KG是由RA韦尔奇基金会授予P-1760支持，数学和科学教育（以KG）的TCU安卓研究所，TCU研究和创新活动格兰特（以KG）和TCU电监会格兰特（以JHS）。

1.合成生物素的Fc半胱氨酸（1） <ol><li>固相方法，以产生树脂结合的1。 <ol><li>放置生物素加载树脂（250毫克，0.145毫摩尔）转化为烧结注射器和通过制定二甲基甲酰胺（5毫升）中，并摇动在实验室摇动器在注射器20分钟使树脂溶胀。排出溶液和二甲基甲酰胺重复肿胀一次。 </li><li>通过加入4-6毫升20％哌啶的二甲基甲酰胺在注射器随后振荡10-15分钟，除去Fmoc保护基。与另一4-6毫升哌啶，重复脱保护过程。甲醇（1：1），3倍的甲醇：二氯甲烷（1：1）的序列的3倍二甲基甲酰胺，3倍二甲基甲酰胺洗涤树脂，3×二氯甲烷，约5 ml的每种。做的小珠的一小部分（〜10）茚三酮试验（+），以确认成功地由脱保护的蓝色的存在下在加热时。 </li><li>混合含有1&#39;-Fmoc-氨基二茂铁-1-羧酸溶液（203.3毫克，0.4350毫摩尔），1-羟基苯并三唑水合物（58.8毫克，0.413毫摩尔），二异丙基碳二亚胺（0.0673毫升，0.435毫摩尔），二异丙基乙基胺（0.0757毫升，0.435毫摩尔），以及一个4：二氯甲烷1:1混合物和二甲基甲酰胺。得出这样的成烧结注射器并轻轻摇动在实验室摇动器6小时。然后排出从注射器的溶液和洗涤如前所述。 </li><li>进行茚三酮试验 - 如上所述，确认耦合（）。茚三酮测试仍然可以在尽管从含有部分铁的附着得到的珠粒的橙色确认耦合有用。 </li><li>然后通过加入二甲基甲酰胺20％哌啶的除去Fmoc基团，并如上所述洗涤。茚三酮试验（+）应当用于确认去除Fmoc。 </li><li>制备的Fmoc-半胱氨酸（TRT）组成的溶液-OH（254.8毫克，0.4350毫摩尔），1-羟基苯并三唑水合物（58.8毫克，0.4125毫摩尔），二异丙基碳二亚胺（0.0673毫升，0.4350毫摩尔），二异丙基乙基胺（0.0757毫升，0.4350毫摩尔），并在4：二氯甲烷和二甲基甲酰胺的1:1混合物。加入该半胱氨酸耦合鸡尾酒烧结注射器，轻轻摇动6小时。洗，用前述的协议。 </li><li>确认使用茚三酮试验耦合（ - ），然后除去，用20％哌啶和洗涤将Fmoc组分。验证的游离末端胺使用茚三酮试验（+）。 </li></ol></li><li>从树脂裂解1。 <ol><li>使TFA溶液（9.45毫升），水（0.25毫升），1,2-乙二硫醇（0.25毫升），三异丙基硅烷（0.1毫升）中，将其添加到注射器并轻轻摇晃4小时。 </li><li>收集在Eppendorf管中所产生的红棕色溶液，蒸发缓慢用空气流为TFA。 </li><li>添加冷二乙醚（约15毫升）至Eppendorf管中，以沉淀1，这将形成温和搅拌。隔离通过离心的产物（1克，5分钟）。牛逼的乙醚洗涤液（〜60毫升数量）和离心机母鸡重复周期，得到1，为红色/褐色固体。 </li></ol></li></ol> 2.表征和分析1 <ol><li>确认该身份用1 H（16次扫描）和13 C NMR（512次扫描）在氘化甲醇（300微升）和ESI-MS分析，在图2所示的连接和组成相匹配。 期望的结果如下： 1 H NMR谱（CD 3 OD）δ/ ppm的：1.407-1.684（米，6H），2.245（T，2H），2.665-3.150（米，12H），4.015（吨，1H），4.104（D，2H ），4.274（Q，1H），4.426（D，2H），4.479（Q，1H），4.595（吨，1H）和13 C NMR谱（CD 3 OD）δ/ ppm的：24.644（CH 2），25.472 （CH 2），28.051（CH 2），28.300（CH 2），35.474（CH 2），38.698（CH 2），39.241（CH 2），39.717（CH 2），55.340 / 55.538（CP-环）， 60.286（C1H），61.964（CH），62.521 / 62.821（CP-环），66.038 / 66.170（CP-环），69.153 / 69.328（CP-环），71.468 / 71.593（CP-环），76.466（CH），171.770 （C = O），175.361（C = O）。 ESI-MS（M / Z）：实测值：639.00 [1 + NA] +，理论值：639.1 [1 + NA] +和HR-MS（M / Z）：实测值：617.2049 [1 + H] +，理论值： 617.1622 [1 + H] +。 </li><li>执行高效液相色谱法，元素分析来确认的孤立1的组合物。 使用C8进行HPLC色谱反相柱用100％甲醇以0.5ml / min的流速。注意：HPLC保留时间分别为：3.198-4.674分钟。 </li></ol>在金表面3.固定的1 <ol><li>切聚合物2支撑的黄金滑入了〜0.25平方。 </li><li>填在50ml的烧杯中的1（〜1毫米）DI水的溶液。 </li><li>金滑动添加到烧杯中，并覆盖用表玻璃。所有流载玻片孵育O / N在室温无搅拌。 </li><li>从溶液中除去金滑动，并允许它在空气中干燥。 </li><li>获得扫 ​​描使用扫描型电子显微镜（或等同物）来观察固定1电子显微镜图像。 </li></ol>

