该专用肥产品公司（SFP）是公认的提供必要的资金用于这项研究。

1。样品的准备<ol><li>制备2毫升酶溶液和0.5ml的每个实验运行的底物溶液。在具有相同的组合物到基片加入过程中尽量减少稀释和混合的热缓冲液稀释浓缩的储备溶液酶和底物。 <ol><li>选择的缓冲液条件是足够的，以防止试验过程中pH值的变化。例如，20mM的HEPES pH值为7是足够的测量值在中性pH值。 注意：如果涉及质子交换，所使用的缓冲液的质子化焓必须考虑的，因为它们会影响该反应的测量ΔH。根据分析系统上的缓冲器或添加剂分子的可能的具体效果，必须考虑到。如果有机溶剂（ 如 DMSO）中包含的酶溶液，在完全相同的浓度添加到他们的底物溶液。 </li><li>对于M1实验，开始与酶浓度在nM范围（ 例如 1 nm）和具有底物浓度的数量级至少三个数量比酶浓度和上面的K M为高。 </li><li>对于M2的实验，开始在PM-nM范围酶浓度（ 如 15分）。在注射器中底物浓度的MM范围（ 如 400毫米）。 注：在单次注射M1实验，浓度应足以达到总底物转化成产物在实验时间。因此，酶的浓度取决于酶速率：如果正在研究中的酶具有低介电常数猫 ，更高的酶浓度（高达10μM）必须被使用。另一方面，在M2实验中所用的酶浓度必须保证注入的基片是仅轻微（少于5％）消耗，并且这种酶反应在稳定状态。出于这个原因，较高的酶效率，所需的酶浓度越低。在实验结束时，在样品池的底物浓度应比K M高。 </li></ol></li><li>仔细检查酶和底物浓度与适当的分析程序（ 如 280nm处的吸光度，比色，BCA检测11，这是需要获得热力学和动力学参数的准确和可靠的计算。 </li><li>测量溶液的pH值，并验证该实验条件（±0.05个pH单位）在这两个酶和底物溶液的pH值不匹配是最小的。 </li></ol> 2。进行实验注意：相同的过程必须既为M1和M2的实验中，其中依次执行的其他应用。 <ol><li>验证样品池和进样注射器，根据制造商的被清洗说明。填装有蒸馏水的仪器装载注射器，轻轻地插入在采样单元的针，填充单元以及使用该注射器除去液体。使用这种方法，两次，用蒸馏水与缓冲洗样品池和的两倍。 </li><li>加载样本细胞用2毫升酶溶液使用装载注射器，小心地避免形成气泡。慢慢地将溶液注入到电池直到它泄漏出来的干细胞的顶部。产生ca的冲刺0.25毫升溶液进入细胞。重复两次。这个步骤除去截留在样品池气泡。 </li><li>放置在装载注射器的针上的细胞干细胞和细胞口之间的壁架和除去任何过量的溶液。 </li><li>启动VP-Viewer程序，并从计算机接口，平衡国际贸易委员会对仪器的温度3°C以下所需的实验温度。这是必要的，以避免长期平衡期间在实验前，由于被动器乐的冷却装置。 </li><li>填充基准单元用蒸馏水与上述相同的过程。当缓冲器具有高离子强度或高重量克分子渗透压浓度是在样品溶液中，在相同的缓冲液应该被用来作为一个参比溶液。 </li><li>链接的塑料注射器注入注射器的填充口，用一个薄的硅管。填充注射器将针尖入水中并制订，直到水从顶部填充端口，表明注射器已满。然后从水中将注射器针尖和制订空气，排空注射器。与此过程中，用缓冲液冲洗注射器，并通过该系统吸入空气。随后，将注射针在含有0.5毫升底物溶液的窄管，小心规划并完全填充注射器，留下少量的溶液在试管的底部。 </li><li>从电脑界面，按“关闭加注口”。除去硅装弹管。按“清除和重新填充”按钮，以允许注射器赶走气泡，并驱逐他们回到本体溶液。重复两次。 </li><li>移动注射器，擦拭在两侧，以消除任何液滴，然后将注射器的针头插入样本单元。 </li><li>在计算机上，设置相应的运行参数。实验酶和底物的浓度可以被显示出来。 <ol><li>在M1的实验中，设置至少两个加法衬底的（5-30微升），以验证重复性。设置每个喷射之间的间隔时间（ 例如 1,000秒）足够大，以确保接下来的加成前的热信号返回到基线。 </li><li>在M2的实验中，设置多个（ 如 15×5-10微升）注射。设置注射（ 例如 180秒）使系统秒之间的时间间隔tabilize热功率为每次注射后的新基准。 注意：在M2实验注射之间的间隔时间应足够短，以避免一个显著量底物的转化，使稳定状​​态的条件下进行测量。 </li><li>在M2的实验中，对在第一次注射，其对应值的随后的数据分析过程中被丢弃使用少量（ 例如 2微升）。事实上，它往往呈现通过注射器尖端由于初始衬底扩散的工件，并用于捕获在注射器针的气泡的存在。 </li><li>设定的基准功率，近似水平，其中基线将被放置在反应开始前，在20的值。然后定义实验的酶和底物浓度，并选择一个名称实验。 </li><li>确定实验温度，通常在25℃下ITC允许2之间的工作温度<html> ℃和80℃。该实验是准备好运行。按“开始”按钮，开始实验。 </li></ol></li><li>一旦实验结束后，根据制造商的说明清洁样品细胞和注射器。 </li><li>重复实验至少一个或两个以上的时间来检查该数据的再现性。 </li></ol> 3。数据分析<ol><li>从分析程序，点击“读数据”按钮，然后导航到M2进行实验的。ITC的文件所在的文件夹。点击“文件类型”的滚动下拉箭头，选择“酶试验（它？）”。随后，点击并打开M2实验。ITC文件。 </li><li>获得从根据等式5整合M1实验的曲线反应的ΔH。 “宽度=”126“/&gt;（5） <ol><li>点击“读数据”按钮，并选择该文件。ITC的货币供应量M1进行实验。使用地，划分轨迹包含每一个峰值不同部分，每个峰值保存为。OPJ文件。打开对应于第一峰的文件，从在单次注射M1实验，结合它并划分所得到的面积值的温谱图基线偏差产生的，表示在μcal，通过在样品池的最终底物浓度，单位为μM，以及由细胞体积表示升，确定，根据方程（5），该反应的ΔH。 </li><li>重复同样的步骤对货币供应量M1实验的第二个高峰，并获得两个ΔH测量的平均值。 </li></ol></li><li>从M2的实验测量的原始基准和新的基线每次注射之后的差额确定的dQ / dt的 。转换电子xperimental数据到反应速率根据公式4，使用在M1实验中得到的焓值和拟合数据的米氏方程。 <ol><li>从分析程序，点击“读数据”按钮，并选择该文件。ITC对M2进行实验。点击“文件类型”的滚动下拉箭头，选择“酶试验（它？）”。 </li><li>酶测定对话框打开，允许选择四种模式之一。从窗口 - 选择“只基板方法2”。 </li><li>在ΔH窗口，表示ΔH的在步骤4.2中得到的值。 </li><li>点击“浓度”按钮，以检查浓度值的拟合程序中被使用。点击“平均时间（P）”按钮。该对话框提供了机会，改变或接受默认值。此值表示每个注射前的时间离子，其中，仪表平均值的功率信号，以确定在每个底物浓度的功率电平。单击确定以确认默认值。 </li><li>点击“零Y轴”按钮。光标变成一个十字，允许双击一个点，放置在y = 0。双击刚刚第一次注射点之前。 </li><li>点击计算费用按钮。率作图的底物浓度，除以滴定样品池体积（考虑到稀释效应）中加入底物而得。此操作提供了可安装以获得K M和 k 猫一个典型的米氏情节。 </li><li>如果某点应予以删除，选择“截断数据”按钮。移动数据标记来排除坏的数据点和双击标记或按回车键之一。 </li><li>使用“调整到模拟”功能，以适应曲线，并获得的动力学常数。 </li></ol></li></ol>

