This research was supported by the National Institutes of Health NIGMS (R01-GM094541) and Boston College.

注:所有氧和水分敏感的操作均在惰性气氛（N 2）使用标准无空气技术或在手套箱中进行。 THF（四氢呋喃），Et 2 O等（乙醚），CH 2 Cl 2中（二氯甲烷），甲苯和戊烷，通过中性氧化铝柱和氩气下经过纯化。乙腈经氢化钙2（氢化钙）干燥，并在使用前在氮气氛下蒸馏。的Pd / C（在碳上的钯）的混合物在100℃下在高真空下加热，使用前12小时。硅胶（230-400目）为在180℃在高真空下12小时干燥。快速色谱法在惰性气氛下与此硅胶进行。所有其它化学品和溶剂均购并原样使用。 注意:注意，使用前请咨询所有相关的材料安全数据表（MSDS）。几个在合成中使用的化学品的重新急性毒性和致癌性。 1. 1,2- Azaborines的制备注:在这项研究中的所有化合物的表征数据先前已经报道12，13，16。 <img alt="图1" src="/files/ftp_upload/55154/55154fig1.jpg" /> 图1:1,2- azaborines合成方案。每个化合物（1-5）的合成详细协议在协议部分中描述。 <a href="http://ecsource.jove.com/files/ftp_upload/55154/55154fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li>制备N -allyl-ñ-TBS- 乙氯烯丙基加合物（1） <ol><li>在手套箱中，量出6.70克（50.0毫摩尔）triallylboraNE 22到烘箱干燥的24/40 1升圆底烧瓶装有搅拌棒并加入250毫升二氯甲烷。密封用橡胶隔膜烧瓶中。外箱，冷却该溶液至-78℃，在真空气体歧管在氮气氛下。 </li><li>加的己烷为1.00μm三氯化硼溶液（100毫摩尔），以通过套管转移在-78℃的triallylborane溶液100毫升。让在该温度4小时，将反应混合物搅拌。 注意:小心，三氯化硼与水高度反应。 </li><li>在一个单独的100 mL容量瓶中，测出25.7克N - （ 叔丁基二甲基） - N -allylamine（150毫摩尔），并添加20.0毫升二氯甲烷。向1升反应烧瓶中，通过套管转移保持在-78℃的反应温度加入该溶液中。 </li><li>加21.0毫升三乙胺（150毫摩尔）到反应中。保持在氮气正流搅拌，并允许它逐渐温热至ロ嗡温度过夜。 </li><li>搅拌18小时后，通过使用带有液氮阱高真空（300-500毫托）的真空气体歧管除去溶剂的大约一半。 </li><li>取反应烧瓶入手套箱并通过配有一个24/40真空适配器烘箱干燥的中等孔隙率玻璃料和24/40的500mL圆底烧瓶中搅拌滤出形成在反应混合物中的白色盐酒吧。使用二氯甲烷转移所有剩余的材料。 </li><li>使用具有高真空的真空气体歧管，箱外滤液删除所有剩余的挥发性物质。 </li><li>在手套箱中，将浓缩的反应混合物转移到烘箱干燥的14/20 100毫升圆底烧瓶装有搅拌棒，以进行蒸馏制备。帽用隔膜烧瓶。 </li><li>在氮气流，迅速打开该隔膜并加入200毫克氢化钙作为干燥剂。 </li><li>通过安装一个oven-设置蒸馏装置干燥附着有温度计14/20蒸馏头，一面带接收14/20 50毫升无空气存储烧瓶中，并在另一侧与含有粗产物的100毫升烧瓶中。装备蒸馏头用冷却水。 （使用润滑脂的蒸馏装置的所有关节。） 注意产品开始时的蒸气温度达到300毫乇60℃以蒸馏。 </li><li>继续通过升高油浴的温度，直到蒸馏头温度计读取75℃收集产品。 注:该产品是一种透明无色液体。 </li><li>通过除去热源停止蒸馏和存储前冷却装置到室温。冷却后，repressurize用氮气的装置和紧紧关闭塞阀上的自由空气存储烧瓶中。存放在手套箱冷冻器中分离出的产物。 注:产率可以有所不同，从65％至84％。 </li></ol></li><li>制备N </eM&gt; -TBS- 乙 -Cl闭环产物（2） <ol><li>在手套箱中，测量在烘箱干燥的24/40 1升圆底烧瓶出56.0克N- -allyl-Ñ-TBS- 乙烯丙基氯化物加合物（1）（217毫摩尔）有搅拌棒，加入500甲苯溶液。 </li><li>测量出1.79克格鲁布斯第一代催化剂（2.17毫摩尔）。 </li><li>添加催化剂分批至在室温下，在手套箱1中的搅拌溶液中。在加入过程中清除框以除去由反应形成的乙烯气体。 注:该反应是气进化停止的30分钟内完成典型。作为完成的另一个指标，反应颜色已经完全从最初的紫色变为褐色。在这一点上，产品可以用真空蒸馏分离;详情请参阅参考12。在这个协议中，为3的直接合成一釜两步过程进行说明。 </li></OL&gt; </li><li> Ñ-TBS- 乙 -Cl -1,2- azaborine的制备（3） <ol><li>到在甲苯中闭环产物（2）的反应溶液中，添加碳（10重量％的Pd，13.2毫摩尔）14.0克钯。 </li><li>从盒中取出烧瓶，并与已用氮气吹扫，并配有冷却水烘箱干燥的24/40回流冷凝器适合它。 </li><li>在搅拌的同时在135℃使用油浴于剧烈回流加热反应。 </li><li>在回流下保持16小时后，冷却通过从油浴中取出烧瓶中的反应。在冷却至室温后，撤回使用装有烘箱干燥的路厄锁定针的注射器，将反应混合物用0.5毫升等分试样。转移0.5mL的等分试样到螺旋盖的NMR管经由图 11B核磁共振12来分析。 注:期望的产物峰出现在约 35.5 ppm的12，而开始脑膜IAL峰出现在加利福尼亚州 。 42.9 ppm的12。通常在这一点上图 11B NMR表明反应是不完全的，具有轻微的原料峰剩余。 </li><li>如果反应不完全，使反应烧瓶入手套箱并碳（10重量％的Pd，2.82毫摩尔）添加一个附加3.00克钯。 </li><li> Resubject反应如1.3.2和1.3.3中所述至回流条件。 </li><li>在回流另外24小时后，分析由图 11B核磁共振反应的另一等分试样。