The authors have nothing to disclose.

不对称二茂铁衍生物的合成是具有挑战性的溶液。例如，曾试图以产生1的溶液导致期望的产物（低于20％）的产量低。同样地，利用1&#39;-氨基二茂铁羧酸（没有的Fmoc）和树脂结合的生物素的反应导致与报道Baristic 等经聚合的产物一致不溶产物。和最小的产品。47这是通过二茂铁进一步复杂化和其衍生物是光敏感的，该氨基同源容易在溶液中二聚化。这些问题使?...

生物传感器是采用生物分子识别技术作为用于选择性分析平台和被用于它们的特异性，速度和低成本的小型设备。电化学生物传感器用于生物分子的检测是在这个领域的前沿，由于其简单性，成本效益和高灵敏度。这些传感器1,3的一般解剖是装备有识别分子的电极的特定的感兴趣的生物标志物。该生物标记物的由识别分子结合导致的电位或电流的局部变化可以通过简单的测量来检测。迄今为止，识别部分的范围可以从酶，抗体，4-8，9-12整个细胞，13-16受体，17-20肽21-23和DNA 24，并 ​​主要集中在大，生物分子25-28研究在这个舞台上的努力主要集中在免疫传感器磨片重的免疫球蛋白是固定有氧化还原活性核（如二茂铁）和用于检测的目标抗体。这些研究被排除在临床应用中，由于精度差，消耗的时间从使用抗原/抗体引起的并发症而产生1,3成长的注意力都集中在小分子检测的生物医学（小于1公斤/摩尔） ，食品和除了国家安全的环境利益。29生物传感器设备的最有名的例子是自测血糖监测仪，其具有耦合到一个口袋大小的安培计丝网印刷酶电极。这些系统通常利用在充电量通过葡萄糖氧化反应产生的总量的测定在一段时间的库仑法。适销对路的设备必须是便携，坚固耐用，手持尽量使用浅显的广大民众。 氧化还原标签，如二茂铁的necessaRY以提供电化学检测生物标志物或小分子的溶液中，因为大多数的生物标志物是无内在电化学活性。30-38二茂是有机金属分子，是金标准电化学，这使得它对于整合到电化学生物传感器的极佳选择。二茂铁基的氧化还原活性种已经获取了相当大的注意，由于它们的尺寸小，稳定性好，方便合成存取，易于化学修饰，相对亲脂性，并且易于氧化还原调谐的。3,30-42小分子基于所述二茂铁芯具有被广泛地用作金属离子和小分子的检测。32-38,43系统靶向较大的品种，如生物分子已经利用大的抗体或免疫球蛋白，以已被嵌入到一个电化学表面二茂铁衍生物的附件。1,3,39 ，44在每一种情况下，该电势和电流的Intensi的铁三 / 二铁氧化还原对TY被改变时分子偶联，从而产生一个新的分光手柄，指示所述分析物分子的存在。这种变化源于该环戊二烯基环的pI系统和铁d轨道之间产生的大量重叠。如果PI-系统被修改， 即衍生或反应，则该轨道相互作用将反过来，变化。这将影响到铁芯，并且可以观察到在的铁III /铁二夫妇的电位的移位。40,45,46这些性质使得用作定量剂在电化学免疫测定或生物传感器这种系统的吸引力。 为了产生特异于生物传感器的能力含二茂铁的系统是最佳​​的修改一个Cp环与生物受体特异于靶分子，并利用其它的Cp环作为分子系绳到电化学读数或ELEC处接上（ 图1）。这些不对称二茂铁衍生物的合成是由副反应并在分子间交联形成的二聚和聚合物质的形成了挑战。47然而，偶联化学制备的酰胺键是最直接的途径，以提供二茂铁的涉及生物组分，例如简单的衍生物肽和它们的代谢物。因此，首先在20世纪50年代开发的Merrifield用于肽合成的固相技术可以应用到含二茂铁的有机金属化合物。通过使用正交取代的1&#39;-Fmoc-氨基二茂铁-1-羧酸分子，二茂铁系统，它可以包含一个受体部分（生物素），电化学读数（二茂铁），并固定化 - 连接体组分（半胱氨酸）的具有已建成和在此详述。此生物缀合物的合成进行了讨论，以及证据固定在金表面上。这项工作represenTS生物素，二茂铁和氨基酸用于固定在金表面的组成的系统的第一表示。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Biotin Novatag Resin</td>
			<td>NovaBiochem</td>
			<td>8550510001</td>
			<td></td>
		</tr>
		<tr>
			<td>TORVIQ 10ML LUER LOCK FRITTED SYRINGE</td>
			<td>Fisher</td>
			<td>NC9299151</td>
			<td></td>
		</tr>