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作者什么都没有透露。

ITC的意义，研究酶的活性就现有的方法 除了其经典应用研究的结合平衡，等温滴定量热法提供了可靠的和快速的方法，使用的反应热作为探针来表征溶液中的​​酶反应，而不需要对系统的修改或标记。动力学参数K 猫和K M分别通过一组时间过程实验，其中的产物的形成（或底物消耗）在连续或不连续测定法进行监测，通常获得的?...

定量测定生化反应提供了深入了解生物过程在生命的基础。量热法提供了一种无标记的方法来定量地表征溶液中的​​几乎所有的化学反应。这种技术测量热释放或吸收随着时间的推移，因此是一种普遍的检测系统和一个非常方便的方法来量化反应的分子（ 即结合热力学），以及测量的反应速率（ 即动力学）的量。特别是，等温滴定量热法（ITC）已被采纳为首选方法来表征生物分子平衡的热力学，涉及蛋白质-配体，蛋白质-蛋白质，蛋白质-金属离子与蛋白-DNA相互作用1-6。此外，国际贸易中心提供动力学信息的能力，使其成为一个非常强大的系统来衡量酶的催化作用，虽然这个应用程序的潜力仍然低估了7-9。 米氏方程10是酶反应的定量描述，因为它提供了反应速率和底物浓度之间的关系，这取决于2动力学参数：米氏常数（K M）和催化速率常数（k 猫 ） 。 第 k 猫 / K M比值被称为一种酶的催化效率。在实践中，对于特定的反应测定K M和 k 猫提供催化的完整描述。 在一个典型的酶反应（ 图1），在衬底（S）相互作用的酶（E）形成的酶-底物（ES）的配合物，其随后被激活进入过渡状态（ES *）。后者被转化成酶产品（EP）的复杂的最终解离。这些步S是由以下的反应说明。 <img fo:content-width="2in" src="/files/ftp_upload/51487/51487eq1.jpg" width="197" /> （1） 其中，k 1是速率常数为ES复合物中，k -1的形成是速率常数为ES复合物的解离，而K是猫的催化速率常数或周转数。 根据米氏方程10中 ，该反应的速率可以计算为： <img fo:content-width="1.5in" src="/files/ftp_upload/51487/51487eq2.jpg" width="144" /> （2） 其中K M =（K-1 + K 猫 ）/ k1 和 k2 的猫 = V 最大 / [E]， 用 v max均当所有的酶结合到基体达到的最大速度。 等温滴定热量计是在本研究中用于表征尿素的酶水解仪器。该仪器由含有2杜撰形细胞（ 图1）的绝热屏蔽的。这些被连接到外部以窄接入管中。样品池（ 约 1.4ml）中装入与酶溶液，同时基准单元通常被充满水或与用于分析的溶剂。一种旋转式注射器用长针和相连的搅拌桨，通常含有约 0.3ml的底物溶液，装在样品池。一种热电装置测量温度的样品和参考单元之间的差异，采用了“小区反馈网络”，它通过增加或减少热保持这种差异为零。在实验过程中，衬底被注入到在一个恒定的温度选择的酶溶液。当ENzymatic发生反应，热释放或吸收的量是成比例的转换成产物的分子底物分子的数量。另外，热气流的速度直接相关，该反应的速率。测得的数据，表现为发热微量的（ 图1）的偏差从初始基线，表示热功率（μcal/秒）供给由仪器的样品池，其正比于所述试样盒中发生的热流量随着时间的推移。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51487/51487fig1highres.jpg" width="500" /> 图1。的等温滴定量热仪来研究酶反应的示意图。发生后的衬底（在注射器）的滴定到酶溶液（在样品池）的结果在该疗法的一个变化的酶促反应由量热仪，需要保持样品池和参比电极常数之间的温度差正常释放动力。 <a href="https://www.jove.com/files/ftp_upload/51487/51487fig1highres.jpg" target="_blank">点击这里查看大图。</a> 总体上，热变化（Q）是正比于反应（ΔH）和生成（n）的，而这又是通过总体积时代赋予的浓度产物的摩尔数的摩尔焓： <img fo:content-width="2in" src="/files/ftp_upload/51487/51487eq3.jpg" width="200" /> （3） 该产物的形成随时间（DP / dt）的，对应于反应速率，因此，可以与以上通过关系在同一时间（的dQ / dt）的产生的热量： <img fo:content-width="2in" src="/files/ftp_upload/51487/51487eq4.jpg" width="150" /> （4） 根据这个公式，以便获得一个米氏绘制它需要测量ⅰ）的总摩尔焓ΔH，以及ii）的热流动的dQ / dt的在不同的底物浓度。通常，这是在两个不同的实验进行：在第一个实验中（ 方法1，M1），其中所述衬底中注入酶溶液，并加热为完整的底物转化的测量;在第二个实验（ 方法2，M2），底物的多次注射执行和热产生率的测量是在不同的底物浓度。这两组数据的数据足以计算得出的动力学参数K M和 k 的猫 。 在本文章中，一般的协议来确定动力学参数，使用国际贸易中心进行酶促反应描述。我们通过刀豆尿素施用的方法，以尿素水解本身作为参照系。使用这种方法得到的结果和文献报道的数据之间的吻合证明了这种方法的可靠性。

