TGK承认国际研究生院慕尼黑工业大学的科学与工程（IGGSE）提供的财政支持。香港承认的鼎力支持中心综合蛋白质科学慕尼黑（CIPSM）。

注:从供应商处获得的所有试剂和溶剂，无需进一步纯化即可使用。 注意:使用前请咨询所有相关材料安全数据表（MSDS）。进行化学合成（ 例如，通风柜，护目镜，手套，实验室外套，全长裤，不露脚趾的鞋子）时，请利用一切适当的安全设备。 1.胍基化的前体的合成<ol><li> 烷基化物种 <ol><li> 甲基化种 <ol><li>溶解将1.0g N，N&#39; -二叔丁氧羰基-1H-在10mL的干-吡唑-1-甲脒（3.2毫摩尔，1当量）和1.0克三苯基膦（PPH 3，3.8毫摩尔，1.2当量）在使用在室温下（RT）橡胶隔膜的惰性气氛的50mL圆底烧瓶中的四氢呋喃（THF）。使用注射器（4.8毫摩尔，1.5当量）和升添加无水甲醇194μL等它搅拌在RT。 </li><li>单独地，制备在小玻璃瓶中与747μL二异丙基偶氮二羧酸酯（DIAD，3.8毫摩尔，1.2当量）在2mL无水THF中的溶液。在搅拌的同时，用注射器（在15分钟）DIAD在THF滴加该溶液添加到的N，N&#39; -二叔丁氧羰甲基-1H-吡唑-1-甲脒，PPH 3和甲醇溶液中。让该溶液在RT下搅拌2个小时。 </li><li>除去使用旋转蒸发器减压除溶剂，将粗产物溶解在1mL的二氯甲烷（DCM）。在戊烷溶液加载闪式柱（2.5厘米×20厘米），在10％乙酸乙酯100g硅胶的（EtOAc）纯化。加载在柱的溶解的粗产物和压力（1.5巴）下加入溶剂。 </li><li>收集的级分（溶剂系统:等度10％的EtOAc在戊烷中），识别由薄层色谱（TLC）的所需化合物斑点，并收集它（R F = 0.4，乙酸乙酯/戊烷1:9，UV）。合并所需使用旋转蒸发器，以得到0.85g标题化合物，为黄色粘性油状物（2.6毫摩尔，81％收率）的级分并在减压下蒸发溶剂。存储在RT进一步使用，得到化合物。 </li></ol></li><li> 己胺改性品种 <ol><li>溶解1.0克N，N&#39; -二丁氧羰基-1H-吡唑-1-甲脒（3.2毫摩尔，1.0当量），将0.9g DDE -6-氨基己醇（3.8毫摩尔，1.2当量）的，和1.0克使用在室温下橡胶隔膜加入PPh 3（3.8毫摩尔，1.2当量）在10毫升在惰性气氛的50mL圆底烧瓶中的干燥THF中的。 </li><li>单独地，制备在小玻璃瓶中与747μL的DIAD（3.8毫摩尔，1.2当量）在2mL无水THF中的溶液。在搅拌的同时，DIAD在THF滴加该溶液添加到N的溶液，用注射器N&#39; -二叔丁氧羰甲基-1H-吡唑-1-甲脒，PPH 3，和DDE -6-氨基己醇（在15分钟内） 。让该溶液在RT下搅拌2个小时。 </li><li>除去使用旋转蒸发器减压除溶剂，采取在1毫升DCM中的粗产物的。在戊烷/ EtOAc中的溶液加载闪式柱（2.5厘米×20厘米）用100克二氧化硅（2:1）。适用于该列的溶解粗产物和压力（1.5巴）下添加溶剂。 </li><li>收集的级分（溶剂系统:等度2:1戊烷/ EtOAc）纯化，识别与TLC所需级分，并收集它们（R F = 0.66，2:1个戊烷/ EtOAc中，UV）。合并所需的部分并使用旋转蒸发器，得到1.6g的标题化合物，为黄色粘性油状物（2.8毫摩尔，产率88％）在减压下蒸发溶剂。存储在RT进一步使用，得到化合物。 </li></ol></li></ol></li><li> 酰化物种（2个反应步骤） <ol><li>溶解1.4克S- -methylisothiuronium半硫酸盐（10毫摩尔，1.0当量）和2.2克二（ 叔丁基）二碳酸酯（10毫摩尔，1.0当量）在剧烈搅拌响DCM的两相溶液/ 饱和水性 。 碳酸氢钠和让它在RT下搅拌24个小时。 </li><li>分离各层在分液漏斗和三次用DCM萃取水相。合并有机相，用Na 2 SO 4干燥它们，并使用旋转蒸发器在减压下除去溶剂。取在1毫升己烷的粗产物的。 </li><li> 1己烷/乙酸乙酯:以4:1的溶液加载闪式柱（2.5厘米×20厘米）用100克二氧化硅。加载在柱的溶解的粗产物。在压力下加入溶剂。识别与TLC（溶剂系统中的所希望的部分:4:1己烷等度/乙酸乙酯（检测UV:254纳米），并收集它们（R = 0.30）。 </li><li>合并所需的部分并使用旋转蒸发器，以获得1.1g标题化合物，为黄色粘性油状物（5.8毫摩尔，58％收率）在减压下蒸发溶剂。存储所述试剂，N - （ 叔丁氧羰） - S- -Methylisothiourea，在RT下供进一步使用。 </li><li>溶解250毫克N - （ 叔丁氧羰） -在10mL无水N，N-二甲基甲酰胺（DMF）s -methylisothiourea（1.3毫摩尔，1.0当量）在50mL的圆底烧瓶中，在惰性气氛中在RT。添加用注射器N，N-二异丙基乙胺（DIPEA）的440μL（2.6毫摩尔，2当量）。在搅拌的同时，添加245μLAC 2 O（5.2毫摩尔，4.0当量）的使用注射器逐滴并让它在RT下搅拌1个小时。 </li><li>过量的甲醇（2毫升）添加到使用注射器的混合物，并让它在RT下搅拌15分钟。除去使用旋转蒸发器在减压下除去溶剂。取在1毫升DCM中的粗产物的。 </li><li> 1己烷/乙酸乙酯3:在3溶液加载闪式柱（2.5厘米×20厘米）用60克二氧化硅1。溶解的粗产物施加到柱上。添加溶剂（3:1己烷/ EtOAc）和压力（1.5巴）。识别所需的级分用TLC（3:1己烷/乙酸乙酯，UV:220纳米），和colLECT他们（R F = 0.62）。 </li><li>合并所需的部分并使用旋转蒸发器，以获得210毫克的标题化合物，为白色固体（0.9毫摩尔，产率70％）在减压下蒸发溶剂。存储在RT以供进一步使用的化合物。 </li></ol></li></ol> 2.环肽前体的合成<ol><li> 装载和封盖TCP树脂 <ol><li>添加1.00克2- chlortriyl酰氯（CTC）树脂（0.9毫摩尔/克）和320毫克使Fmoc-Gly-OH（1.2当量）中的入塑料20毫升的注射器用玻璃料。制备313μLDIPEA（2个当量）在5mL无水DCM中的溶液并将其添加到所述注射器。让它转动，以便使用旋转单元在RT 1个小时。 </li><li>同时，准备5的2-mL溶液:1个甲醇/ DIPEA（V / V;封盖溶液）。添加封盖解决注射器，让它旋转，另外的15分钟。用DCM（5×）和DMF（3×）洗涤树脂。 </li></ol></li><li> 树脂上Fmoc去保护<ol><li>用DMF中的20％哌啶处理树脂结合使Fmoc-Gly 10分钟，然后5分钟。用DMF（3×）洗涤树脂。 </li></ol></li><li> 树脂上的Fmoc-ORN（DDE）的耦合-OH <ol><li>添加934毫克的Fmoc-L-ORN（DDE）-OH（2当量），684毫克1- [双（二甲基氨基）亚甲基] -1H-1,2,3-三唑并[4,5-B]吡啶鎓-3- OXID六氟磷酸盐（HATU）（2当量），和245毫克1-羟基-7-氮杂苯并三唑（加入HOAt）（2当量），以一个小的玻璃小瓶中，并在5毫升DMF中溶解。加入DIPEA的814μL。 </li><li>添加该溶液至Fmoc-脱保护的，洗涤和树脂结合的甘氨酸并让它旋转1个小时。用DMF（3×）洗涤树脂。 </li></ol></li><li> 树脂上Fmoc去保护 <ol><li>处理树脂结合的Fmoc-ORN（DDE）-Gly用在DMF中20％哌啶10分钟，然后5分钟。用DMF（3×）洗涤树脂。 </li></ol></li><li> 树脂上的Fmoc-VAL-OH的偶联 <ol><li>添加610毫克的Fmoc-VAL-OH（2当量），684毫克的HATU（2当量），和245毫克的加入HOAt（2当量），以一个小的玻璃小瓶中，并在5毫升DMF中溶解。加入DIPEA的814μL。 </li><li>此溶液添加到Fmoc基脱保护，洗涤和树脂结合的ORN（DDE）-Gly并让它旋转1个小时。 </li></ol></li><li> 树脂上Fmoc去保护 <ol><li>处理树脂结合的Fmoc-VAL-ORN（DDE）-Gly用在DMF中20％哌啶10分钟，然后5分钟。用DMF（3×）洗涤树脂。 </li></ol></li><li> 树脂上甲基化的N- <ol><li>用DCM（3×）洗涤树脂。在DCM中溶解887毫克2- nitrobenzenesulfonylchloride（ 邻 -NBS-CL，4当量），并添加1.2毫升2,4,6-三甲基吡啶（10当量）。 </li><li>添加该溶液到树脂结合的肽，并让它在室温下孵育20分钟。 </li><li>用CH 2 Cl 2（3×），并用THF（5×）洗涤树脂。制备具有1.18克的PPh 3（5当量）和在THF中的甲醇的365μL的溶液。收藏此解决注射器。 </li><li>在2mL THF中制备具有DIAD的883μL的溶液，并把它添加到注射器中。让它孵育15分钟。用THF（5×），用DMF（5×）洗涤树脂。 </li></ol></li><li> Ø-NS脱保护 <ol><li>准备用570μL巯基乙醇（10.0当量）和672μLdiazabicycloundecen的（DBU）在2mL DMF中的溶液。收藏此解决方案的注射器，让它孵化5分钟。 </li><li>重复脱保护步骤，然后用DMF（5×）洗涤树脂。 </li></ol></li><li> 树脂上的Fmoc-D-的Phe-OH的偶联 <ol><li>添加697毫克的Fmoc-D-PHE-OH（2当量），684毫克的HATU（2当量），和245毫克加入HOAt（2当量）的至小玻璃瓶中，并在5毫升DMF中的溶解。加入DIPEA的814μL。 </li><li>添加该溶液至Fmoc-脱保护的，洗涤和树脂结合的肽，并让它旋转1个小时。 </li></ol></li><li> 树脂上Fmoc去保护 <ol><li>治疗树脂-Bound用DMF中的20％哌啶10分钟，然后进行5分钟的肽。用DMF（3×）洗涤树脂。 </li></ol></li><li> 树脂上的Fmoc-天冬氨酸（OT丁基）-OH的偶联 <ol><li>添加741毫克的Fmoc-天冬氨酸（OT卜）-OH（2当量），（2当量），684毫克的HATU和245毫克加入HOAt（2当量）至小玻璃瓶中，将其溶解在5mL的DMF。加入DIPEA的814μL。 </li><li>添加该溶液至Fmoc-脱保护的，洗涤和树脂结合的肽，并让它旋转1个小时。 </li></ol></li><li> 从树脂上的线性肽的裂解 <ol><li>用DCM洗涤树脂（3x）和随后制备六氟异丙醇（HFIP）的DCM溶液的10毫升（1:4; V / V）。 </li><li>添加该溶液至树脂，让它旋转15分钟。收集在一个圆底烧瓶中的溶液。重复切割和使用旋转蒸发器蒸发溶剂。 </li></ol></li><li> 线性肽的环化 <ol><li>迪ssolve 384毫克的NaHCO 3（5.0当量）和叠氮磷酸二苯的582μL（DPPA）（3.0当量）在50mL DMF中的并将其添加到粗产物。让它搅拌过夜，或直到没有线性肽与ESI-MS（10小时）17观察到。 </li><li>在减压下，减少溶剂使用旋转蒸发器小体积。制备氯化钠（盐水）的饱和水溶液。使用巴斯德吸管，所述环化肽滴添加至40毫升饱和NaCl水溶液在一个离心管中。 </li><li>离心悬浮液（5000×g下，5分钟），并用HPLC级水（2×）洗涤沉淀物。冻干产品。 </li></ol></li></ol> 3.胍基化和脱保护解决方案<ol><li> 胍基与量身订做的前体 <ol><li>溶解在DMF中的小体积少量（ 例如，25毫克）的脱保护的正交环肽（ 例如，2米L）。添加2当量。的胍基化前体和2当量。 DIPEA的溶液中，并让它在RT下搅拌2个小时。 </li><li>监视与HPLC-MS的反应的进展。反应完成后，除去溶剂，再溶解guanidinylated环状在乙腈肽，并进行半制备型HPLC纯化16。 </li></ol></li><li> 酸不稳定的侧链保护基团的去除 <ol><li>制备2毫升含三氟乙酸（TFA），水和三异丙基硅烷（TIPS）的溶液（95 / 2.5 / 2.5; V / V / V）。添加该溶液到纯化的产物在一个小的玻璃瓶中并让它在室温搅拌至少1小时。观察与ESI-MS 17的完全脱保护。 </li><li>在减压下蒸发溶剂至小体积。制备冰冷醚和在50毫升离心管中加入10毫升。通过添加它沉淀使用巴氏吸管在冰冷醚去保护的肽滴。离心悬浮液（5000×g下，5分钟），得到颗粒。 </li><li>洗用冰冷的乙醚沉淀和离心它（5000×g下，5分钟，2次）。溶解产物在2毫升的HPLC级水中，用半制备型HPLC纯化16它。 </li></ol></li></ol> 4.分析数据和用于纯化参数<ol><li> 分析性HPLC-ESI-MS <ol><li>使用C18柱（12纳米孔径3μm的粒径，125毫米×2.1毫米）或C8柱（20纳米的孔径，5μm的粒子的LCQ质谱仪进行分析型HPLC-ESI-MS尺寸，250毫米×2.1毫米），用H 2 O（0.1％v / v甲酸）/乙腈（0.1％v / v甲酸）作为洗脱剂。 </li></ol></li><li> 半制备性HPLC <ol><li>用ODS-A柱（20×250毫米，5微米），8毫升/分钟的流速，和线性渐变执行使用制备型HPLC仪器半制备性HPLC的H 2 O（0.1％V / V TFA）和乙腈（0.1％体积/体积TFA）。 </li></ol></li></ol>