		<tr>
			<td>piperdine</td>
			<td>Acros</td>
			<td>P/3520/PB05</td>
			<td></td>
		</tr>
		<tr>
			<td>ninhydrin test</td>
			<td>Sigma-Aldrich</td>
			<td>60017-1ea</td>
			<td></td>
		</tr>
		<tr>
			<td>1&#8217;-Fmoc-amino-ferrocene-1-carboxylic acid</td>
			<td>Omm Scientific</td>
			<td>Special Order</td>
			<td></td>
		</tr>
		<tr>
			<td>1-&#8203;Hydroxybenzotriazole hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>157260-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#8242;-Diisopropylcarbodiimide</td>
			<td>Sigma-Aldrich</td>
			<td>D125407-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-&#8203;Diisopropylethylamine</td>
			<td>Sigma-Aldrich</td>
			<td>496219-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Cys(Trt)-OH</td>
			<td>Novabiochem</td>
			<td>8520080025</td>
			<td></td>
		</tr>
		<tr>
			<td>trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T5408</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-ethanedithiol</td>
			<td>Sigma-Aldrich</td>
			<td>2930</td>
			<td></td>
		</tr>
		<tr>
			<td>triisopropyl silane</td>
			<td>Sigma-Aldrich</td>
			<td>233781</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes (20 mL)</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>any source</td>
			<td></td>
			<td>dry with molecular sieves prior to use &#38; store in 100 mL media bottle for easy usage.</td>
		</tr>
		<tr>
			<td>dichloromethane</td>
			<td>any source</td>
			<td></td>
			<td>dry with molecular sieves prior to use &#38; store in 100 mL media bottle for easy usage.</td>
		</tr>
		<tr>
			<td>dimethylformamide</td>
			<td>any source</td>
			<td></td>
			<td>dry with molecular sieves prior to use &#38; store in 100 mL media bottle for easy usage.</td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

synthetic methodology for asymmetric ferrocene derived bio-conjugate systems via solid phase resin-based methodology

