Li Q X, Casida J E
Department of Environmental Science, Policy and Management, University of California, Berkeley 94720-3112, USA.
Bioorg Med Chem. 1995 Dec;3(12):1675-84. doi: 10.1016/0968-0896(95)00153-0.
Affinity probes for the noncompetitive blocker or picrotoxinin site of the gamma-aminobutyric acid (GABA)-gated chloride channel were designed for four types of applications: photoaffinity reagents to covalently label the binding site; fluorescent probes for receptor analysis; biotinylated compounds and agarose/sepharose conjugates for affinity chromatography; ligand-protein/ enzyme conjugates for immunoassay. These 5e-tert-butyl-2e-[4-(substituted-ethynyl) phenyl]-1,3-dithianes were optimized by structure-activity studies for potency as inhibitors of 3H ethynylbicycloorthobenzoate binding to bovine brain membranes, measured as the concentration for 50% inhibition (IC50). Preferred compounds are 5e-(CH3)3CCH(CH2S)2CH-2e-C6H6-4-C = CCH2OCH2C(O)R, wherein R confers the following properties and IC50 values: R = SCH2CH2SCH2C(O)C6H4-4-N3, photoaffinity, 9 nM; R = NHCH2CH2NHC(O)C6H2-2-OH,5-1,4-N3, photoaffinity, 105 nM: R = SCH2CH2S-4-benzofurazan-7-NO2, fluorescent, 13 nM: R = SCH2CH2SCH2-5-fluorescein, fluorescent, 27 nM; R = NHCH2CH2NH[C(O)(CH2)5NH]2-biotin, affinity chromatography, 190 nM. The most potent photoaffinity ligand (IC50 9 nM) was labeled at 7 Ci mmol-1 by reacting the appropriate thiol with 3H 4-azidophenacyl bromide (obtained by alumina-catalyzed tritium exchange of its enolizable hydrogens). The first steps have been taken in using the NCB site for affinity chromatography of the GABAA receptor in CHAPS-solubilized bovine brain membranes with the dithiane-biotin probe and an avidin-acrylic bead system or with an analogous dithiane-agarose/ sepharose column eluting with GABA or dithiane as above (R = OH). A protein conjugate of a related dithiane-monosulfone elicited production of specific antisera in rabbits. These findings illustrate the diversity and utility of new affinity probes prepared in the alkynylphenyldithiane series.
针对γ-氨基丁酸（GABA）门控氯离子通道的非竞争性阻断剂或印防己毒素位点设计了亲和探针，用于四种类型的应用：用于共价标记结合位点的光亲和试剂；用于受体分析的荧光探针；用于亲和色谱的生物素化化合物和琼脂糖/琼脂糖凝胶缀合物；用于免疫测定的配体-蛋白质/酶缀合物。这些5e-叔丁基-2e-[4-（取代乙炔基）苯基]-1,3-二硫烷通过结构活性研究进行了优化，以提高其作为3H乙炔基双环邻苯二甲酸酯与牛脑膜结合抑制剂的效力，以50%抑制浓度（IC50）来衡量。优选的化合物为5e-（CH3）3CCH（CH2S）2CH-2e-C6H6-4-C = CCH2OCH2C（O）R，其中R赋予以下性质和IC50值：R = SCH2CH2SCH2C（O）C6H4-4-N3，光亲和性，9 nM；R = NHCH2CH2NHC（O）C6H2-2-OH，5-1,4-N3，光亲和性，105 nM；R = SCH2CH2S-4-苯并呋喃-7-NO2，荧光性，13 nM；R = SCH2CH2SCH2-5-荧光素，荧光性，27 nM；R = NHCH2CH2NH[C（O）（CH2）5NH]2-生物素，亲和色谱，190 nM。通过使适当的硫醇与3H 4-叠氮苯甲酰溴（通过其烯醇化氢的氧化铝催化氚交换获得）反应，将最有效的光亲和配体（IC50 9 nM）标记为7 Ci/mmol-1。已经采取了第一步措施，使用NCB位点，通过二硫烷-生物素探针和抗生物素蛋白-丙烯酸珠系统，或使用上述用GABA或二硫烷洗脱的类似二硫烷-琼脂糖/琼脂糖凝胶柱（R = OH），对CHAPS溶解的牛脑膜中的GABAA受体进行亲和色谱分析。一种相关二硫烷-单砜的蛋白质缀合物在兔体内引发了特异性抗血清的产生。这些发现说明了炔基苯基二硫烷系列中制备的新型亲和探针的多样性和实用性。