在这一点上，反应应当是完整的。 </li><li>反应烧瓶转移到手套箱中，并通过填充有纸湿巾一次性快速色谱柱通过反应混合物。收集洗涤滤液用50.0毫升甲苯。 </li><li>禁区外的滤液上配备了高真空的真空气体歧管移除所有挥发性物质。 注:在除去挥发物可以采取40分钟吨直径:90分钟根据真空压力。当真空计中读取的最大压力，它表明除去溶剂完全。同时除去溶剂，确保该烧瓶下方的水浴保持在室温下的散热。 </li><li>在手套箱中，将浓缩反应混合物用搅拌棒转移到烘箱干燥的24/40的250mL圆底烧瓶中。帽用隔膜烧瓶。 </li><li>迅速除去隔膜和加入200毫克氢化钙作为干燥剂。 </li><li>通过拟合安装有温度计烘箱干燥的24/40蒸馏头，一面带接收24/40 100毫升无空气存储烧瓶中，并在另一侧与含有粗产物的100mL烧瓶设置蒸馏装置。装备蒸馏头用冷却水。使用润滑脂的蒸馏装置的所有关节。 注意产品开始时蒸汽温度达到65至70℃，在1000毫托与油以蒸90-100℃之间浴温度。该产品是一种无色透明的液体。 </li><li>通过除去热源停止蒸馏和存储前冷却装置到室温。冷却后，repressurize用氮气的装置和紧紧关闭塞阀上的自由空气存储烧瓶中。在-30℃储存在手套箱冷冻器分离的产物。 注:一锅，两步过程中的收益率是52％。 </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/55154/55154fig2.jpg" /> 图2:对于N -TBS- 乙 -Cl -1,2- azaborine监测形成图 11B NMR谱（3）。 a）将 正 -Allyl-Ñ-TBS-B-烯丙基氯加合物（1），开始用于闭环复分解的材料。 B）16小时后氧化。 （未反应的N- -TBS- 乙 -Cl闭环产物（2）和异构化乙 -乙烯基闭环产物（2&#39;）与N- TBS-B-氯-1,2- azaborine沿（3）被示出。）c）另外24小时后氧化。通过真空蒸馏纯化后D）的分离的产物（3）。 <a href="http://ecsource.jove.com/files/ftp_upload/55154/55154fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="4"><li> ñ-H- 乙 -OBu -1,2- azaborine的制备（4） <ol><li>在手套箱中，量出4.00克N- TBS -B-氯-1,2- azaborine （3）（17.6毫摩尔）在烘箱干燥的100毫升圆底烧瓶装有搅拌棒并加入35毫升乙腈。量出1.14克乙酰胺（19.3毫摩尔）和转移到反应烧瓶中。密封用橡胶隔膜烧瓶中。 </li><li>从包装盒中取出反应瓶，适合采用OVEN干燥已用氮气吹扫，并配有冷却水24/40回流冷凝器。 </li><li>加热反应在85℃的油浴中搅拌下回流。 </li><li>在回流3小时后，冷却通过从油浴中取出烧瓶中的反应。在冷却至室温后，除去上配备有高真空的真空气体歧管溶剂。 </li><li>在真空下除去挥发物后，再溶解于15.0毫升THF中的反应残基。在该溶液中添加正丁醇（35.1毫摩尔）的3.21毫升。 </li><li>在室温下搅拌1小时进行反应。 </li><li>在配备有高真空，得到粗产物真空气体歧管除去溶剂。 </li><li>在手套箱中，通过硅胶柱色谱法分离出产物，用戊烷和乙醚的混合物（5:1）作为洗脱剂。从用装有高真空的真空气体歧管的含产物的级分除去挥发物，得到纯的产物，为白色固体。 注:产量可能会有所不同，从82％到87％。 </li></ol></li><li> Ñ-H- 乙 -乙基-1,2- azaborine的制备（5） <ol><li>在手套箱中，量出1.00克N-H -B-在烘箱干燥的100毫升圆底烧瓶OBU -1,2- azaborine（4）（6.62毫摩尔）有搅拌棒，并添加25毫升的Et 2 O.印章上的真空气体歧管的 N 2下的橡胶隔膜和地点烧瓶。 </li><li>在一个单独的烘箱干燥的100mL圆底烧瓶中，加入26.5毫升的乙基锂溶液（0.5M的苯/环己烷，13.2毫摩尔）在氮气氛下。从乙基锂试剂除去溶剂（苯/环己烷）的混合物，以避免与分离所需azaborine产物的问题。重新溶解固体乙基锂13.0毫升的Et 2 O. 注意:注意:有机锂试剂是自燃（点燃与空气或水接触）。所需产物（5 </strong&gt;）特别挥发性（MW = 106.96）。因此，需要从乙基锂试剂除去苯/环己烷溶剂中的纯产物的有效的隔离，由于与所需产物的类似的波动。 </li><li>冷却含有4至-30℃的冷却浴的反应烧瓶中。 </li><li>添加通过套管转移乙基锂溶液至反应烧瓶中在-30℃。保持在此温度下搅拌1小时，并允许它慢慢温热至室温。 </li><li>监测由图 11B核磁共振反应。 （所希望的中间峰出现在约 39.8 ppm的）。 </li><li>反应完成后，冷却反应至-30℃，并添加6.62毫升的HCl溶液（在Et 2 O等2.0M，13.2毫摩尔）。搅拌1小时的反应，同时允许其缓慢温热至室温。 </li><li>在装有低真空（20-25托），得到粗产物混合物在真空气体歧管除去溶剂。 </li><li>在手套箱中，通过硅胶柱色谱法分离出产物，用2-甲基丁烷作为洗脱剂。除去上配备有一个衰减真空，得到纯产物，为无色液体的真空气体歧管挥发。 注:产量是56％。 </li></ol></li></ol> 2.蛋白质制备及T4溶菌酶突变体的结晶<ol><li>蛋白表达和纯化<ol><li>生长与亚克隆质粒（L99A或L99A / M102Q）在200毫升的LB 17，20（LB培养基）在37℃下补充有40.0毫克氨苄青霉素12小时媒体转化的大肠杆菌 RR1细胞。 注:LB培养基的组成为12g胰蛋白胨，5g酵母提取物，10g氯化钠和每1 LH 2 O 1克葡萄糖</li><li>接种4.00大号LB培养基（补充有800毫克的氨苄青霉素）与160毫升过夜培养，并在37℃用filtere孵育以240rpmD气供应。 </li><li>当光密度在600nm达到0.7，通过从孵化器中取出冷却该细菌培养至室温。 </li><li>在一圆锥形离心管量出695毫克异丙基β-D-1-硫代半乳糖苷（IPTG）（0.7毫摩尔最终浓度）并加入4毫升的LB培养基溶解。 </li><li>该解决方案IPTG加入到培养诱导蛋白的表达。孵育在110转21小时的培养物在25℃。 </li><li>离心文化在4,412 XG在4℃下30分钟。弃上清，重悬沉淀在300毫升的0.1M磷酸钠（pH 6.6），0.2M NaCl和添加0.5M的乙二胺四乙酸（EDTA）（pH8.0）中30.0毫升。搅拌在4℃下17小时的悬浮液。 </li><li>加30.0毫升脱氧核糖核酸酶的1.0M的MgCl 2和0.500毫升我（DNA酶I）到悬浮液中，并在室温下搅拌8小时。 </li><li>离心裂解细胞悬液在30913 XG在4 30分钟<html> 76℃。收集含有表达的蛋白的上清液并弃沉淀。 </li><li>在4℃下透析对20mM磷酸钠（pH 6.5 L99A和pH 6.3 L99A / M102Q）的裂解物过夜。 </li><li>洗涤和平衡的用在一柱平衡缓冲液（50mM的Tris，1mM EDTA中（pH为7.3））羧甲基离子交换珠20.0毫升。装载含溶液的蛋白质到珠子和用100mL的平衡缓冲液洗柱。洗脱用300mM NaCl的的平衡缓冲液中的600毫升线性梯度的列。 </li><li>使用低压层析系统的柱纯化。使用1毫升/分钟的泵速率，并收集10毫升馏分。收集含有纯蛋白质的级分。 注意:在洗脱，监测280nm处的光密度（OD）来验证蛋白质的存在。确定通过SDS-PAGE各馏分的纯度和仅收集最纯的级分。 </li></ol></li></ol>"FO:保together.within页="1"&gt; <img alt="图1" src="/files/ftp_upload/55154/55154fig3.jpg" /> 图3:T4溶菌酶突变体L99A / M102Q的蛋白质纯化的代表性结果。 A）色谱表示在AU所测量的UV吸光度在280nm（蓝线）和电导率在毫秒/厘米（红线）。级分20-23合并，并且纯度是通过SDS-PAGE测定。 B）的 15％SDS-PAGE凝胶显示了在18.6 kDa的级分20-23中的纯化的蛋白的存在。 <a href="http://ecsource.jove.com/files/ftp_upload/55154/55154fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="2"><li>蛋白质结晶<ol><li>转移纯化的蛋白质溶液至透析膜管（12,000 14,000 MWCO）。放置透析管到含有4.00 L 100 mM磷酸钠，550 mM氯化钠，0.02％NaN 3的（为L99A pH值6.5和pH值6.3 L的烧杯99A / M102）。搅拌在4℃下的4升溶液过夜。 </li><li>加入2-巯基乙醇（5毫米的终浓度）的透析的样品，并集中到〜40.0毫克/毫升使用离心浓缩（10,000 MWCO）。通过测量280nm处的吸收确定的蛋白浓度。通过结晶前纺去除任何沉淀。 </li><li>使用了蛋白质结晶的蒸汽扩散悬滴法。混合使用上的硅化玻璃盖玻片贮存溶液（2.0-2.2酸钠/磷酸钾，pH 6.7-7.1，50毫摩尔2-巯基乙醇，50mM的2-羟乙基二硫化物）5.00微升的浓缩的蛋白质溶液的5.00微升。 </li><li>通过将盖玻片倒置在每口井含贮备溶液顶部平衡对1.00毫升贮备溶液混合下降。使用油脂与滑封紧每口井。使用与贮存溶液24孔板在2.2.3中提到设置的各种条件。在4℃晶体生长存储盘。 注:晶体通常在封面上的幻灯片上的平衡缓冲液1-2周之内增长。在步骤2.2.3贮存溶液混合蛋白溶液中析出立即指示结晶失败。 </li></ol></li></ol><img alt="图4" src="/files/ftp_upload/55154/55154fig4.jpg" /> 图4:与配体络合之前T4溶菌酶突变体L99A的晶体的代表性图片。晶体在母液（2.1米钠/磷酸钾，pH 6.9，50mM的2-巯基乙醇，50mM的2-羟乙基二硫化物）。 <a href="http://ecsource.jove.com/files/ftp_upload/55154/55154fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="3"><li>复合制剂<ol><li>挑选使用塑造成一个循环低温管晶体（见材料清单）和将它们放置在一个0.6毫升离心管中。添加母液50.0μL到所述管，以避免干燥和保存的晶体。 注:单晶选择用显微镜的帮助。 </li><li>传送含有晶体管进入的手套箱。取azaborine配体（5）和地点的5.00微升管的卡扣帽的内部。帽与管存储在2-7天通过蒸汽扩散平衡的手套箱内冰箱（4℃）。 注:azaborine配体（5）的物理性能（高挥发性和溶解性在水溶液中）使与通过蒸汽扩散法的预制蛋白质晶体的络合。 </li><li>转移使用成形为一个循环来含氮paratone的在液氮中快速冷冻之前的液滴载玻片低温管道的晶体。由暴跌对晶体挑选具有N-paratone并迅速闪冷冻保护的结晶循环到含有液氮的杜瓦瓶。通过使用低温钳安装在环路的结晶。 </li><li>收集的同步辐射的单晶的数据集。使用公开的结构20,24进行分子置换;由电子密度的未建模的斑点验证蛋白结合位点内的配体的存在。 </li></ol></li></ol><img alt="图5" src="/files/ftp_upload/55154/55154fig5.jpg" /> 图5:T4溶菌酶突变L99A的原子模型与电子密度的结合口袋。 A）L99A腔的结合位点的未建模的电子密度斑点。 B）L99A腔在两个备选构建模azaborine配体5。 <a href="http://ecsource.jove.com/files/ftp_upload/55154/55154fig5large.jpg" target="_blank">请CL</a>ICK这里查看该图的放大版本。