<table border="0" cellpadding="0" cellspacing="0" width="796">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">HEPES</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td style="width:323px;">dissolving in water and adjusting pH with NaOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">TRIZMA-Base</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">T1503</td>
			<td>dissolving in water and adjusting pH with HCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sodium dihydrogen phosphate</td>
			<td style="width:111px;">Riedel-de-Haen</td>
			<td style="width:119px;">4270</td>
			<td>dissolving in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sodium phosphate dibasic</td>
			<td style="width:111px;">Riedel-de-Haen</td>
			<td style="width:119px;">30427</td>
			<td>dissolving in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Urea</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">U4128</td>
			<td>dissolving in water at 40 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Canavalia ensiformis urease (type C-3)</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">U0251</td>
			<td>dissolving in 20 mM HEPES pH 7 and stored at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;"></td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">VP-ITC on Origin 7.0</td>
			<td style="width:111px;">MicroCal (GE Healthcare)</td>
			<td style="width:119px;">SYS13901</td>
			<td>instrument&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">VPViewer2000 1.30.00 on Origin 7.0</td>
			<td style="width:111px;">MicroCal (GE Healthcare)</td>
			<td style="width:119px;"></td>
			<td>data acquisition software supplied with the instrument</td>
		</tr>
	</tbody>
</table>

hot biological catalysis: isothermal titration calorimetry to characterize enzymatic reactions

等温滴定量热法（ITC）是一个良好描述的技术，其测量过程中的化学反应的热释放或吸收，用它作为一个内在探头几乎以表征每一个化学过程。如今，这种技术被广泛地应用，以确定生物分子的结合平衡的热力学参数。此外，ITC已经被证明是能够直接测量酶促反应的动力学和热力学参数（K 猫 ，K M，△H），即使该应用程序仍是开发不足的。作为热变化过程中酶催化自发地发生，ITC，不需要该系统的下分析的任何修改或标签，并且可以在溶液中进行。此外，该方法需要的材料少量。这些特性使得德祥一个非常宝贵的，强大的和独特的工具来研究酶动力学的几个应用，如，例如，药物发现。 K值猫和尿素由刀豆 （刀豆）脲酶酶水解的K M。进行反应的内在摩尔焓（ΔHint）的计算。由此得到的值与文献中报道的先前的数据相一致，这表明该方法的可靠性。

