Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric Gloeobacter ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.

中文翻译：

---

三氟甲基苯基二氮丙啶胆固醇类似物作为胆固醇模拟物和光标记试剂的验证

胆固醇的脂肪族二氮丙啶类似物以前已被用于详细说明胆固醇蛋白质组并确定蛋白质上的胆固醇结合位点。含有三氟甲基苯基二氮丙啶 (TPD) 基团的胆固醇类似物尚未见报道。两类二氮丙啶都已准备好用于神经类固醇光标记研究，它们的组合使用提供了单独使用任一二氮丙啶类都无法获得的信息。因此，我们准备了胆固醇TPD类似物，并用它们与先前报告的脂族二吖丙因类似物作为光亲和标记试剂一起以获得关于胆固醇结合五聚体的位点的附加信息Gloeobacter配体门控离子通道 (GLIC)。我们首先验证了 TPD 类似物作为胆固醇替代品，并将它们的作用与先前报道的脂肪族二氮丙啶在细胞培养试验中的作用进行了比较。所有探针都与 GLIC 上的相同胆固醇结合位点结合，但在光标记效率和识别的残留物方面存在差异。哺乳动物 (HEK) 细胞膜的光标记表明两类探针标记的蛋白质模式存在差异。总的来说，这些日期表明含有脂肪族二氮丙啶或 TPD 基团的胆固醇光亲和标记试剂提供了补充信息，并且都将成为未来胆固醇生物学研究的有用工具。