早期检测是治疗成功的关键大多数疾病的，并且是许多类型的癌症的诊断和治疗尤其迫切。所使用的最常见的技术是成像方式如磁共振成像（MRI），正电子发射断层（PET）和计算机辅助断层扫描（CT），并且最佳为了解该疾病的物理结构，但只能每四到执行一次由于使用的成像剂和总成本的六个星期。考虑到这一点，对“点的关怀”技术，如生物传感器，其评估疾病和/或治疗在临床医生的办公室疗效的阶段，做到及时，将彻底改变治疗方案的发展。1作为一种手段来探索基于二茂铁的生物传感器用于生物相关分子2的检测，方法被开发以产生本文所述的二茂铁-生物素的生物共轭物。本报告将重点放在可在金表面被固定生物素二茂铁半胱氨酸系统。

1，树脂结合形式示于图2。二茂铁组分的共价连接产生了一种橙色色调的树脂珠粒是持久连续洗涤和表示含有复杂的固定化铁如由相对于铁的吸收树脂珠粒的PEG部分。的1对无树脂形式是彩色的，以使树脂珠粒相同。以下除去从树脂珠的化合物，纯度和产率（68％），从所述方法得到的是远远优于典型的解决方案的方法。产品的元素分析表明，1被?...

Watch this Scientific Journal Video about Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology at JoVE.com

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

合成方法通过固相树脂为基础的方法不对称二茂铁产生的生物共轭系统

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

synthetic-methodology-for-asymmetric-ferrocene-derived-bio-conjugate

Research

JoVE Journal

Chemistry

9.6K 次观看.  Texas Christian University. The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

视频: Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. Specifically, the sapphire cell enables visual data collection from multiple loadings to solve a set of material balances to precisely determine phase composition. Ternary phase diagrams can then be established to determine the proportion of each component in each phase at a given condition. In principle, any ternary system can be studied although ternary systems (gas-liquid-liquid) are the specific examples discussed herein. For instance, the ternary THF-Water-CO2 system was studied at 25 and 40&#160;&#176;C and is described herein. Of key importance, this technique does not require sampling. Circumventing the possible disturbance of the system equilibrium upon sampling, inherent measurement errors, and technical difficulties of physically sampling under pressure is a significant benefit of this technique. Perhaps as important, the sapphire cell also enables the direct visual observation of the phase behavior. In fact, as the CO2 pressure is increased, the homogeneous THF-Water solution phase splits at about 2 MPa. With this technique, it was possible to easily and clearly observe the cloud point and determine the composition of the newly formed phases as a function of pressure.
The data acquired with the sapphire cell technique can be used for many applications. In our case, we measured swelling and composition for tunable solvents, like gas-expanded liquids, gas-expanded ionic liquids and Organic Aqueous Tunable Systems (OATS)1-4. For the latest system, OATS, the high-pressure sapphire cell enabled the study of (1) phase behavior as a function of pressure and temperature, (2) composition of each phase (gas-liquid-liquid) as a function of pressure and temperature and (3) catalyst partitioning in the two liquid phases as a function of pressure and composition. Finally, the sapphire cell is an especially effective tool to gather accurate and reproducible measurements in a timely fashion.

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

The high pressure sapphire cell apparatus is a unique tool to study, without sampling, phase behavior under a wide range of pressures. Using a cathetometer, very precise volume measurements can be recorded to measure liquid expansion and phase composition. Thus, this synthetic method enables the study of (1) phase equilibria of multi-component mixtures and (2) the partition behavior of catalyst or model compounds as a function of pressure.