<ol>
	<li>Saczewski, F., Balewski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+activities+of+guanidine+compounds.">Biological activities of guanidine compounds.</a> Exp. Opin. Ther. Patents. 19 (10), 1417-1448 (2009).</li><li>Saczewski, F., Balewski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+activities+of+guanidine+compounds,+2008-2012+update.">Biological activities of guanidine compounds, 2008-2012 update.</a> Exp. Opin. Ther. Patents. 23 (8), 965-995 (2013).</li><li>Wirth, T. H., Davidson, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mercury+(II)+Comlexes+of+Guanidine+and+Ammonia,+and+a+general+discussion+of+the+Complexing+of+Mercury+(II)+by+Nitrogen+Bases.">Mercury (II) Comlexes of Guanidine and Ammonia, and a general discussion of the Complexing of Mercury (II) by Nitrogen Bases.</a> J. Am. Chem. Soc. 86 (20), 4325-4329 (1964).</li><li>Berlinck, R. G., Burtoloso, A. C., Kossuga, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemistry+and+biology+of+organic+guanidine+derivatives.">The chemistry and biology of organic guanidine derivatives.</a> Nat. Prod. Rep. 25, 919-954 (2008).</li><li>Peterlin-Ma&#353;i&#269;, L., Kikelj, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+mimetics.">Arginine mimetics.</a> Tetrahedron. 57 (33), 7073-7105 (2001).</li><li>Peterlin-Ma&#353;i&#269;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+mimetic+structures+in+biologically+active+antagonists+and+inhibitors.">Arginine mimetic structures in biologically active antagonists and inhibitors.</a> Curr. Med. Chem. 13 (30), 3627-3648 (2006).</li><li>Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins:+bidirectional,+allosteric+signaling+machines.">Integrins: bidirectional, allosteric signaling machines.</a> Cell. 110 (6), 673-687 (2002).</li><li>Liddington, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+aspects+of+integrins.">Structural aspects of integrins.</a> Adv. Exp. Med. Biol. 819, 111-126 (2014).</li><li>Plow, E. F., Haas, T. A., Zhang, L., Loftusi, J., Smith, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+binding+to+integrins.">Ligand binding to integrins.</a> J. Biol. Chem. 275 (29), 21785-21788 (2000).</li><li>Kapp, T. G., Fottner, M., Maltsev, O. V., Kessler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+cause,+great+impact+-+modification+of+guanidine+group+in+RGD+controls+subtype+selectivity.">Small cause, great impact - modification of guanidine group in RGD controls subtype selectivity.</a> Angew. Chem. Int. Ed. 55, 1540-1543 (2016).</li><li>Xiong, J. P., 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=Crystal+structure+of+the+extracellular+segment+of+integrin+alpha+Vbeta3.">Crystal structure of the extracellular segment of integrin alpha Vbeta3.</a> Science. 294, 339-345 (2001).</li><li>Xiong, J. P., 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=Crystal+structure+of+the+extracellular+segment+of+integrin+alpha+Vbeta3+in+complex+with+an+Arg-Gly-Asp+ligand.">Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with an Arg-Gly-Asp ligand.</a> Science. 296 (5565), 151-155 (2002).</li><li>Nagae, M., 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=Crystal+structure+of+&#945;5&#946;1+integrin+ectodomain:+atomic+details+of+the+fibronectin+receptor.">Crystal structure of &#945;5&#946;1 integrin ectodomain: atomic details of the fibronectin receptor.</a> J. Cell Biol. 197 (1), 131-140 (2012).</li><li>Hersel, U., Dahmen, C., Kessler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGD+modified+polymers:+biomaterials+for+stimulated+cell+adhesion+and+beyond.">RGD modified polymers: biomaterials for stimulated cell adhesion and beyond.</a> Biomaterials. 24 (24), 4385-4415 (2003).</li><li>Auernheimer, J., Dahmen, C., Hersel, U., Bausch, A., Kessler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoswitched+Cell+Adhesion+on+Surfaces+with+RGD+Peptides.">Photoswitched Cell Adhesion on Surfaces with RGD Peptides.</a> J. Am. Chem. Soc. 127 (46), 16107-16110 (2005).</li><li>Corradini, D., Eksteen, E., Eksteen, R., Schoenmakers, P., Miller, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of HPLC. , (2011).</li><li>de Hoffmann, E., Stroobant, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mass Spectrometry: Principles and Applications. , (2007).</li><li>Frank, A. O., 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=Conformational+Control+of+Integrin-Subtype+Selectivity+in+isoDGR+Peptide+Motifs:+A+Biological+Switch.">Conformational Control of Integrin-Subtype Selectivity in isoDGR Peptide Motifs: A Biological Switch.</a> Angew. Chem. Int. Ed. 49 (48), 9278-9281 (2010).</li><li>Rossiter, S., 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=Selective+substrate-based+inhibitors+of+mammalian+dimethylarginine+dimethylaminohydrolase.">Selective substrate-based inhibitors of mammalian dimethylarginine dimethylaminohydrolase.</a> J. Med. Chem. 48 (14), 4670-4678 (2005).</li><li>Weiss, S., Keller, M., Bernhardt, G., Buschauer, A., K&#246;nig, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N(G)-Acyl-argininamides+as+NPY+Y(1)+receptor+antagonists:+Influence+of+structurally+diverse+acyl+substituents+on+stability+and+affinity.">N(G)-Acyl-argininamides as NPY Y(1) receptor antagonists: Influence of structurally diverse acyl substituents on stability and affinity.</a> Bioorg. Med. Chem. 18 (17), 6292-6304 (2010).</li><li>Hammerschmidt, F., Kvaternik, H., Schweifer, A., Mereiter, K., Aigner, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Synthesis+of+No-Carrier-Added+[*I]MIBG+and+Its+Precursor.">Improved Synthesis of No-Carrier-Added [*I]MIBG and Its Precursor.</a> Synthesis. 44 (21), 3387-3391 (2012).</li></ol>

