财政支持，经费赞助Ministero大学dell&#39;Istruzione，德拉找实习（PRIN 2009K3RH7N_002）和玛丽·居里欧洲内部的奖学金（CYCLOGUO-298555），以及赞助COST行动CM0603“自由基化学生物学与COST行动CM1201“仿生自由基化学”上表示诚挚的感谢。

1。单声道 - 反式异构体的胆甾醇酯的合成<ol><li>溶解的胆甾醇酯（亚油酸或花生四烯酸胆固醇酯）在2 - 丙醇（15毫摩尔）。为了更好的增溶，将样品超声处理15分钟，在氩气下。 </li><li>将该溶液转移到石英光化学反应器中，添加2 - 巯基乙醇，2 - 丙醇（达到700 mM的浓度，从一个2 M原液的硫醇）。为了消除溶液中的氧的存在下，20分钟，将反应混合物用氩气冲洗。 </li><li>照射反应混合物中使用5.5W的低压汞灯的UV光，在22±2℃下进行4分钟。监控的银银薄层色谱法（TLC）分析的证据的单-反的胆甾醇酯的形成（参见图1的化学式）。的TLC染色进行浇注板铈铵molibdate（CAM）的溶液中，并出现斑点在加热板。在早期阶段，淬灭反应，为了回收的起始原料，可以重复使用，以执行其他轮的异构化，得到的总产率增加。 </li><li>收集的反应混合物在一个圆底烧瓶中，用几毫升的2 - 丙醇洗涤装置。除去溶剂，纯化单-胆甾醇酯的反式异构体，如在文献中描述的由Ag-TLC。5使用己烷-乙醚（9:1 V / V）作为洗脱剂为单-反式胆甾醇亚油酸酯的异构体，而使用己烷 - 乙醚 - 乙酸（9:1:0,1体积/体积）为单 - 反式胆甾醇花生四烯酸酯的异构体。 </li></ol> 2。人血清胆固醇酯组分分离<ol><li>稀释1毫升人血清（的血液通过离心分离得到的），用1毫升盐水洗涤，并倒入到一个分液漏斗中的溶液在氩气气流下，为了避免工件（ 例如氧化加合物）。加入10毫升氯仿 - 甲醇（2：1 V / V）三次。摇动分液漏斗中非常缓慢，以限制由于白蛋白存在下形成的乳液。 </li><li>一次，用盐水（10ml）洗涤有机层，然后收集在锥形烧瓶中在氩气流下，干燥，用无水硫酸钠干燥。用旋转蒸发器除去挥发物，得到黄色油状物（血浆总脂质成分）。 </li><li>占用的粗品，用1毫升氯仿 - 甲醇（2:1体积/体积），在氩气气流下负载到制备TLC并用己烷 - 乙醚（9:1体积/体积）的混合物作为洗脱剂。刮去硅胶部分含有胆固醇酯组分，倒入一小瓶。然后，提取的二氧化硅（3×5毫升），用氯仿 - 甲醇（2:1体积/体积）中，收集的有机层，除去溶剂蒸发后，得到的胆甾醇酯（通常是在〜1.5毫克）的纯馏分。 </li><li>查看由铝箔覆盖于-20℃下在氩气下和存储在一个黑暗的小瓶的胆甾醇酯胆固醇Ë光线和氧气sters敏感的。 </li></ol> 3。单跨胆固醇酯的拉曼光谱表征<ol><li>提取从人血清（≥0.7毫克），如在第2条中所述的胆甾醇酯，溶解于少量的四氯化碳（≤10微升）和在小瓶中。 CCl 4中被选择作为溶剂，因为它的拉曼信号与胆固醇酯的分析感兴趣的区域不重叠。 </li><li>将该溶液转移到样本保持器通过一次性玻璃吸管，然后小心地除去溶剂，由缓慢的氩气流中。一旦形成均匀的油膜的样本保持器的内壁上，后者放置到仪器进行测量。 </li><li>傅里叶变换拉曼光谱上直接获得的胆甾醇酯的脂质提取物，没有任何衍生反应。样品上的激光功率是&lt;100毫瓦，以避免样品的损害。的总的扫描数为每个频谱为≥800，以最大限度地减少背景噪音。 </li><li> 1,700-1,630厘米-1范围内的拉曼光谱进行了分析，从C = C双键的分子（C = C伸缩振动模式），因为它反映了贡献。甲进行曲线拟合分析该区域中，从而允许区分中的脂肪酸的烷基链和双键中胆固醇（参见图2）的B环的顺式和反式双键的叠加的贡献。 </li><li>为了适应正常的振动带，一些参数应该是固定的，或限制在合理的范围内。的组分峰公司被描述为洛伦兹和高斯函数的线性组合，而在半高带宽最好是由计算机确定的优化例程。的组分峰的数目和位置的，得到通过使用第四微分光谱，这允许检测也有一些微弱的单位频带。由于信号信噪比劣化后，分化的信号可以排除使用第四衍生物，平滑第四衍生物，具有13点Savitsky-格雷（Golay）功能，有优势，因此，一个合适的分辨率和信噪比之间的妥协得到。在尽可能低的c 2值获得最佳曲线拟合。 </li><li>反式异构体的存在下，发现由该组件在1671±1cm -1处 ，此外，至少两种组分，稍移向较低的频率，由于脂肪酸链和胆固醇的C = C双键。血浆胆甾醇酯的代表性的拉曼光谱示于图9。在插图中示出与胆甾醇亚油酸酯和单 - 反式胆甾醇亚油酸异构体的特定区域的比较。 </li></ol> 4。衍生脂肪酸甲酯（FAME）和气相色谱分析<ol><li>溶解胆甾醇酯（〜1.5毫克），用0.5毫升的1M氢氧化钠溶液中，苯 - 甲醇（2:3体积/体积）和地点所涵盖的铝箔在氩气下在一个黑暗的小瓶中。 </li><li>保持该混合物在搅拌下于室温，并用TLC显示器用己烷 - 乙醚（9:1体积/体积）的混合物作为洗脱剂。反应时间通常是30分钟但仔细的反应监测需要。事实上，一旦形成相应的甲基酯，该反应必须被骤冷，以避免降解和副产物的形成。 TLC表明甲基酯的定量生成。 </li><li>淬灭反应混合物，通过添加1毫升的盐水洗涤，并提取与n-己烷（3×2毫升）的甲基酯。收集的有机层到小瓶中，并通过旋转蒸发除去溶剂，得到的甲基酯的馏分，然后将其不经进一步纯化用于GC分析。 </li><li>的甲基酯溶解在n-己烷的最小量的（通常为1毫克，5个0微升）和注入的60米×0.25毫米×0.25微米（50％ - 氰基丙基） - 甲基聚硅氧烷柱配备在GC。使用氢火焰离子化检测器（FID），以下烤箱计划：在165°C的低温启动，保持3分钟，增加1°C /分钟高达195°C，持续40分钟，随后的第二增加为10°C / min升温至240℃，保持10分钟。选择一个恒压模式（29 PSI）。氦气或氢气作为载气，可以使用。甲基酯比较，与真实样品的保留时间确定的， 图10示出了代表性的GC分析，获得的血浆胆甾醇酯的FAME。 </li></ol> 5。合成（5&#39;R） - 和（5&#39;S）-5&#39;-1,8 - 环-2&#39;-脱氧腺苷<ol><li>将33毫克的8 - 溴-2&#39;-脱氧腺苷（8 - 溴-墙裙）的乙腈（100毫升）溶解，以达到浓度为1mM。在光催化反应器，调节气体流量（Ar或N2），填补了瑞星千克基本与8 - 溴-墙裙溶液。 </li><li>准备一个隔垫适当大小的光反应器的输入，并通过连接到惰性气体通过它的中心的线的针。关闭系统的隔膜。的惰性气体进行冲洗30分钟。 </li><li>通过UV光的照射，使用125W中压汞灯，在22±2℃下进行15分钟，收集到一个250毫升的圆底烧瓶中的溶液。 10毫升乙腈和洗净的光催化反应器，在相同的烧瓶收集洗涤。 </li><li>淬火的粗反应混合物用1M NH 4 OH溶液，并在旋转蒸发器中蒸发掉溶剂。 </li><li>在已知的条件下运行的高效液相色谱分析法（HPLC-UV）分析柱（见插装部分），并比较参照化合物的档案。 </li><li>运行高性能液相色谱分析（HPLC-UV）用分析柱（见插装部分），通过使用以下的溶胶喷口和梯度：2 mM的甲酸铵作为溶剂A和乙腈作为溶剂B设定流速为1毫升/分钟，要达到在2.2分钟的梯度从0％溶剂B，0.3％溶剂B。 0.8％的溶剂B在4.0分钟，4.8分钟1％溶液中的B。维持在1％的溶剂B为9分，然后在6分钟到8％溶剂B。在4分钟到10％溶剂B，30％溶剂B中5分钟，并保持在30％溶剂B，其他5分钟后。收集对应于在两个不同的小瓶中的两个非对映体产物的色谱峰。 </li><li>两个收集的馏分在紫外分光光度计测量吸光度和计算是以Lamber-Beer定律（A =εlC，其中A为吸光度，ε的消光系数和升细胞的长度）的基础上的准确浓度。使用2&#39;-脱氧腺苷（墙裙）（15,400 M -1厘米-1在260 nm）的消光系数ε。 </li></ol> 6。合成（5&#39;R） -D（5&#39;S）-5&#39;-1,8 - 环-2&#39;-脱氧鸟苷<ol><li>为了达到1mM和2mM浓度，分别将35毫克的8 - 溴-2&#39;-脱氧鸟苷（8 - 溴-dGuo）在蒸馏水（100毫升）和30毫克的碘化钠（NaI）溶解。规管的惰性气体（Ar或N 2）连接到的光反应器的流和填充的光反应器与反应溶液。 </li><li> Degass程序5（步骤b）中所描述的反应混合物。 </li><li>通过UV光的照射，使用125W中压汞灯，在22±2℃30分钟，收集到250毫升圆底烧瓶中加入该溶液。用10ml的水和洗净的光反应器中的相同的烧瓶中收集洗液。 </li><li>骤冷的粗反应混合物用1M NH 4 OH溶液，并在真空下蒸除溶剂。 </li><li>中描述的步骤5（步骤E至G）进行分析。使用2&#39;-脱氧鸟苷（dGuo）（11,700 M -1厘米-1在260 nm）的消光系数ε。 </li></ol> 7。合成的同位素标记的嘌呤（5&#39;R） - 和（5&#39;S）-5&#39;-1,8 - cyclonucleosides <ol><li>准备2毫升1mM的水溶液（蒸馏水），15 N同位素标记的嘌呤核苷（[15N 5] DADO或[15N 5] dGuo）用隔膜帽（4毫升）在玻璃样品瓶中。 </li><li>小瓶用的隔膜帽连接，并连接到通过气体管线（N 2 O的溶液的饱和度），到达底部的小瓶中的隔垫的针。在隔垫盖的另一个短针作为气体出口。 </li><li>冲洗30分钟，该溶液与N 2 O。的气体流量调节是非常低的。 </li><li>首先退出针取出后1&#39;的长1如下，以便有一个小的内部压力小瓶。 </li><li> 8小时（计算的基础上的剂量率约4.5戈瑞/分），将溶液中的装置的伽马辐。 </li><li>反应后骤冷的粗品，用1M NH 4 </sUB&gt; OH的解决方案。 </li><li>已知的条件下，6运行高性能液相色谱分析（HPLC-UV）和比较色谱图与标准的参考文献。 </li></ol> 8。 DNA水溶液中的伽玛辐<ol><li>准备1毫升0.5毫克/毫升的小牛胸腺DNA的溶液（蒸馏水）中，在一个玻璃小瓶（2毫升），并把用隔膜帽。 </li><li> Degass反应步骤7中描述的（步骤BD）。 </li><li>将溶液中的γ辐解装置30分钟（计算剂量/速率约4.5戈瑞/分的基础上）。 </li></ol> 9。酶消化DNA <ol><li>含有80微克的DNA到Eppendorf管中，添加8 U的核酸酶P1，0.01 U的磷酸二酯酶Ⅱ，300 mM乙酸钠（pH 5.6）中和10 mM的氯化锌在20微升20 nmol的EHNA。将混合物在37℃下孵育48小时。 </li><li>接下来，添加8 U碱性磷酸酶的混合物，0.02 U phosphodiesterase口，在40微升的0.5M的Tris-HCl缓冲液（pH值8.9）中的混合物的消化。将样品在37℃下孵育2小时。 </li><li>通过加入10％甲酸中的混合物。 </li><li>转移的Amicon Ultra-0.5离心过滤装置（截止3 kDa）的样品，加入200μltridistilled H 2 O在14,000 xg离心15分钟将设过滤器进入离心机转子。超过滤装置离心管，然后分开。在离心管，冷冻干燥的酶的解决方案。 </li></ol> 10。在此之前分析的DNA样品脱盐<ol><li>运行高效液相色谱分析法（HPLC-UV）分析柱（见插装部分）的已知条件。 </li><li>冻干消化的DNA溶解在20μl的H 2 O中，并注入HPLC-UV。 </li><li>收集样品后，一小瓶盐洗脱（第一分钟）和前洗脱添e的第一核苷（5&#39;R-cdGuo），，直到色谱节目结束。 </li><li>将样品冻干20μl水resolubilized中。 </li></ol> 11。 LC-MS/MS定量分析<ol><li>准备的的HPLC-MS/MS（三重四极杆）之前加载的分析方法和MS检测方法（ESI）开始分析。 </li><li>在程序7中制备的样品中的同位素标记的化合物的混合物中加入1μl。 </li><li>注入20微升消化的DNA溶液在蒸馏水中加标。 </li><li>以前的校准参照化合物的基础上，得到的色谱数据进行了阐述。一位代表HPLC运行含有腺苷，鸟苷2&#39;-脱氧核糖核苷和它们的氧化和环衍生物， 如图11所示。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(5'S)-+and+(5'R)-5',8-cyclo-2'-deoxyguanosine:+mechanistic+insights+on+the+2'-deoxyguanosin-5'-yl+radical+cyclization.">(5'S)- and (5'R)-5',8-cyclo-2'-deoxyguanosine: mechanistic insights on the 2'-deoxyguanosin-5'-yl radical cyclization.</a> Chemical Research in Toxicology. 20 (12), 1820-1824 (2007).</li><li>Wang, J., Clauson, C. L., Robbins, P. D., Niedernhofer, L. J., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oxidative+DNA+lesions+8,5'-cyclopurines+accumulate+with+aging+in+a+tissue-specific+manner.">The oxidative DNA lesions 8,5'-cyclopurines accumulate with aging in a tissue-specific manner.</a> Aging Cell. 11 (4), 714-716 (2012).</li><li>Ferreri, C., Chatgilialoglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+fatty+acid-based+functional+lipidomics+in+the+development+of+molecular+diagnostic+tools.">The role of fatty acid-based functional lipidomics in the development of molecular diagnostic tools.</a> Expert Review of Molecular Diagnostics. 12 (7), 767-780 (2012).</li></ol>

