) to form a covalently bonded hydrogel allows for the reduction of metal ions such as palladium on the CNFs with control over both nanostructure and macroscopic aerogel monolith shape after supercritical drying. Crosslinking the carboxymethylated cellulose nanofibers is achieved using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in the presence of ethylenediamine. The CNF hydrogels maintain their shape throughout synthesis steps including covalent crosslinking, equilibration with precursor ions, metal reduction with high concentration reducing agent, rinsing in water, ethanol solvent exchange, and CO2 supercritical drying. Varying the precursor palladium ion concentration allows for control over the metal content in the final aerogel composite through a direct ion chemical reduction rather than relying on the relatively slow coalescence of pre-formed nanoparticles used in other sol-gel techniques. With diffusion as the basis to introduce and remove chemical species into and out of the hydrogel, this method is suitable for smaller bulk geometries and thin films. Characterization of the cellulose nanofiber-palladium composite aerogels with scanning electron microscopy, X-ray diffractometry, thermal gravimetric analysis, nitrogen gas adsorption, electrochemical impedance spectroscopy, and cyclic voltammetry indicates a high surface area, metallized palladium porous structure.

A synthesis method for cellulose nanofiber biotemplated palladium composite aerogels is presented.The resulting composite aerogel materials offer potential for catalysis, sensing, and hydrogen gas storage applications.

纤维素纳米纤维生物模板复合气凝胶的合成方法

Here, a method to synthesize cellulose nanofiber biotemplated palladium composite aerogels is presented. Noble metal aerogel synthesis methods often ...