作者什么都没有透露。

为胍基化的前体是正交脱保护的环肽衍生物，（C（OrnD（OT丁基）GF（N Me）的V）），这是通过固相肽合成（SPPS）的标准Fmoc方案合成。鸟氨酸被用作正交保护的衍生物，羰基（Fmoc-ORN（DDE）-OH），其可以与肼在DMF中的肽支架的环化反应后进行脱保护。该肽的前体是由化合物沉淀，并通过随后的冷冻干燥纯化。 为胍基烷基化的前体可以在在一步反应良好的产?...

其中在天然配体中最丰富的药效基团是胍基，其参与多种相互作用1,2。例如，它可作为在氢键相互作用的电位四倍氢供体并参与静电相互作用，例如盐桥或阳离子-π相互作用。在药物化学，该组中的药物和药物候选4，经常发现，虽然很常作为胍模拟物5,6。其原因胍模拟物的开发是去除普遍存在的，带正电荷的胍基团，以及作为配体的亲脂性的调整。在肽配体，唯一的胍基的氨基酸是精氨酸，其因此通常在肽配体的生物活性区域中发现。 一个非常PR用于含精氨酸的配体家族ominent例子是RGD结合整联亚家族。通常，整联是一类细胞粘附受体，它们接管重要的功能在所有高等生物的。其中的一些功能，包括细胞粘附，迁移和细胞存活。因此，它们也参与病理指示，诸如癌症和纤维化。整合素是选自由α-和β亚单位构成24个目前已知的整联亚型的异源二聚体跨膜蛋白;其中8识别的三肽序列精氨酸-甘氨酸-天冬氨酸（RGD =）在它们的配体7。所述结合区域位于在细胞外部分，即所谓的整头组8这两个亚型之间的接口。 RGD由两个共同相互作用确认:金属离子依赖性的粘附位点（MIDAS）区域，该区域位于β亚基和结合在配体的羧酸（侧茶在ASP的）;和胍组中的配体，其位于α亚基。大多数整联亚型是混杂和共享至少它们的天然细胞外基质（ECM）配体9的一部分。因此，人工整合素配体的发展，主要的焦点是，除了具有高结合亲和力的亚型选择性。最近，我们能够出台针对亚型选择性配体的产生的关键因素:胍组。通过不同的修饰，所述αv-和双选择性配体α5含整亚型可以通过简单的修改的胍基团，然后它可以区分不同的α亚基10变成选择性的化合物。 在αV的口袋，所述胍基经由二齿盐桥与Asp218 11,12相互作用侧面上 。这种相互作用Ç一个也α5β1可以观察到（在此，用Asp227在α5），但是另外，端部上有Gln残基（Gln221）的胍基团的相互作用，观察到有13。因此，我们修改了胍组中的两个相对的方式:在一种情况下，通过与胍基的Nδ的甲基化阻止侧上相互作用，而在其他情况下，与胍Nω的甲基化，阻断端上的相互作用。出人意料的是，这个小的修改导致了配体完整的选择性转变。除了烷基化，新功能化方法本出版物中介绍。对于这种类型的配体pentapeptidic的经典官能的方法是通过不参与结合的氨基酸的侧链共轭（ 例如，K在C（RGDfK））14，15。这里，我们表明，官能化也可以通过修改胍 - 这对于结合至关重要 - 与酰基或烷基化连接基。上的正电荷，对于结合是必需被保留，并且模型表明，长链分出来的结合口袋的，因此提供进一步的接头的附接的理想可能性和标记单元（ 例如，荧光标记物或分子螯合剂成像）。 在这项工作中，我们集中在对于胍基的含精氨酸的配体的变形例的制备步骤。这涉及Nω甲基化的物质的合成，以及与较长的接头单元胍。不同的修饰包括酰基和烷基。