脲酶（EC 3.5.1.5;尿素水解酶）是古细菌，细菌，单细胞真核生物和植物中发现一个多亚基含镍酶。这种蛋白作用于有机氮矿化的最后步骤，催化尿素水解为氨和氨基甲酸盐，其自发地分解，得到氨和碳酸氢钠（ 公式6）12的第二分子。 <img fo:content-width="4in" src="/files/ftp_upload/51487/51487eq6.jpg" width="450" /> （6） 该反应导致的环境中，这是负?...

Watch this Scientific Journal Video about Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions at JoVE.com

Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

等温滴定量热法测量热流释放或吸收的化学反应。该方法可用于定量酶催化。在本文中，该协议有助于建立，运行试验和数据分析通常被描述，并应用于酶水解尿素由刀豆脲酶的特性。

热生物催化：等温滴定量热法表征酶促反应

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as ...

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20.4K 次观看.  University of Bologna. 等温滴定量热法测量热流释放或吸收的化学反应。该方法可用于定量酶催化。在本文中，该协议有助于建立，运行试验和数据分析通常被描述，并应用于酶水解尿素由刀豆脲酶的特性。 

视频: Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan &#948; solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials&#39; morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by St&#246;ber&#39;s methods, which have smooth surfaces.

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

科研

化学

Retropinacol/Cross-pinacol Coupling Reactions - A Catalytic Access to 1,2-Unsymmetrical Diols

Unsymmetrical 1,2-diols are hardly accessible by reductive pinacol coupling processes. A successful execution of such a transformation is bound to a clear recognition and strict differentiation of two similar carbonyl compounds (aldehydes &#8594; secondary 1,2-diols or ketones &#8594; tertiary 1,2-diols). This fine-tuning is still a challenge and an unsolved problem for an organic chemist. There exist several reports on successful execution of this transformation but they cannot be generalized. Herein we describe a catalytic direct pinacol coupling process which proceeds via a retropinacol/cross-pinacol coupling sequence. Thus, unsymmetrical substituted 1,2-diols can be accessed with almost quantitative yields by means of an&#160;operationally simple performance under very mild conditions. Artificial techniques, such as syringe-pump techniques or delayed additions of reactants are not necessary. The procedure we describe provides a very rapid access to cross-pinacol products (1,2-diols, vicinal diols). A further extension of this new process, e.g. an enantioselective performance could provide a very useful tool for the synthesis of unsymmetrical chiral 1,2-diols.

A novel account for the synthesis of unsymmetrical 1,2-diols based on a retropinacol/cross-pinacol coupling mechanism is described. Due to the catalytic execution of this reaction a considerable improvement compared to conventional cross-pinacol couplings is achieved.

Retropinacol /跨频哪醇偶联反应 - 催化访问1,2  - 非对称二醇

Unsymmetrical 1,2-diols are hardly accessible by reductive pinacol coupling processes. A successful execution of such a transformation is bound to a clear ...

Mizoroki-Heck Cross-coupling Reactions Catalyzed by Dichloro{bis[1,1',1''-(phosphinetriyl)tripiperidine]}palladium Under Mild Reaction Conditions