没有利益冲突的声明。

自然发生的几何反式异构体的顺式不饱和脂肪酸的转化是在生物环境中的自由基应力与生产连接的变换。细胞膜的脂质，其中含有不饱和脂肪酸，是一个有关生物目标为激进的压力，并在细胞培养，动物和人类的评估分析协议，在每一种情况下，我们首先研究了内源性的顺反磷脂异构化。8-10表明，这种转变的发生可通过各种各样的含S-的化合物，包括硫醇，硫醚，二硫化物，其中不同的自由基应力条件下能够生成生成硫中心自由基， 即异构化剂（ 图3）。在这篇文章中所显示的示例集中在胆固醇酯之类的，它代表的是知名的一部分，参与脂蛋白代谢血脂，严格。之间的酯键的脂肪酸和町lesterol生物合成的脂肪酸由转印从磷脂酰胆碱的甘油部分的位置2的胆固醇，通过酶卵磷脂胆固醇酰基转移酶（LCAT）催化步骤。因此，血浆胆甾醇酯是严格连接与膜脂周转，含有相对较高比例的多不饱和脂肪酸（PUFA），通常存在于磷脂酰胆碱， 即亚油酸和花生四烯酸。脂蛋白的形成涉及心血管疾病和代谢性疾病。天然胆甾醇酯与自由基的反应性，可以发生在双键的亚油酸和花生四烯酸酯的残基，它可以在相应的反式几何异构体转化（参见图1的结构）。生物样品中的反式胆甾醇酯的含量是表征有趣的生物标志物的发展。间接方法包括改造cholestery升酯从血浆中分离出相应的脂肪酸甲基酯（FAME）和分离的气相色谱协议。在这种情况下，被执行时，顺式和反式脂肪酸甲基酯的标准参考文献的校准，以便使样品中的反式含量的定量分析。上的胆固醇酯库进行分析研究的基础上，我们提出，也适用于基于拉曼光谱的方法，可以直接进行从血浆中分离的胆固醇酯馏分，没有进一步的衍生的相应的FAME（见图2和9）。这是值得注意的，到现在为止没有成功的方法描述为独立的顺式和反式异构体通过HPLC含有脂质的脂肪酸，而不是所描述的胆甾醇酯的氢过氧化物。到目前为止，间接气相色谱测定方法至今仍然是最为有效的方法。通过该方法的第一定量评估通报BULLETIN来自孤立从健康受试者血浆中的胆甾醇酯的单 - 反式含量提供。使用火焰离子化检测器（FID）的可探测的极限是令人满意的分之一（ppb）和已被检测到的化合物的毫微摩尔数量。5。对于不同的检测系统，这个限制可以在更低。电离辐射对胆甾醇酯的效果进一步的研究中，无论是一个线性响应是否得到相对于所施加的剂量。 作为第二个例子，我们选择了可以产生自由基损伤的DNA的修饰核苷。羟基自由基（HO•）是已知的最有害的活性氧（ROS）的能力，导致化学修饰DNA。单个或多个病变可能发生在真核细胞中DNA，位于细胞核和线粒体。识别和测量DNA的氧化损害的主要类需要appropr第十一条分子库，以建立解析协议。我们的兴趣集中在最小的串联病变，是嘌呤5&#39;-1,8 - 环-2&#39;-脱氧核糖核苷，具有一个额外的由自由基攻击创建的基极和糖部分之间的共价键。本发明的化合物是5&#39;-1,8 -环-2&#39; -脱氧腺苷和5&#39;-1,8 -环-2&#39; -脱氧鸟苷的5&#39;R和5&#39;S非对映体形式（ 图5）中存在。它们可能成为自由基应激标记是物质的基础研究。事实上，当DNA暴露在HO•自由基氢抽象的糖C5&#39;的位置是导致这些串联病变的形成可能发生的事件之一。嘌呤5&#39;-1,8 - cyclonucleosides可作为非对映异构体的总和，通过酶促消化γ照射的DNA样品，不同的从1至12的病变/ 10 6核苷/戈瑞HPLC-MS/MS测定氧的形式没有去氧气的生理水平我Ň组织，非对映体比率5&#39;R / 5&#39;S〜4和5〜3&#39;-1,8 - cdAdo和5&#39;-1,8 - cdGuo，分别为（ 图11）11，值得注意的是，辐射剂量之间的关系和5&#39;,8-cdAdo和5&#39;,8-cdAdo病变细胞的DNA中检测到的是被理解。不能被视为基于2kGy照射单实验报告在实验定论。这种和分析定量的四个病变11的进一步的实验，用于定义这种关系是必要的。这些病灶的检测和更流行的氧化变换（如8 -氧代-2&#39;-脱氧鸟苷，8-oxodGuo的）物质的激烈调查，证明两个病灶的氧化代谢过程中的重要性。6,13使用HPLC -MS/MS（三重四极杆）的检出限30fmol个病灶。最后的改进声称的工具机生产线达到渺摩尔水平的检测限为核证减排量。在最近的文献中，分析程序必须包括适当的清理，以满足MS / MS / MS（离子阱）或MS / MS（三重四极杆）的检出限的样品和浓缩在我们的例子。 仿生的化合物1-4的合成方法，从8-bromopurine的衍生物下或光解开始开发。7,12这些程序涉及的自由基级联反应，模仿的DNA损伤的机制形成5星&#39;-1,8 - cdAdo和5&#39; ,8-cdGuo病变。从生物学的角度，发现这些病变积聚老化的组织特异性的方式（肝&gt;肾&gt;脑），提供的证据表明，DNA修复机制是不足以维持这些病变的遗传物质从13事实上，核苷酸切除修复（NER）是唯一的途径目前已确定的这些病变的修复。 这两个类在图1和图5中所示的化合物的上课不是市售的时刻，但是，在文献中描述的合成策略它不会是用于商业用途，难以制备这些化合物。 化学生物学研究提供了多学科的方法不仅具有识别的新的机制，在生物环境发生了巨大的价值，而且还给出了一个根本性的贡献的生物标志物的发现和诊断，最终带来新颖的保健和预防策略。14化学的贡献是一个成功的分子医学的发展需要，创建集成的平台和面板的代谢分析，预计允许，治疗和营养干预的最佳合理化设计，减少不确定性和失败时，他们可以预测的。

自由基的反应显示其巨大的重要性，许多生物的活动，包括老龄化和炎症。如今，它是越来越明显，每个参与此反应的化学步骤是必要的澄清，以了解底层机制和设想有效的战略控制的自由基，修复的损坏。从化学研究的贡献是根本，但在生物环境中可以直接研究是困难的，因为不同的进程复杂化的叠加扰动检查的结果及相关结论。因此，战略模拟生物相关条件下的自由基反应，在生物学的化学机制的研究已成为一个基本步骤。 在过去的十年，我们的小组研制仿生条件下的自由基过程的模型。特别是，我们EN面孔的不饱和脂肪酸，核苷和含硫氨基酸的生物相关的转换，在轨道上，并把它们作为生物标志物的健康状况进行评估和验证。1-4 我们一般的做法是由三个模块组成： <ul><li>有机合成，它提供了访问到适当修改的生物分子。合成的计划也可以设计以模拟在生物环境产生的自由基过程，因此适用条件，能够模拟生物过程的原则，仿生自由基化学的兴趣，这是。在仿生模型的反应介质是水或异构的介质，由于共存的亲水性和疏水性的舱室。与仿生模型的优点是有一个简化的环境，可能与已知反应的合作伙伴，反应性和产品的详细信息，可以检查发现新的化学途径。从这个步骤可以收集机制和动力学信息; </li><li>纯化，分析和鉴定的产品，提供修改后的生物分子结构和化学信息，以组织分子库和促进recognition.in更复杂的生物环境。协议被建立也鉴于解决复杂的混合物的化合物，如那些衍生自生物样本; </li><li>使用衍生的生物样品，可以通过在体外实验中使用的细胞培养，并从动物和人类体内研究的生物标志物的发展。仿生模型开发的协议，然后应用到复杂的分析样品，在不同的条件下设想自由基变换。分子库开发的合成，是生命科学的发现有很大的帮助。收集到的信息的自由的根本转变GIVe的机会，修复和预防战略。仔细评估的结果可用于数据库的生物标志物的意义以及可能的相关因素的多因素分析。 </li></ul>我们选择了两个类相关的生物标记认可这样的做法：胆甾醇酯和嘌呤5&#39;,8-环-2&#39;-脱氧核糖核苷。