<table border="0" cellpadding="0" cellspacing="0" width="758">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">N,N&#8242;-Di-Boc-1H-pyrazole-1-carboxamidine, 98%&#160;</td>
			<td style="width:151px;">Sigma Aldrich</td>
			<td style="width:176px;">434167&#160;ALDRICH</td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Triphenylphosphine, 99%</td>
			<td>Sigma Aldrich</td>
			<td>T84409&#160;SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrahydrofuran, &#62;99.5%</td>
			<td>Carl Roth</td>
			<td>4745</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrahydrofuran anhydrous, 99.8%</td>
			<td>Carl Roth</td>
			<td>5182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol anhydrous, 99.8%</td>
			<td>Sigma Aldrich</td>
			<td>322415&#160;SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diisopropyl azodicarboxylate, 98%</td>
			<td>Sigma Aldrich</td>
			<td>225541&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dichlormethan, for synthesis, 99.5%</td>
			<td>Carl Roth</td>
			<td>8424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silica gel for flash chromtaography</td>
			<td>Sigma Aldrich</td>
			<td>60738&#160;SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-Pentane, 99%</td>
			<td>Carl Roth</td>
			<td>8720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-Hexane, 99%</td>
			<td>Carl Roth</td>
			<td>CP47</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylacetate, 99.5%</td>
			<td>Carl Roth</td>
			<td>7338</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aminohexanol, 95%</td>
			<td>Sigma Aldrich</td>
			<td>A56353&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S-Methylisothiourea hemisulfate, 98%</td>
			<td>Sigma Aldrich</td>
			<td>M84445 ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Di-tert-butyl dicarbonate, 99%</td>
			<td>Sigma Aldrich</td>
			<td>205249&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N-Dimethylformamid, 99.8%</td>
			<td>Carl Roth</td>
			<td>A529</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N-Diisopropylethylamin, 99.5%</td>
			<td>Carl Roth</td>
			<td>2474</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic anhydrid, 99%</td>
			<td>Carl Roth</td>
			<td>4483</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chlortrityl resin</td>
			<td>Carbolution</td>
			<td>CC11006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-Gly-OH, 98%</td>
			<td>Carbolution</td>
			<td>CC05014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Piperidin, 99%</td>
			<td>Sigma Aldrich</td>
			<td>104094&#160;SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-Orn(Dde)-OH</td>
			<td>Iris-Biotech</td>
			<td>FAA1502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HATU, 99%</td>
			<td>Carbolution</td>
			<td>CC01011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HOAt, 99%</td>
			<td>Carbolution</td>
			<td>CC01004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-Val-OH</td>
			<td>Carbolution</td>
			<td>CC05028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Nitrobenzenesulfonyl chloride, 97%</td>
			<td>Sigma Aldrich</td>
			<td>N11507&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,4,6-Collidine, 99%</td>
			<td>Sigma Aldrich</td>
			<td>27690&#160;SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mercaptoethanol, 99%&#160;</td>
			<td>Sigma Aldrich</td>
			<td>M6250&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diazabicycloundecen, 98%</td>
			<td>Sigma Aldrich</td>
			<td>139009&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-D-Phe-OH, 98%</td>
			<td>Sigma Aldrich</td>
			<td>47378&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-Asp(OtBu)-OH, 98%</td>
			<td>Carbolution</td>
			<td>CC05008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hexafluoroisopropanol</td>
			<td>Carbolution</td>
			<td>CC03056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diphenylphosphoryl azide, 97%</td>
			<td>Sigma Aldrich</td>
			<td>178756&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TFA, 99.9%</td>
			<td>Carl Roth</td>
			<td>P088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triisopropylsilan, 98%</td>
			<td>Sigma Aldrich</td>
			<td>233781&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetonitrile, HPLC grade</td>
			<td>Carl Roth</td>
			<td>HN44</td>
			<td></td>
		</tr>
	</tbody>
</table>

modification and functionalization of the guanidine group by tailor-made precursors