Dichloro-bis(aminophosphine) complexes of palladium with the general formula of [(P{(NC5H10)3-n(C6H11)n})2Pd(Cl)2] (where n = 0-2), belong to a new family of easy accessible, very cheap, and air stable, but highly active and universally applicable C-C cross-coupling catalysts with an excellent functional group tolerance. Dichloro{bis[1,1',1''-(phosphinetriyl)tripiperidine]}palladium [(P(NC5H10)3)2Pd(Cl)2] (1), the least stable complex within this series towards protons; e.g. in the form of water, allows an eased nanoparticle formation and hence, proved to be the most active Heck catalyst within this series at 100 &#176;C and is a very rare example of an effective and versatile catalyst system that efficiently operates under mild reaction conditions. Rapid and complete catalyst degradation under work-up conditions into phosphonates, piperidinium salts and other, palladium-containing decomposition products assure an easy separation of the coupling products from catalyst and ligands. The facile, cheap, and rapid synthesis of 1,1',1"-(phosphinetriyl)tripiperidine and 1 respectively, the simple and convenient use as well as its excellent catalytic performance in the Heck reaction at 100 &#176;C make 1 to one of the most attractive and greenest Heck catalysts available.
We provide here the visualized protocols for the ligand and catalyst syntheses as well as the reaction protocol for Heck reactions performed at 10 mmol scale at 100 &#176;C and show that this catalyst is suitable for its use in organic syntheses.

Mizoroki-Heck Cross-coupling Reactions Catalyzed by Dichloro{bis[1,1&#039;,1&#039;&#039;-phosphinetriyltripiperidine]}palladium Under Mild Reaction Conditions

Dichloro{bis[1,1',1''-(phosphinetriyl)tripiperidine]}palladium [(P(NC5H10)3)2Pd(Cl)2] (1) is an easy accessible, cheap, and air stable, but highly active Heck catalyst with an excellent functional group tolerance that efficiently operates under mild reaction conditions to give the coupling products in very high yields.

Mizoroki-哎呀交叉偶联反应由二氯{双[1,1&#39;，1&#39;&#39;  - （phosphinetriyl）tripiperidine]}钯催化在温和的反应条件

Dichloro-bis(aminophosphine) complexes of palladium with the general formula of [(P{(NC5H10)3-n(C6H11)n})2Pd(Cl)2] (where n = 0-2), belong to a new family ...

Light-driven Enzymatic Decarboxylation

Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. Recycling of the electron donors NADH or NADPH requires the use of additional enzymes and sacrificial substrates. Interestingly, several oxidoreductases accept hydrogen peroxide as electron donor. While being inexpensive, this reagent often reduces the stability of enzymes. A solution to this problem is the in situ generation of the cofactor. The continuous supply of the cofactor at low concentration drives the reaction without impairing enzyme stability. This paper demonstrates a method for the light-catalyzed in situ generation of hydrogen peroxide with the example of the heme-dependent fatty acid decarboxylase OleTJE. The fatty acid decarboxylase OleTJE was discovered due to its unique ability to produce long-chain 1-alkenes from fatty acids, a hitherto unknown enzymatic reaction. 1-alkenes are widely used additives for plasticizers and lubricants. OleTJE has been shown to accept electrons from hydrogen peroxide for the oxidative decarboxylation. While addition of hydrogen peroxide damages the enzyme and results in low yields, in situ generation of the cofactor circumvents this problem. The photobiocatalytic system shows clear advantages regarding enzyme activity and yield, resulting in a simple and efficient system for fatty acid decarboxylation.

We describe a protocol for the light-catalyzed generation of hydrogen peroxide &#8212; a cofactor for oxidative transformations.

光驱动脱羧酶

Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. ...

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

溶出动态核极化仪器用NMR实时酶反应速率测量

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of ...

A Protocol for Safe Lithiation Reactions Using Organolithium Reagents

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents warrants proper technique, thorough training, and proper personal protective equipment. To aid in the training of researchers using organolithium reagents, a thorough, step-by-step protocol for the safe and effective use of tert-butyllithium on an inert gas line or within a glovebox is described. As a model reaction, preparation of lithium tert-butyl amide by the reaction of tert-butyl amine with one equivalent of tert-butyl lithium is presented.

The safe and proper use of organolithium reagents is described.

使用有机锂试剂的一个协议安全的锂化反应

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents ...

Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. Their synthesis by conventional organic chemistry often requires tedious multi-step synthesis processes, with difficult control of the stereochemical outcome. We present a protocol to enzymatically synthetize amino alcohols starting from the readily available L-lysine in 48 h. This protocol combines two chemical reactions that are very difficult to conduct by conventional organic synthesis. In the first step, the regio- and diastereoselective oxidation of an unactivated C-H bond of the lysine side-chain is catalyzed by a dioxygenase; a second regio- and diastereoselective oxidation catalyzed by a regiodivergent dioxygenase can lead to the formation of the 1,2-diols. In the last step, the carboxylic group of the alpha amino acid is cleaved by a pyridoxal-phosphate (PLP) decarboxylase (DC). This decarboxylative step only affects the alpha carbon of the amino acid, retaining the hydroxy-substituted stereogenic center in a beta/gamma position. The resulting amino alcohols are therefore optically enriched. The protocol was successfully applied to the semipreparative-scale synthesis of four amino alcohols. Monitoring of the reactions was conducted by high performance liquid chromatography (HPLC) after derivatization by 1-fluoro-2,4-dinitrobenzene. Straightforward purification by solid-phase extraction (SPE) afforded the amino alcohols with excellent yields (93% to &#62;95%).

Chiral amino alcohols are versatile molecules for use as scaffolds in organic synthesis. Starting from L-lysine, we synthesize amino alcohols by an enzymatic cascade reaction combining diastereoselective C-H oxidation catalyzed by dioxygenase followed by cleavage of the carboxylic acid moiety of the corresponding hydroxyl amino acid by a decarboxylase.

酶联反应合成 l-赖氨酸手性氨基醇的研究

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. ...

Employing Pressurized Hot Water Extraction (PHWE) to Explore Natural Products Chemistry in the Undergraduate Laboratory

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products research has also found applications as an effective teaching tool. Specifically, this technique has been used to introduce second- and third-year undergraduates to aspects of natural products chemistry in the laboratory. In this report, two experiments are presented: the PHWE of eugenol and acetyleugenol from cloves and the PHWE of seselin and (+)-epoxysuberosin from the endemic Australian plant species Correa reflexa. By employing PHWE in these experiments, the crude clove extract, enriched in eugenol and acetyleugenol, was obtained in 4-9% w/w from cloves by second-year undergraduates and seselin and (+)-epoxysuberosin were isolated in yields of up to 1.1% w/w and 0.9% w/w from C. reflexa by third-year students. The former exercise was developed as a replacement for the traditional steam distillation experiment providing an introduction to extraction and separation techniques, while the latter activity featured guided-inquiry teaching methods in an effort to simulate natural products bioprospecting. This primarily derives from the rapid nature of this PHWE technique relative to traditional extraction methods that are often incompatible with the time constraints associated with undergraduate laboratory experiments. This rapid and practical PHWE method can be used to efficiently isolate various classes of organic molecules from a range of plant species. The complementary nature of this technique relative to more traditional methods has also been demonstrated previously.

Employing Pressurized Hot Water Extraction PHWE to Explore Natural Products Chemistry in the Undergraduate Laboratory

Here, we employ a pressurized hot water extraction (PHWE) method, which utilizes an unmodified household espresso machine to introduce undergraduates to natural products chemistry in the laboratory. Two experiments are presented: PHWE of eugenol and acetyleugenol from cloves and PHWE of seselin and (+)-epoxysuberosin from the Australian plant Correa reflexa.

利用加压热水提取 (phwe) 在本科实验室中探索天然产物化学

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products ...