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>MATERIALS</td>
 </tr>
 <tr>
 <td>Cholesteryl linoleate &ge;98%</td>
 <td>Sigma-Aldrich</td>
 <td>C0289-100 mg</td>
 <td></td>
 </tr>
 <tr>
 <td>Cholesteryl arachidonate&ge;95% </td>
 <td>Sigma-Aldrich</td>
 <td>C8753-25mg</td>
 <td></td>
 </tr>
 <tr>
 <td> 2-mercaptoethanol</td>
 <td>Sigma-Aldrich</td>
 <td>M6250-100 ml</td>
 <td></td>
 </tr>
 <tr>
 <td> 2-propanol</td>
 <td>Sigma-Aldrich</td>
 <td>34965-1L</td>
 <td></td>
 </tr>
 <tr>
 <td> Methanol 215 SpS </td>
 <td>Romil</td>
 <td> H409-2,5 L</td>
 <td></td>
 </tr>
 <tr>
 <td> Ethanol</td>
 <td>Sigma-Aldrich</td>
 <td>02860-2.5L</td>
 <td></td>
 </tr>
 <tr>
 <td> Chloroform SpS</td>
 <td>Romil</td>
 <td>H135 2,5 L</td>
 <td></td>
 </tr>
 <tr>
 <td> n-Hexane 95% SpS</td>
 <td>Romil</td>
 <td> H389 2,5 L</td>
 <td></td>
 </tr>
 <tr>
 <td> Acetonitrile 230 SpS</td>
 <td>Romil</td>
 <td>H047 2,5 L</td>
 <td></td>
 </tr>
 <tr>
 <td> Dichloromethane SpS</td>
 <td>Romil</td>
 <td>H2022,5 L</td>
 <td></td>
 </tr>
 <tr>
 <td> Carbon tetrachloride</td>
 <td>Sigma-Aldrich</td>
 <td>107344-1L </td>
 <td></td>
 </tr>
 <tr>
 <td> Sodium iodide</td>
 <td>Sigma-Aldrich</td>
 <td>383112-100G</td>
 <td></td>
 </tr>
 <tr>
 <td> Sodium hydrogen carbonate</td>
 <td>Carlo Erba</td>
 <td>478536-500 g </td>
 <td></td>
 </tr>
 <tr>
 <td> Diethyl ether</td>
 <td>Sigma-Aldrich</td>
 <td> 309966-1L</td>
 <td></td>
 </tr>
 <tr>
 <td> NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>S7653-5KG</td>
 <td></td>
 </tr>
 <tr>
 <td> NaOH solid</td>
 <td>Sigma-Aldrich</td>
 <td>221465-25G</td>
 <td></td>
 </tr>
 <tr>
 <td> NH4OH sol. 28%-30%</td>
 <td>Sigma-Aldrich</td>
 <td>221228-1L-A</td>
 <td></td>
 </tr>
 <tr>
 <td> Acetic acid</td>
 <td>Sigma-Aldrich</td>
 <td>320099-500ML</td>
 <td></td>
 </tr>
 <tr>
 <td> Ammonium Cerium(IV)sulfate dihydride</td>
 <td>Sigma-Aldrich</td>
 <td>221759-100G</td>
 <td></td>
 </tr>
 <tr>
 <td> Ammonium Molybdate tetrahydrate</td>
 <td>Sigma-Aldrich</td>
 <td>A7302-100G</td>
 <td></td>
 </tr>
 <tr>
 <td> Sulfuric Acid 95%-98%</td>
 <td>Sigma-Aldrich</td>
 <td>320501-1L</td>
 <td></td>
 </tr>
 <tr>
 <td> Silver Nitrate</td>
 <td>Sigma-Aldrich</td>
 <td>209139-25G</td>
 <td></td>
 </tr>
 <tr>
 <td> Sodium sulfate anhydrous</td>
 <td>Sigma-Aldrich</td>
 <td>238597-500G</td>
 <td></td>
 </tr>
 <tr>
 <td> Nuclease P-I from penicillium citrinum</td>
 <td>Sigma-Aldrich</td>
 <td>N8630-1VL</td>
 <td></td>
 </tr>
 <tr>
 <td> Phosphodiesterase II type I-sa </td>
 <td>Sigma-Aldrich</td>
 <td>P9041-10UN</td>
 <td></td>
 </tr>
 <tr>
 <td> Erythro-9-(2-hydroxy-3-nonyl)adenine, hc</td>
 <td>Sigma-Aldrich</td>
 <td>E114-25MG</td>
 <td></td>
 </tr>
 <tr>
 <td> Phosphatase alkaline type VII-t from*bov</td>
 <td>Sigma-Aldrich</td>
 <td>P6774-1KU</td>
 <td></td>
 </tr>
 <tr>
 <td> Phosphodiesterase I type VI</td>
 <td>Sigma-Aldrich</td>
 <td>P3134-100MG</td>
 <td></td>
 </tr>
 <tr>
 <td> Deoxyribonuclease II type IV from*porcin</td>
 <td>Sigma-Aldrich</td>
 <td>D4138-20KU</td>
 <td></td>
 </tr>
 <tr>
 <td> Trizma(r) base, biotechnology performanc ce</td>