<ol>
	<li>Ducruix, A., Gi&#233;ge, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Crystallization of Nucleic Acids and Proteins, A Practical Approach. , (1992).</li><li>Bosdet, M. J. D., Piers, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-N+as+a+C-C+substitute+in+aromatic+systems.">B-N as a C-C substitute in aromatic systems.</a> Can. J. Chem. 87 (1), 8-29 (2009).</li><li>Campbell, P. G., Marwitz, A. J., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Advances+in+Azaborine+Chemistry.">Recent Advances in Azaborine Chemistry.</a> Angew. Chem. Int. Ed. 51 (25), 6074-6092 (2012).</li><li>Wang, X. -. Y., Wang, J. -. Y., Pei, J. B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterosuperbenzenes:+Synthesis+and+Properties.">Heterosuperbenzenes: Synthesis and Properties.</a> Chem. Eur. J. 21 (9), 3528-3539 (2015).</li><li>Zhou, H. -. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elemental+isomerism:+a+boron-nitrogen+surrogate+for+a+carbon-carbon+double+bond+increases+the+chemical+diversity+of+estrogen+receptor+ligands.">Elemental isomerism: a boron-nitrogen surrogate for a carbon-carbon double bond increases the chemical diversity of estrogen receptor ligands.</a> Chem. Biol. 14 (6), 659-669 (2007).</li><li>Ito, H., Yumura, K., Saigo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+characterization,+and+binding+property+of+isoelectronic+analogues+of+nucleobases,+B(6)-substituted+5-aza-6-borauracils+and+-thymines.">Synthesis, characterization, and binding property of isoelectronic analogues of nucleobases, B(6)-substituted 5-aza-6-borauracils and -thymines.</a> Org. Lett. 12 (15), 3386-3389 (2010).</li><li>Vlasceanu, A., Jessing, M., Kilburn, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BN/CC+isosterism+in+borazaronaphthalenes+towards+phosphodiesterase+10A+(PDE10A)+inhibitors.">BN/CC isosterism in borazaronaphthalenes towards phosphodiesterase 10A (PDE10A) inhibitors.</a> Bioorg. Med. Chem. 23 (15), 4453-4461 (2015).</li><li>Rombouts, F. J., Tovar, F., Austin, N., Tresadern, G., Trabanco, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzazaborinines+as+Novel+Bioisosteric+Replacements+of+Naphthalene:+Propranolol+as+an+Example.">Benzazaborinines as Novel Bioisosteric Replacements of Naphthalene: Propranolol as an Example.</a> J. Med. Chem. 58 (23), 9287-9295 (2015).</li><li>Culling, G. C., Dewar, M. J. S., Marr, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Heteroaromatic+Compounds.+XXIII.+Two+Analogs+of+Triphenylene+and+a+Possible+Route+to+Borazarene.">New Heteroaromatic Compounds. XXIII. Two Analogs of Triphenylene and a Possible Route to Borazarene.</a> J. Am. Chem. Soc. 86 (6), 1125-1127 (1964).</li><li>White, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-Phenyl-2,1-borazarene+and+Derivatives+of+1,2-Azaboracycloalkanes.">2-Phenyl-2,1-borazarene and Derivatives of 1,2-Azaboracycloalkanes.</a> J. Am. Chem. Soc. 85 (22), 3634-3636 (1963).</li><li>Ashe III, A. J., Fang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Synthesis+of+Aromatic+Five-+and+Six-Membered+B&#8722;N+Heterocycles+via+Ring+Closing+Metathesis.">A Synthesis of Aromatic Five- and Six-Membered B&#8722;N Heterocycles via Ring Closing Metathesis.</a> Org. Lett. 2 (14), 2089-2091 (2000).</li><li>Marwitz, A. J. V., Matus, M. H., Zakharov, L. N., Dixon, D. A., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Hybrid+Organic/Inorganic+Benzene.">A Hybrid Organic/Inorganic Benzene.</a> Angew. Chem. Int. Ed. 48 (5), 973-977 (2009).</li><li>Abbey, E. R., Lamm, A. N., Baggett, A. W., Zakharov, L. N., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+Group-Free+Synthesis+of+1,2-Azaborines:+A+Simple+Approach+to+the+Construction+of+BN-Benzenoids.">Protecting Group-Free Synthesis of 1,2-Azaborines: A Simple Approach to the Construction of BN-Benzenoids.</a> J. Am. Chem. Soc. 135 (34), 12908-12913 (2013).</li><li>Rudebusch, G. E., Zakharov, L. N., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodium-catalyzed+boron+arylation+of+1,2-azaborines.">Rhodium-catalyzed boron arylation of 1,2-azaborines.</a> Angew. Chem. Int. Ed. 52 (35), 9316-9319 (2013).</li><li>Brown, A. N., Li, B., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negishi+Cross-Coupling+Is+Compatible+with+a+Reactive+B-Cl+Bond:+Development+of+a+Versatile+Late-Stage+Functionalization+of+1,2-Azaborines+and+Its+Application+to+the+Synthesis+of+New+BN+Isosteres+of+Naphthalene+and+Indenyl.">Negishi Cross-Coupling Is Compatible with a Reactive B-Cl Bond: Development of a Versatile Late-Stage Functionalization of 1,2-Azaborines and Its Application to the Synthesis of New BN Isosteres of Naphthalene and Indenyl.</a> J. Am. Chem. Soc. 137 (28), 8932-8935 (2015).</li><li>Knack, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BN/CC+Isosteric+Compounds+as+Enzyme+Inhibitors:+N-+and+B-Ethyl-1,2-azaborine+Inhibit+Ethylbenzene+Hydroxylation+as+Non-Convertible+Substrate+Analogs.">BN/CC Isosteric Compounds as Enzyme Inhibitors: N- and B-Ethyl-1,2-azaborine Inhibit Ethylbenzene Hydroxylation as Non-Convertible Substrate Analogs.</a> Angew. Chem. Int. Ed. 52 (9), 2599-2601 (2013).</li><li>Eriksson, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+a+Protein+Structure+to+Cavity-Creating+Mutations+and+Its+Relation+to+the+Hydrophobic+Effect.">Response of a Protein Structure to Cavity-Creating Mutations and Its Relation to the Hydrophobic Effect.</a> Science. 255 (5041), 178-183 (1992).</li><li>Eriksson, A. E., Baase, W. A., Wozniak, J. A., Matthews, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cavity-containing+mutant+of+T4+lysozyme+is+stabilized+by+buried+benzene.">A cavity-containing mutant of T4 lysozyme is stabilized by buried benzene.</a> Nature. 355 (6358), 371-373 (1992).</li><li>Morton, A., Baase, W. A., Matthews, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetic+Origins+of+Specificity+of+Ligand+Binding+in+an+Interior+Nonpolar+Cavity+of+T4+Lysozyme.">Energetic Origins of Specificity of Ligand Binding in an Interior Nonpolar Cavity of T4 Lysozyme.</a> Biochemistry. 34 (27), 8564-8575 (1995).</li><li>Wei, B. Q., Baase, W. A., Weaver, L. H., Matthews, B. W., Shoichet, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Model+Binding+Site+for+Testing+Scoring+Functions+in+Molecular+Docking.">A Model Binding Site for Testing Scoring Functions in Molecular Docking.</a> J. Mol. Biol. 322 (2), 339-355 (2002).</li><li>Lee, H., Fischer, M., Shoichet, B. K., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+Bonding+of+1,2-Azaborines+in+the+Binding+Cavity+of+T4+Lysozyme+Mutants:+Structures+and+Thermodynamics.">Hydrogen Bonding of 1,2-Azaborines in the Binding Cavity of T4 Lysozyme Mutants: Structures and Thermodynamics.</a> J. Am. Chem. Soc. 138 (37), 12021-12024 (2016).</li><li>Brown, H. C., Racherla, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoboranes.+43.+A+Convenient,+Highly+Efficient+Synthesis+of+Triorganylboranes+via+a+Modified+Organometallic+Route.">Organoboranes. 43. A Convenient, Highly Efficient Synthesis of Triorganylboranes via a Modified Organometallic Route.</a> J. Org. Chem. 51 (4), 427-432 (1986).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Liu, L., Baase, W. A., Matthews, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Halogenated+Benzenes+Bound+within+a+Non-polar+Cavity+in+T4+Lysozyme+Provide+Examples+of+I.S+and+I.Se+Halogen-bonding.">Halogenated Benzenes Bound within a Non-polar Cavity in T4 Lysozyme Provide Examples of I.S and I.Se Halogen-bonding.</a> J. Mol. Biol. 385 (2), 595-605 (2009).</li></ol>