高压蓝宝石细胞对CO的相平衡测量<sub&gt; 2</sub&gt; /有机/水系统

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. ...

科研

化学

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.

稳定肝细胞表型采用优化合成表面

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing ...

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

X射线粉末衍射在保护科学：迈向文物艺术品腐蚀产物的常规晶体结构测定

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

通过动态光散射显微镜混浊解决方案粒度分布的测定

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

微型固相萃取和LC-MS / MS检测人尿中3-硝基酪氨酸的临床应用

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the average content of fluorinated drugs in their formulated drug product forms: tablets or capsules. This method is specific to fluorinated drugs because it detects only the content of fluorine, avoiding interference from the excipients that lack fluorine. The advantages of measuring the active content of fluorinated drugs using low-field 19F TD-NMR versus high-field 19F solid-state (SS) NMR are the simplicity of the method; the low cost; and the non-destructive nature of the technique, with all samples recoverable in intact forms (e.g., powders, tablets, and capsules), making this technique affordable for any laboratory.
We have tested the method with three fluorinated drug products available on the market - cinacalcet, lansoprazole, and ciprofloxacin - with doses ranging from 15 to 500 mg. The results of the analyses, measured by low-field 19F TD-NMR, supported the reported label claims for the average drug content.
Based on the simplicity and reproducibility of the analysis, we envision this methodology being implemented in any laboratory, including manufacturing plants, as a process analytical technology (PAT) tool in the pharmaceutical industry.

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

A simple and non-destructive technique that measures the average content of drug substances in formulated drug products containing fluorine using low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) is presented here. The technique can be applied to the development and manufacturing of drugs in the pharmaceutical industry.

应用程序和非破坏性19F 时间域核磁共振技术的方法来衡量中含氟药物产品的内容

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the ...

Phase Behavior of Charged Vesicles Under Symmetric and Asymmetric Solution Conditions Monitored with Fluorescence Microscopy

Phase-separated giant unilamellar vesicles (GUVs) exhibiting coexisting liquid-ordered and liquid-disordered domains are a common biophysical tool to investigate the lipid raft hypothesis. Numerous studies, however, neglect the impact of physiological solution conditions. On that account, the current work presents the effect of high-salinity buffer and trans-membrane solution asymmetry on liquid-liquid phase separation in charged GUVs grown from dioleylphosphatidylglycerol, egg sphingomyelin, and cholesterol. The effects were studied under isothermal and varying temperature conditions.
We describe equipment and experimental strategies applicable for monitoring the stability of coexisting liquid domains in charged vesicles under symmetric and asymmetric high-salinity solution conditions. This includes an approach to prepare charged multicomponent GUVs in high-salinity buffer at high temperatures. The protocol entails the option to perform a partial exchange of the external solution by a simple dilution step while minimizing the vesicle dilution. An alternative approach is presented utilizing a microfluidic device that allows for a complete external solution exchange. The solution effects on phase separation were also studied under varying temperatures. To this end, we present the basic design and utility of an in-house built temperature control chamber. Furthermore, we reflect on the assessment of the GUV phase state, pitfalls associated with it and how to circumvent them.

Experiments on phase separated giant unilamellar vesicles (GUVs) frequently neglect physiological solution conditions. This work presents approaches to study the effect of high-salinity buffer on liquid-liquid phase separation in charged multicomponent GUVs as a function of trans-membrane solution asymmetry and temperature.

用荧光显微镜监测对称和非对称溶液条件下带电泡的相行为

Phase-separated giant unilamellar vesicles (GUVs) exhibiting coexisting liquid-ordered and liquid-disordered domains are a common biophysical tool to ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

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 methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2&#039;-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

用于固相合成含有2&#39;-<em&gt; 0</em通过圆二色性修饰和表征

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 ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

基质辅助激光解吸电离飞行时间 (MALDI) 质谱法表征合成聚合物

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

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 ...