Binding assays are widely used to study the estrogenic activity of compounds targeting the estrogen receptor (ER). The fluorescence properties of benzofurazan (BD), an environmentally sensitive fluorophore, are affected by solvent polarity. In this study, we synthesized BD-labeled estradiol (E2) derivatives hoping to develop a fluorescent ligand to be used in ER binding assays, without the separation of free- from bound-ligand. Three fluorescent ligands with a BD skeleton were obtained and their fluorescence properties were investigated. Analysis of the fluorescent ligands and human recombinant ERα (hr-ERα) interactions revealed that the fluorescence intensity increased in hydrophobic environments, such as the receptor-binding site. In saturation binding assays, ABD-E2 derivative 2c showed positive cooperative binding, and its dissociation constant (K_d) and Hill coefficient were 23.4 nM and 1.34, respectively. The estrogenic compounds affinity, assessed by competitive binding assays was well correlated with the results obtained by conventional studies, using the fluorescence polarization method. Overall, the developed assay using BD-labeled ligands was a simple, rapid, and reliable method for the evaluation of ER binding affinity.

结合测定法广泛用于研究靶向雌激素受体（ER）的化合物的雌激素活性。对环境敏感的荧光团苯并呋喃（BD）的荧光性质受溶剂极性的影响。在这项研究中，我们合成了BD标记的雌二醇（E2）衍生物，希望开发出可用于ER结合测定的荧光配体，而无需从结合配体中分离出来。获得了三个具有BD骨架的荧光配体，并对其荧光性质进行了研究。荧光配体和人类重组ERα（hr-ERα）相互作用的分析表明，在疏水环境（例如受体结合位点）中，荧光强度增加。在饱和结合测定中，ABD-E2衍生物2c表现出积极的合作结合，其解离常数（K _d）和希尔系数分别为23.4 nM和1.34。使用荧光偏振法，通过竞争性结合测定法评估的雌激素化合物亲和力与通过常规研究获得的结果高度相关。总体而言，使用BD标记的配体开发的检测方法是一种简单，快速且可靠的方法，用于评估ER结合亲和力。