All authors declare no competing financial interests in this manuscript.

在这个协议中的第一部分中，我们描述的基于先前报道的方法12,13 1,2- azaborines修饰合成。 Triallylborane 22被用作使用allyltriphenyl锡或钾allyltrifluoroborate制备路线的替代Ñ-allyl-Ñ-TBS- 乙烯丙基氯化物加合物（1）。这种方法允许更原子经济和环保的做法。对于N -TBS- 乙 -Cl -1,2- azaborine（3）</st...

含杂环（ 即 1,2- azaborines）硼-氮最近已经引起显著关注，芳烃等排。此isosterism可导致现有的结构图案的多样化，扩大化学空间2，3，4。 Azaborines具有在生物医学研究5，6，7，8为应用潜在效用，尤其是在药物化学中化学家进行的结构库和功能相关的分子的合成的区域。显著，然而，尽管有无数发达合成路线可用含芳烃分子，只对azaborines的合成方法有限数量已报道9，10， </s达&gt; 11，12，13。这主要是由于对于硼源和空气和该分子的湿气敏感性质在合成序列的早期阶段的选择是有限的。 在本文的第一部分，我们将介绍使用标准无空气技术（3）N -TBS- 乙 -Cl-1,2-azaborine的多克规模的合成。该化合物作为可被进一步官能化以在结构上更复杂的分子14,15一种多用途的中间体。从3开始，N- -H- 乙 -乙基-1,2- azaborine（5）在蛋白结合研究使用的合成和纯化将描述。由于5的波动，其高效分离需要的反应温度，时间，和DIST的精确控制推理的条件。 在第二部分中，用于蛋白质表达和T4溶菌酶突变体（L99A和L99A / M102Q）17，18，19的分离方案，20将呈现，接着蛋白质结晶和制备蛋白质-配体晶体复合物。 T4溶菌酶突变体L99A和L99A / M102Q被选为生物模型系统来检查包含azaborine分子17 NH的氢键能力。使用标准的分子生物学协议，该蛋白在大肠杆菌 RR1菌株表达并用异丙基β-D-1-硫代半乳糖苷（IPTG）诱导。蛋白质纯化进行使用离子交换柱层析。蛋白质结晶使用悬高度浓缩纯化的蛋白质溶液（&gt; 95％纯度通过凝胶电泳）上进行滴气相扩散法。由于本研究的配位体对氧的敏感性，蛋白质 - 配体配合物是无空气的条件下制备。