 <td>Sigma-Aldrich</td>
 <td>T6066-100G</td>
 <td></td>
 </tr>
 <tr>
 <td> EDTA</td>
 <td>Sigma-Aldrich</td>
 <td>E1644-100G</td>
 <td></td>
 </tr>
 <tr>
 <td> Succinic acid bioxtra</td>
 <td>Sigma-Aldrich</td>
 <td>S3674-250G</td>
 <td></td>
 </tr>
 <tr>
 <td> Calcium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>C5670-100G</td>
 <td></td>
 </tr>
 <tr>
 <td> Formic acid, 98 %</td>
 <td>Sigma-Aldrich</td>
 <td>06440-100ML</td>
 <td></td>
 </tr>
 <tr>
 <td> Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-3 membrane</td>
 <td>Millipore</td>
 <td>UFC500324</td>
 <td></td>
 </tr>
 <tr>
 <td> 8-Bromo-2'-deoxyguanosine</td>
 <td>Berry &amp; Associates</td>
 <td>PR3290-1 g</td>
 <td></td>
 </tr>
 <tr>
 <td> 8-Bromo-2'-deoxyadenosine</td>
 <td>Berry &amp; Associates</td>
 <td>PR3300-1 g</td>
 <td></td>
 </tr>
 <tr>
 <td> Sodium iodide</td>
 <td>Sigma-Aldrich</td>
 <td>383112-100G</td>
 <td></td>
 </tr>
 <tr>
 <td> Sodium hydrogen carbonate</td>
 <td>Carlo Erba</td>
 <td>478536-500 g </td>
 <td></td>
 </tr>
 <tr>
 <td> 2'-deoxyguanosine:H2O (U-15N5, 96-98%)</td>
 <td>Cambridge Isotope Laboratories, Inc</td>
 <td>CILNLM-3899-CA-0.1</td>
 <td></td>
 </tr>
 <tr>
 <td> 2'-deoxyadenosine (U-15N5, 98%) 95%+ CHEMICAL PURITY</td>
 <td>Cambridge Isotope Laboratories, Inc</td>
 <td>CILNLM-3895-0.1</td>
 <td></td>
 </tr>
 <tr>
 <td> Nitrous oxide (N2O) </td>
 <td>Air Liquide </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td> Deoxyribonucleic acid from calf thymus</td>
 <td>Sigma Aldrich</td>
 <td>D4522-5MG</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>60Co-Gammacell</td>
 <td>AECL- Canada </td>
 <td>220</td>
 <td></td>
 </tr>
 <tr>
 <td>Immersion well reaction medium pressure 125 watts </td>
 <td>Photochemical reactors ltd</td>
 <td>Model 3010</td>
 <td></td>
 </tr>
 <tr>
 <td>Evaporating flask 250 ml</td>
 <td>Heidolph</td>
 <td>P/N NS 29/32 514-72000-00</td>
 <td></td>
 </tr>
 <tr>
 <td>Luna 5 &mu;m C18(2) 100 &Aring;, LC Column 250 x 4.6 mm </td>
 <td>Phenomenex</td>
 <td>00A-4252-E0 </td>
 <td></td>
 </tr>
 <tr>
 <td>Alltima C8 Column 250 x 10 mm 5 &mu;m</td>
 <td>Grace</td>
 <td>88081</td>
 <td>Semipreparative</td>
 </tr>
 <tr>
 <td>SecurityGuard Kit</td>
 <td>Phenomenex</td>
 <td>KJ0-4282</td>
 <td>Analytical holder kit and accessories</td>
 </tr>
 <tr>
 <td>Holder for 10.0 mm ID cartridges</td>
 <td>Phenomenex</td>
 <td>AJ0-7220</td>
 <td>Semipreparative holder </td>
 </tr>
 <tr>
 <td>10.0 mm ID cartridges </td>
 <td>Phenomenex</td>
 <td>AJ0-7221 </td>
 <td></td>
 </tr>
 <tr>
 <td>High-performance liquid chromatography (HPLC) </td>
 <td>Agilent</td>
 <td>1100</td>
 <td></td>
 </tr>
 <tr>
 <td>LC/MS/MS </td>
 <td>Applied Biosystems</td>
 <td>4000QTRAP System</td>
 <td></td>
 </tr>
 <tr>
 <td>Tandem mass ESI spectrometer </td>
 <td>(Bruker Daltonics) </td>
 <td>Esquire 3000 plus </td>
 <td></td>
 </tr>
 <tr>
 <td>Vial 2-4 ml </td>
 <td>SUPELCO </td>
 <td>Cod 27516</td>
 <td></td>
 </tr>
 <tr>
 <td>Vial 4 ml </td>
 <td>SUPELCO </td>
 <td>Cod 27517</td>
 <td></td>
 </tr>
</tbody>
</table>