胍组是在药物化学中最重要的药效基团之一。携带胍基的唯一的氨基酸是精氨酸。在本文中，提供了一种用于在肽配体的胍基的修饰的简单方法，用RGD结合整联配体的一个例子。据最近表明，胍基团在这些配位体的不同修改允许亚型的选择性调节（ 例如，αV亚型和α5之间）。此外，通过胍基的功能化以前未知的策略被证明，而合成的方法在本文件中审查。这里所描述的修改涉及末端（Nω）烷基化和乙酰化胍基。用于合成，量身定做的前体分子被合成，然后将其进行反应与正交脱保护的胺来传送预修饰胍组。对于烷基化胍的合成中，基于N-前体，N&#39; -二叔丁氧羰甲基-1H-吡唑-1-甲脒用于合成酰化化合物，选择为N -Boc- S的相应的酰化衍生物的前体-甲基异硫脲，其可以在一个和两个步骤的反应来获得。

环肽前体合成的线性肽，环化，和正交DDE脱保护。在沉淀后，所述化合物的纯度用HPLC-MS（ 图1）进行分析。以监测反应的进展，2小时的反应时间（ 图2）之后进行的HPLC分析。 有关胍基残基较大，在2小时反应时间通常是不够的。在这种情况下，继续反应，并用LC-MS监测。进行反应和最终的脱保护后，使...

Watch this Scientific Journal Video about Modification and Functionalization of the Guanidine Group by Tailor-made Precursors at JoVE.com

Modification and Functionalization of the Guanidine Group by Tailor-made Precursors

一种用于基于使用所述相应的前体的烷基改性胍的合成方案被呈现。

修改和胍集团的官能通过量身定制的前兆

The guanidine group is one of the most important pharmacophoric groups in medicinal chemistry. The only amino acid carrying a guanidine group is arginine. ...

modification-functionalization-guanidine-group-tailor-made

Research

JoVE Journal

Biochemistry

10.5K 次观看.  Technische Universität München. 一种用于基于使用所述相应的前体的烷基改性胍的合成方案被呈现。 

视频: Modification and Functionalization of the Guanidine Group by Tailor-made Precursors

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

用磷酸盐结合标记的 SDS-页 Rab10 磷酸化检测 LRRK2 活性

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

原子力显微镜对细胞外囊泡的成像

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

线粒体和内质视网膜成像由相关光和体积电子显微镜

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

一次人气道上皮细胞气道表面液体 pH 值的实时、半自动荧光测量

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

使用光子芯片的高通量总内反射荧光和直接随机光学重建显微镜

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

通过基因表达分析的改良方法从谷物种子获取高质量的转录数据

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Analysis of SEC-SAXS data via EFA deconvolution and Scatter

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ratio and conformational changes of macromolecules and macromolecular complexes. A high quality SAXS dataset for structural modeling must be from monodisperse, homogeneous samples and this is often only reached by a combination of inline chromatography and immediate SAXS measurement. Most commonly, size-exclusion chromatography is used to separate samples and exclude contaminants and aggregations from the particle of interest allowing SAXS measurements to be made from a well-resolved chromatographic peak of a single protein species. Still, in some cases, even inline purification is not a guarantee of monodisperse samples, either because multiple components are too close to each other in size or changes in shape induced through binding alter perceived elution time. In these cases, it may be possible to deconvolute the SAXS data of a mixture to obtain the idealized SAXS curves of individual components. Here, we show how this is achieved and the practical analysis of SEC-SAXS data is performed on ideal and difficult samples. Specifically, we show the SEC-SAXS analysis of the vaccinia E9 DNA polymerase exonuclease minus mutant.

SEC-BioSAXS measurements of biological macromolecules are a standard approach for determining solution structure of macromolecules and their complexes. Here, we analyze SEC-BioSAXS data from two types of commonly encountered SEC traces&#8212;chromatograms with fully resolved and partially resolved peaks. We demonstrate the analysis and deconvolution using scatter and BioXTAS RAW.

通过 EFA 去卷和散射分析 SEC-SAXS 数据

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

从脂质绑定蛋白和过敏原中去除和替换内源性利根

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...