<table><tbody><tr><td>Tetrahydrofuran (THF), inhibitor-free, for HPLC, &amp;ge;99.9%</td><td>Sigma Aldrich</td><td>34865</td><td></td></tr><tr><td>Diethyl ether (Et&lt;sub&gt;2&lt;/sub&gt;O), for HPLC, &amp;ge;99.9%, inhibitor-free</td><td>Sigma Aldrich</td><td>309966</td><td></td></tr><tr><td>Methylene chloride&amp;nbsp; (CH&lt;sub&gt;2&lt;/sub&gt;Cl&lt;sub&gt;2&lt;/sub&gt;), (Stabilized/Certified ACS)</td><td>Fisher</td><td>D37-20</td><td></td></tr><tr><td>Toluene</td><td>Fisher</td><td>T290-4</td><td></td></tr><tr><td>Pentane, HPLC</td><td>Fisher</td><td>P399-4</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher</td><td>A21-4</td><td></td></tr><tr><td>Calcium hydride (CaH&lt;sub&gt;2&lt;/sub&gt;), reagent grade, 95%</td><td>Sigma Aldrich</td><td>208027</td><td>Pyrophoric</td></tr><tr><td>Palladium on activated carbon (Pd/C), 10 wt% Pd</td><td>Strem</td><td>46-1900</td><td></td></tr><tr><td>1.0 M Boron trichloride solution in hexane</td><td>Sigma Aldrich</td><td>211249</td><td>Highly toxic/Pyrophoric</td></tr><tr><td>Triethylamine, &amp;ge;99.5%</td><td>Sigma Aldrich</td><td>471283</td><td></td></tr><tr><td>Grubbs 1&lt;sup&gt;st&lt;/sup&gt; generation catalyst&amp;nbsp;</td><td>materia</td><td>C823</td><td></td></tr><tr><td>Acetamide</td><td>Sigma Aldrich</td><td>A0500</td><td></td></tr><tr><td>n-Butanol, anhydrous, 99.8%</td><td>Sigma Aldrich</td><td>281549</td><td></td></tr><tr><td>Ethyllithium solution, 0.5&amp;nbsp;M in benzene/cyclohexane</td><td>Sigma Aldrich</td><td>561452</td><td>Highly toxic/ Pyrophoric</td></tr><tr><td>HCl solution, 2.0 M in Et&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>455180</td><td></td></tr><tr><td>2-Methylbutane, anhydrous, &amp;ge;99%</td><td>Sigma Aldrich</td><td>277258</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt;, (Migula) Castellani and Chalmers (ATCC&amp;reg; 31343&amp;trade;)</td><td>ATCC</td><td>31343</td><td></td></tr><tr><td>T4 lysozyme WT* (L99A)</td><td>Addgene</td><td>18476</td><td></td></tr><tr><td>T4 lysozyme mutant (S38D L99A M102Q N144D)</td><td>Addgene</td><td>18477</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma Aldrich</td><td>A0166</td><td></td></tr><tr><td>isopropyl-&amp;beta;-D-1-thiogalactopyranoside (IPTG)&amp;nbsp;</td><td>Invitrogen</td><td>AM9464</td><td></td></tr><tr><td>Sodium phosphate monobasic anhydrous</td><td>Fisher</td><td>BP329</td><td></td></tr><tr><td>Sodium Phosphate dibasic anhydrous</td><td>Fisher</td><td>BP332</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher</td><td>S642212&amp;nbsp;</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Fisher</td><td>BP118</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma Aldrich</td><td>M4880</td><td>Corrosive</td></tr><tr><td>Thermo scientific pierce DNaseI</td><td>Fisher</td><td>PI-90083</td><td></td></tr><tr><td>GE Healthcare&amp;nbsp;Sepharose Fast Flow Cation Exchange Media</td><td>Fisher</td><td>45-002-931</td><td></td></tr><tr><td>Tris-base</td><td>Fisher</td><td>BP152-500&amp;nbsp;</td><td></td></tr><tr><td>Sodium azide</td><td>TCI</td><td>S0489</td><td>Highly toxic</td></tr><tr><td>2-Mercaptoethanol</td><td>Fisher</td><td>ICN806443&amp;nbsp;</td><td></td></tr><tr><td>Sartorius&amp;nbsp;Vivaspin 20 Centrifugal Concentrators</td><td>Fisher</td><td>14-558-501</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P5379</td><td></td></tr><tr><td>2-Hydroxyethyl disulfide</td><td>Sigma Aldrich</td><td>380474</td><td></td></tr><tr><td>N-paratone&amp;nbsp;</td><td>Hampton Research</td><td>HR2-643</td><td></td></tr><tr><td>4 RC Dialysis Membrane Tubing 12,000 to 14,000 Dalton MWCO&amp;nbsp;</td><td>Fisher</td><td>08-667E</td><td></td></tr><tr><td>&amp;nbsp;CryoLoop</td><td>Hampton Research</td><td></td><td>cryogenic tubing shaped into a loop</td></tr><tr><td>CryoTong</td><td>Thermo Fisher</td><td></td><td>cryogenic tong</td></tr><tr><td>Coot</td><td></td><td></td><td>Electron density images are generated from the software</td></tr></tbody></table>

synthesis of 1,2-azaborines and the preparation of their protein complexes with t4 lysozyme mutants