free radicals in chemical biology: from chemical behavior to biomarker development

随着时间的推移不断增加的自由基生命科学的参与，并已连接到多种生理和病理过程。这个主题包含了不同的科学领域，包括物理，生物和生物有机化学，生物学和医学上的应用程序的生命，健康和老龄化的质量的改善，。多学科的技能都需要在充分调查的激进过程的许多方面环境的生物和化学知识在公布的基本过程和机制中起着至关重要的作用。我们开发出一种化学生物学的方法能够连接自由基化学反应的生物过程，机制的途径和产品的信息。这种方法的核心是设计的仿生模型来研究生物分子的行为（脂类，核酸和蛋白质），在水性体系中，获得的见解的反应途径，以及一个S大厦的自由基的反应产物的分子库。此范围内可成功地用于生物标志物发现，实施例中所提供的两个类的化合物：单 - 反式异构体的胆甾醇酯，其中的合成和使用用于检测人血浆中的参考，和嘌呤5&#39;-1,8 - 环-2 &#39; - 脱氧核糖​​，准备在这种病变的DNA样本中检测的协议作为参考使用，经过电离辐射或者获得不同的健康状况。

特别是得到的亚油酸和花生四烯酸的单-反式异构体在图1中所示，作为这种攻击的第一个产品，它可以发生自由基应力的条件下，在生物环境中的胆甾醇酯的异构化反应过程中已被描述。 在图3中示出的顺式-反式的双键异构化中涉及的化学机制。激进的源显示在一个通用的方法来负担S-为中心的自由基。在所描述的协议的自由基源是UV光，是能够打破homolitically SH键中存在的硫醇分子RSH的。 图4总结了协议，用于合成的改性的脂质类和它们的检测人血浆中的三个步骤：在合成代表仿生自由基的过程，而且还提供了一锅煮的方便的入口的GEOMETrical的同分异构体，位置异构体没有任何污染随后纯化和分离协议。 几种分析方法可以应用于一种高灵敏度检测的反式异构体的含量和表征的单 - 反式胆固醇酯图书馆。特别是，可以进行拉曼光谱上直接非衍生化的胆甾醇酯馏分（参见图2和图9）。 第二个例子涉及嘌呤5&#39;-1,8 - 环-2&#39;-脱氧核糖核苷，这是创建的DNA损伤的自由基攻击的位置C5&#39;的糖部分和随后形成一个共价键之间的糖和碱部分。可以产生四种结构，即，5&#39;-1,8 -环-2&#39; -脱氧腺苷和5&#39;-1,8 -环-2&#39; -脱氧鸟苷，现有在5&#39;R和5&#39;S非对映异构体的形式（ 图5）。在图6吨中他的反应机理示出，涉及5的8 bromopurine衍生物光解，得到相应的C8自由基6，该分子内抽象从C5的位置选择性地，得到的2&#39;-deoxyadenosin-5&#39;-基的自由基7为氢原子。自由基7进行环化反应的速率常数的范围为10 5 -10 6 -1，2，然后通过氧化的杂芳族基团8，以使最终产品9中。羟基自由基的反应，所产生的辐解水，用2&#39;-脱氧腺苷，2&#39;-脱氧鸟苷（10），发现发生约。 10％的氢抽象C5&#39;的位置。 我们的化学生物学方法图书馆嘌呤5&#39;-1,8 -环-2&#39;-脱氧核糖核苷（包括标记的化合物）中示出在图7中，与这些病变在寡核苷酸的识别，以及在获得的DNA样品从弗吉尼亚州rious源，例如，例如，在电离辐射的条件下，处理，作为模仿自由基的应力条件。 在图8中示出一个典型的光反应器设备。该装置允许在适当的溶剂中溶解的化合物的照射。它包括：i）在反应腔室的入口为惰性气体（在底部）和气体出口的两个入口和试剂此外配备;ⅱ）含有适当的水银灯的一个内部腔室的冷却系统连接和电功率，其被插入到反应室，通过一个玻璃接头。 <img alt="图1" fo:content-width="6.5in" fo:src="/files/ftp_upload/50379/50379fig1highres.jpg" src="/files/ftp_upload/50379/50379fig1.jpg" /> 图1。单-反式异构体的，胆固醇亚油酸和花生四烯酸。 图2。胆甾醇酯在人血清和直接分析为单-反式异构体的拉曼光谱。 <img alt="图3" fo:content-width="4in" fo:src="/files/ftp_upload/50379/50379fig3highres.jpg" src="/files/ftp_upload/50379/50379fig3.jpg" /> 图3。加成-消除过程，导致双键的顺反异构化，生成硫中心自由基。 <img src="/files/ftp_upload/50379/50379fig4.jpg" alt="图4" /> 图4。三个步骤协议开发的单跨胆固醇酯作为生物标志物自由基的压力。 <img alt="图5" fo:content-width="6.5in" fO：SRC =“/ files/ftp_upload/50379/50379fig5highres.jpg”SRC =“/ files/ftp_upload/50379/50379fig5.jpg”/&gt; 图5。四嘌呤5&#39;,8-环-2-脱氧核糖核苷的非对映异构体。 <a href="https://www.jove.com/files/ftp_upload/50379/50379fig5large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图6" fo:content-width="6.5in" fo:src="/files/ftp_upload/50379/50379fig6highres.jpg" src="/files/ftp_upload/50379/50379fig6.jpg" /> 图6。 C5的自由基的生成，通过光解5或10 HO•自由基反应，嘌呤5&#39;,8-环-2&#39;脱氧核糖形成的机制。 <a href="https://www.jove.com/files/ftp_upload/50379/50379fig6large.jpg" target="_blank">点击此处查看大图</a> 。 <img src="/files/ftp_upload/50379/50379fig7.jpg" alt="图7" /> 图7。协议f或识别一些激进的基于DNA病变线粒体DNA：线粒体DNA，nDNA的核DNA。 <img src="/files/ftp_upload/50379/50379fig8.jpg" alt="图8" /> 图8。光化学反应器。 <img src="/files/ftp_upload/50379/50379fig9.jpg" alt="图9" /> 图9所示。血浆胆甾醇酯的代表性的拉曼光谱在插图与胆甾醇亚油酸酯，胆甾醇单-反亚油酸异构体的特定区域的比较。 <img src="/files/ftp_upload/50379/50379fig10.jpg" alt="图10" /> 图10。的FAME写照GC分析得到的从血浆中胆甾醇酯。 <img alt="图11" fo:content-width="4.5in" fo:src="/files/ftp_upload/50379/50379fig11highres.jpg" src="/files/ftp_upload/50379/50379fig11.jpg" /> &lt;强大的图11。代表HPLC含有2&#39;-脱氧腺苷运行，和2&#39;deoxyguanosine连同他们的氧化和嘌呤5&#39;,8-环-2&#39;-脱氧核糖核苷。 <a href="https://www.jove.com/files/ftp_upload/50379/50379fig11large.jpg" target="_blank">点击此处查看大图</a> 。

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Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development

自由基基于仿生化学已被应用到建筑物库所需的生物标志物的发展。

自由基化学生物学的化学行为，生物标记开发

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological ...

free-radicals-chemical-biology-from-chemical-behavior-to-biomarker

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Alkene Structure and Reactivity

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20.1K 次观看.  Consiglio Nazionale delle Ricerche. 自由基基于仿生化学已被应用到建筑物库所需的生物标志物的发展。