Imine Metathesis by Silica-Supported Catalysts Using the Methodology of Surface Organometallic Chemistry

With this protocol, a well-defined singlesite silica-supported heterogeneous catalyst [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] is designed and prepared according to the methodology developed by surface organometallic chemistry (SOMC). In this framework, catalytic cycles can be determined by isolating crucial intermediates. All air-sensitive materials are handled under inert atmosphere (using gloveboxes or a Schlenk line) or high vacuum lines (HVLs, &#60;10-5 mbar). The preparation of SiO2-700 (silica dehydroxylated at 700 &#176;C) and subsequent applications (the grafting of complexes and catalytic runs) requires the use of HVLs and double-Schlenk techniques. Several well-known characterization methods are used, such as Fourier-transform infrared spectroscopy (FTIR), elemental microanalysis, solid-state nuclear magnetic resonance spectroscopy (SSNMR), and state-of-the-art dynamic nuclear polarization surface enhanced NMR spectroscopy (DNP-SENS). FTIR and elemental microanalysis permit scientists to establish the grafting and its stoichiometry. 1H and 13C SSNMR allows the structural determination of the hydrocarbon ligands coordination sphere. DNP SENS is an emerging powerful technique in solid characterization for the detection of poorly sensitive nuclei (15N, in our case). SiO2-700 is treated with about one equivalent of the metal precursor compared to the amount of surface silanol (0.30 mmol&#183;g-1) in pentane at room temperature. Then, volatiles are removed, and the powder samples are dried under dynamic high vacuum to afford the desired materials [(&#8801;Si-O-)Hf(&#951;2&#960;-MeNCH2)(&#951;1-NMe2)(&#951;1-HNMe2)]. After a thermal treatment under high vacuum, the grafted complex is converted into metal imido silica complex [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)]. [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] effectively promotes the metathesis of imines, using the combination of two imine substrates, N-(4-phenylbenzylidene)benzylamine, or N-(4-fluorobenzylidene)-4-fluoroaniline, with N-benzylidenetert-butylamine as substrates. A significantly lower conversion is observed with the blank runs; thus, the presence of the imido group in [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] is correlated to the catalytic performance.

A new group IV metal catalyst for imine metathesis is prepared by grafting amine metal complex onto dehydroxylated silica. Surface metal fragments are characterized using FT-IR, elemental microanalysis, and solid-state NMR spectroscopy. Further dynamic nuclear polarization surface enhanced NMR spectroscopy experiments complement the determination of the coordination sphere.

利用表面有机金属化学方法，利用硅支持的催化剂进行Imine金属分析

With this protocol, a well-defined singlesite silica-supported heterogeneous catalyst [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] is designed and prepared ...

A Microwave-Assisted Direct Heteroarylation of Ketones Using Transition Metal Catalysis

Heteroarylation introduces heteroaryl fragments to organic molecules. Despite the numerous available reactions reported for arylation via transition metal catalysis, the literature on direct heteroarylation is scarce. The presence of heteroatoms such as nitrogen, sulfur and oxygen often make heteroarylation a challenging research field due to catalyst poisoning, product decomposition and the rest. This protocol details a highly efficient direct &#945;-C(sp3) heteroarylation of ketones under microwave irradiation. Key factors for successful heteroarylation include the use of XPhos Palladacycle Gen. 4 Catalyst, excess base to suppress side reactions and the high temperature and pressure achieved in a sealed reaction vial under microwave irradiation. The heteroarylation compounds prepared by this method were fully characterized by proton nuclear magnetic resonance spectroscopy (1H NMR), carbon nuclear magnetic resonance spectroscopy (13C NMR) and high-resolution mass spectrometry (HRMS). This methodology has several advantages over literature precedents including broad substrate scope, rapid reaction time, greener procedure and operational simplicity by eliminating the preparation of intermediates such as silyl enol ether. Possible applications for this protocol include, but are not limited to, diversity-oriented synthesis for the discovery of biologically active small molecules, domino synthesis for the preparation of natural products and ligand development for new transition metal catalytic systems.

Heteroaryl compounds are important molecules utilized in organic synthesis, medicinal and biological chemistry. A microwave-assisted heteroarylation using palladium catalysis provides a rapid and efficient method to attach heteroaryl moieties directly to ketone substrates.

利用过渡金属催化的酮的微波辅助直接异化

Heteroarylation introduces heteroaryl fragments to organic molecules. Despite the numerous available reactions reported for arylation via transition metal ...