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor diffusion. Oxygen- and moisture-sensitive compounds are prepared and isolated under an inert atmosphere (N2) using either a vacuum gas manifold or a glove box. As an example of azaborine synthesis, we demonstrate the synthesis and purification of the volatile N-H-B-ethyl-1,2-azaborine by a five-step sequence involving distillation and column chromatography for the isolation of products. T4 lysozyme mutants L99A and L99A/M102Q are expressed with Escherichia coli RR1 strain. Standard protocols for chemical cell lysis followed by purification using carboxymethyl ion exchange column affords protein of sufficiently high purity for crystallization. Protein crystallization is performed in various concentrations of precipitant at different pH ranges using the hanging drop vapor diffusion method. Complex preparation with the small molecules is carried out by vapor diffusion method under an inert atmosphere. X-ray diffraction analysis of the crystal complex provides unambiguous structural evidence of binding interactions between the protein binding site and 1,2-azaborines.

1,2- azaborines概略合成路线示于图1。该协议适用于五种不同的含硼氮分子的合成。 图2表示11步骤1.3的过程期间测量并监控所需产物（3）的生成乙NMR谱。蛋白纯化通过使用低压层析系统和有代表性的色谱如图3所示进行。所收集的级分的纯度通过SDS-PAGE测定。 T4溶菌酶突变体L99A的晶体在图4中被描绘。 <st...

Watch this Scientific Journal Video about Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants at JoVE.com

Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants

A protocol for the synthesis of 1,2-azaborines and the preparation of their protein complexes with T4 lysozyme mutants is presented.

1,2- Azaborines的合成及其蛋白复合物的用T4溶菌酶突变体的制备

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor ...

synthesis-12-azaborines-preparation-their-protein-complexes-with-t4

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

7.5K 次观看.  Boston College. A protocol for the synthesis of 1,2-azaborines and the preparation of their protein complexes with T4 lysozyme mutants is presented.

视频: Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants

Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other fields. For instance, trehalose analogues bearing detectable tags have been used to detect Mycobacterium tuberculosis and may have applications as tuberculosis diagnostic imaging agents. Hydrolytically stable versions of trehalose are also being pursued due to their potential for use as non-caloric sweeteners and bioprotective agents. Despite the appeal of this class of compounds for various applications, their potential remains unfulfilled due to the lack of a robust route for their production. Here, we report a detailed protocol for the rapid and efficient one-step biocatalytic synthesis of trehalose analogues that bypasses the problems associated with chemical synthesis. By utilizing the thermostable trehalose synthase (TreT) enzyme from Thermoproteus tenax, trehalose analogues can be generated in a single step from glucose analogues and uridine diphosphate glucose in high yield (up to quantitative conversion) in 15-60 min. A simple and rapid non-chromatographic purification protocol, which consists of spin dialysis and ion exchange, can deliver many trehalose analogues of known concentration in aqueous solution in as little as 45 min. In cases where unreacted glucose analogue still remains, chromatographic purification of the trehalose analogue product can be performed. Overall, this method provides a &#34;green&#34; biocatalytic platform for the expedited synthesis and purification of trehalose analogues that is efficient and accessible to non-chemists. To exemplify the applicability of this method, we describe a protocol for the synthesis, all-aqueous purification, and administration of a trehalose-based click chemistry probe to mycobacteria, all of which took less than 1 hour and enabled fluorescence detection of mycobacteria. In the future, we envision that, among other applications, this protocol may be applied to the rapid synthesis of trehalose-based probes for tuberculosis diagnostics. For instance, short-lived radionuclide-modified trehalose analogues (e.g., 18F-modified trehalose) could be used for advanced clinical imaging modalities such as positron emission tomography-computed tomography (PET-CT).

Trehalose analogues are emerging as important molecules for bio(techno)logical and biomedical applications. We describe an optimized protocol for enzymatically synthesizing and purifying trehalose analogues that is simple, efficient, fast, and environmentally friendly. Its application to the rapid production and administration of a probe for the detection of mycobacteria is demonstrated.

快速一步酶法合成海藻糖类似物的全净化水

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other ...

科研

生物化学

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

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

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

合成和Iodoaziridines纯化涉及定量选择最优固定相的色谱

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

化学

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

通过Bioorthogonal点击化学化学选择性病毒表面改性

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

免疫与感染

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the aziridine ring. The utility of bicyclic aziridinium ions, specifically 1-azoniabicyclo[3.1.0]hexane and 1-azoniabicyclo[4.1.0]heptane tosylate highlighted in the aziridine ring openings by the nucleophile with the release of the ring strain to yield the corresponding ring-expanded azaheterocycles such as pyrrolidine, piperidine and azepane with diverse substituents on the ring in regio- and stereospecific manner. Herein, we report a simple and convenient method for the preparation of the stable 1-azabicyclo[4.1.0]heptane tosylate followed by selective ring opening via a nucleophilic attack either at the bridge or at the bridgehead carbon to yield piperidine and azepane rings, respectively. This synthetic strategy allowed us to prepare biologically active natural products containing piperidine and azepane motif including sedamine, allosedamine, fagomine and balanol in highly efficient manner.

Bicyclic aziridinium ions such as 1-azoniabicyclo[4.1.0]heptane tosylate were generated from 2-[4-tolenesulfonyloxybutyl]aziridine, which was utilized for the preparation of substituted piperidines and azepanes via regio- and stereospecific ring-expansion with various nucleophiles. This highly efficient protocol allowed us to prepare diverse azaheterocycles including natural products such as fagomine, febrifugine analogue and balanol.