视频: Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

隔离和脂质A的化学表征革兰氏阴性菌

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

科研

化学

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

增强型氟烷烃的化学气相沉积等离子通过疏水金属 - 有机骨架的制备氨的去除

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in understanding contaminant mass transport and the ability to decontaminate materials. If a material is contaminated, over time, the transport of highly toxic chemicals (such as chemical warfare agent species) out of the material can result in vapor exposure or transfer to the skin, which can result in percutaneous exposure to personnel who interact with the material. Due to the high toxicity of chemical warfare agents, the release of trace chemical quantities is of significant concern. Mapping subsurface concentration distribution and transport characteristics of absorbed agents enables exposure hazards to be assessed in untested conditions. Furthermore, these tools can be used to characterize subsurface reaction dynamics to ultimately design improved decontaminants or decontamination procedures. To achieve this goal, an inverse analysis mass transport modeling approach was developed that utilizes time-resolved mass spectroscopy measurements of vapor emission from contaminated paint coatings as the input parameter for calculation of subsurface concentration profiles. Details are provided on sample preparation, including contaminant and material handling, the application of mass spectrometry for the measurement of emitted contaminant vapor, and the implementation of inverse analysis using a physics-based diffusion model to determine transport properties of live chemical warfare agents including distilled mustard (HD) and the nerve agent VX.

In this paper, a procedure for quantifying the mass transport parameters of chemicals in various materials is presented. This process involves employing an inverse-analysis based diffusion model to vapor emission profiles recorded by real-time, mass spectrometry in high vacuum.

一个反分析方法化工运输的油漆表征

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in ...

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

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is a major effort to target PPIs in basic research and in the pharmaceutical industry. Protein-protein interfaces are usually large, flat, and often lack pockets, complicating the discovery of small molecules that target such sites. Alternative targeting approaches using antibodies have limitations due to poor oral bioavailability, low cell-permeability, and production inefficiency.
Using peptides to target PPI interfaces has several advantages. Peptides have higher conformational flexibility, increased selectivity, and are generally inexpensive. However, peptides have their own limitations including poor stability and inefficiency crossing cell membranes. To overcome such limitations, peptide cyclization can be performed. Cyclization has been demonstrated to improve peptide selectivity, metabolic stability, and bioavailability. However, predicting the bioactive conformation of a cyclic peptide is not trivial. To overcome this challenge, one attractive approach it to screen a focused library to screen in which all backbone cyclic peptides have the same primary sequence, but differ in parameters that influence their conformation, such as ring size and position.
We describe a detailed protocol for synthesizing a library of backbone cyclic peptides targeting specific parasite PPIs. Using a rational design approach, we developed peptides derived from the scaffold protein Leishmania receptor for activated C-kinase (LACK). We hypothesized that sequences in LACK that are conserved in parasites, but not in the mammalian host homolog, may represent interaction sites for proteins that are critical for the parasites&#39; viability. The cyclic peptides were synthesized using microwave irradiation to reduce reaction times and increase efficiency. Developing a library of backbone cyclic peptides with different ring sizes facilitates a systematic screen for the most biological active conformation. This method provides a general, fast, and facile way to synthesize cyclic peptides.

A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.

一个主干环肽库作为潜在的抗寄生虫治疗微波辐射的发展

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

From Molecules to Materials: Engineering New Ionic Liquid Crystals Through Halogen Bonding

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid crystals (ILCs). Taking advantages of the high specificity of XB for haloperfluorocarbons and the ability of anions to act as XB-acceptors, we obtained supramolecular complexes based on 1-alkyl-3-methylimidazolium iodides and iodoperfluorocarbons, overcoming the well-known immiscibility between hydrocarbons (HCs) and perfluorocarbons (PFCs). The high directionality of the XB combined with the fluorophobic effect, allowed us to obtain enantiotropic liquid crystals where a rigid, non-aromatic, XB supramolecular anion acts as mesogenic core.
X-ray structure analysis of the complex between 1-ethyl-3-methylimidazolium iodide and iodoperfluorooctane showed the presence of a layered structure, which is a manifestation of the well-known tendency to segregation of perfluoroalkyl chains. This is consistent with the observation of smectic mesophases. Moreover, all the reported complexes melt below 100 &#176;C, and most are mesomorphic even at room temperature, despite that the starting materials were non-mesomorphic in nature.
The supramolecular strategy reported here provides new design principles for mesogen design allowing a totally new class of functional materials.

A protocol for the synthesis of a new type of mesogens, based on the halogen-bonded supramolecular anion [CnF2n+1-I&#183;&#183;&#183;I&#183;&#183;&#183;I-CnF2n+1]&#8722;, is reported.

从分子到材料: 通过卤素键合工程新的离子液体晶体

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid ...

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

糖注射水凝胶的制备及其在3D 细胞培养中的应用

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

One-pot Microwave-assisted Conversion of Anomeric Nitrate-esters to Trichloroacetimidates

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate glycosyl donor. Following azido-nitration of a glycal, the product 2-azido-1-nitrate ester can be hydrolyzed under microwave-assisted irradiation. This transformation is usually achieved using strongly nucleophilic reagents and extended reaction times. Microwave irradiation induces hydrolysis, in the absence of reagents, with short reaction times. Following denitration, the intermediate anomeric alcohol is converted, in the same pot, to the corresponding 2-azido-1-trichloroacetimidate.

A 2-azido-1-nitrate-ester can be converted to the corresponding 2-azido-1-trichloroacetimidate in a one-pot procedure. The goal of the manuscript is to demonstrate utility of the microwave reactor in carbohydrate synthesis.

硝酸酯 Anomeric Trichloroacetimidates 的单锅微波辅助转化

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate ...

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

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

利用光致硫醇-烯/yne Hydrothiolation 构造硫醚类/乙烯基硫化物栓系螺旋肽

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...