稳定笼 Aziridinium 离子的制备及其开环 Azaheterocycles 的合成

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

OaAEP1介导酶合成和聚合蛋白固定，用于单分子力光谱

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

利用时间分辨蛋白诱导的荧光增强来鉴定稳定的局部构象，一次一α-突触核蛋白单体

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

Preparation of Enantiopure Non-Activated Aziridines and Synthesis of Biemamide B, D, and epiallo-Isomuscarine

Nitrogen-containing heterocycle aziridines are synthetically very valuable for the preparation of azacyclic and acyclic molecules. However, it is very difficult and laborious to make aziridines in optically pure forms on a large scale to apply asymmetric synthesis of aza compounds. Fortunately, we successfully achieved both enantiomers (2R)- and (2S)-aziridine-2-carboxylates with the electron-donating &#945;-methylbenzyl group at the ring nitrogen as non-activated aziridines. These starting aziridines have two distinct functional groups-highly reactive three-membered ring and versatile carboxylate. They are applicable in ring-opening or ring-transformation with aziridine and in functional group transformation to others from carboxylate. Both of these enantiomers were utilized in the preparation of biologically important amino acyclic and/or aza-heterocyclic compounds in an asymmetric manner. Specifically, this report describes the first expedient asymmetric synthesis of both enantiomers of 5, 6-dihydrouracil-type marine natural products biemamide B and D as potential TGF-&#946; inhibitors. This synthesis consisted of regio- and the stereoselective ring-opening reaction of aziridine-2-carboxylate and subsequent formation of 4-aminoteterahydropyrimidine-2,4-dione. One more example in this protocol dealt a highly stereoselective Mukaiyama reaction of aziridine-2-carboxylate and silyl enol ether, following intramolecular aziridine ring-opening to provide easy and facile access to (-)-epiallo-isomuscarine.

Preparation of Enantiopure Non-Activated Aziridines and Synthesis of Biemamide B, D, and epiallo-Isomuscarine

In this study, we prepare both enantiomers of aziridine-2-carboxylate, which are used in the asymmetric synthesis of alkaloids, including biemamide B and D, and (-)-epiallo-isomuscarine.

对映草非活化氮丙啶的制备及比咪咪唑B、D和 表皮异毒羯碱的合成

Nitrogen-containing heterocycle aziridines are synthetically very valuable for the preparation of azacyclic and acyclic molecules. However, it is very ...

Preparation of Contiguous Bisaziridines for Regioselective Ring-Opening Reactions

Aziridines, a class of reactive organic molecules containing a three-membered ring, are important synthons for the synthesis of a large variety of functionalized nitrogen-containing target compounds through the regiocontrolled ring-opening of C-substituted aziridines. Despite the tremendous progress in aziridine synthesis over the past decade, accessing contiguous bisaziridines efficiently remains difficult. Therefore, we were interested in synthesizing contiguous bisaziridines bearing an electronically diverse set of N-substituents beyond the single aziridine backbone for regioselective ring-opening reactions with diverse nucleophiles. In this study, chiral contiguous bisaziridines were prepared by organocatalytic asymmetric aziridination of chiral (E)-3-((S)-1-((R)-1-phenylethyl)aziridin-2-yl)acrylaldehyde with N-Ts-O-tosyl or N-Boc-O-tosyl hydroxylamine as the nitrogen source in the presence of (2S)-[diphenyl(trimethylsilyloxy)methyl]pyrrolidine as a chiral organocatalyst. Also demonstrated here are representative examples of regioselective ring-opening reactions of contiguous bisaziridines with a variety of nucleophiles such as sulfur, nitrogen, carbon, and oxygen, and the application of contiguous bisaziridines to the synthesis of multi-substituted chiral pyrrolidines by Pd-catalyzed hydrogenation.

Contiguous bisaziridines containing non-activated and activated aziridines were synthesized by asymmetric organocatalytic aziridinations and then subjected to chemoselective ring-opening reactions under acidic or basic conditions. The non-activated aziridine ring opens with less reactive nucleophiles under acidic conditions, whereas the activated aziridine ring opens with more reactive nucleophiles under basic conditions.

用于区域选择性开环反应的连续双氮丙啶的制备

Aziridines, a class of reactive organic molecules containing a three-membered ring, are important synthons for the synthesis of a large variety of ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

使用荧光抗生素探针对细菌耐药性的可视化

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

细菌利用试金石比色法检测

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

1,2- Azaborines的合成及其蛋白复合物的用T4溶菌酶突变体的制备

摘要

探索更多视频

此视频中的章节

快速一步酶法合成海藻糖类似物的全净化水

合成和Iodoaziridines纯化涉及定量选择最优固定相的色谱

通过Bioorthogonal点击化学化学选择性病毒表面改性

稳定笼 Aziridinium 离子的制备及其开环 Azaheterocycles 的合成

OaAEP1介导酶合成和聚合蛋白固定，用于单分子力光谱

利用时间分辨蛋白诱导的荧光增强来鉴定稳定的局部构象，一次一α-突触核蛋白单体

对映草非活化氮丙啶的制备及比咪咪唑B、D和表皮异毒羯碱的合成

用于区域选择性开环反应的连续双氮丙啶的制备

使用荧光抗生素探针对细菌耐药性的可视化

细菌利用试金石比色法检测

1,2- Azaborines的合成及其蛋白复合物的用T4溶菌酶突变体的制备

摘要

探索更多视频

此视频中的章节

快速一步酶法合成海藻糖类似物的全净化水

合成和Iodoaziridines纯化涉及定量选择最优固定相的色谱

通过Bioorthogonal点击化学化学选择性病毒表面改性

稳定笼 Aziridinium 离子的制备及其开环 Azaheterocycles 的合成

OaAEP1介导酶合成和聚合蛋白固定，用于单分子力光谱

利用时间分辨蛋白诱导的荧光增强来鉴定稳定的局部构象，一次一α-突触核蛋白单体

对映草非活化氮丙啶的制备及比咪咪唑B、D和 表皮异毒羯碱的合成

用于区域选择性开环反应的连续双氮丙啶的制备

使用荧光抗生素探针对细菌耐药性的可视化

细菌利用试金石比色法检测

对映草非活化氮丙啶的制备及比咪咪唑B、D和表皮异毒羯碱的合成