Confromational analysis of fluorovitamin D analogs interacting with vitamin D receptor using ^<19>F-NMR spectroscopy

活性维生素D通过与介导基因表达的维生素受体（VDR）结合，通过基因组机制诱导生物学应答。维生素D-VDR复合物的形成是表达生物学作用的第一步，活性维生素D与VDR相互作用时的三维结构至今尚未确定。为了用~ 1H-NMR直接检测维生素D在维生素D-配体复合物中的构象<19>，我们设计了含氟维生素D类似物作为探针化合物，成功地合成了4，4-二氟维生素D和19-氟维生素D类似物作为探针，分别用于检测维生素D的A环和共轭三烯构象。4，4-二氟维生素D和19-氟维生素D类似物与牛胸腺VDR的结合亲和力分别约为活性维生素D的1/100和1/10。为了分析A环和共轭三烯构象平衡，我们研究了<19>氟维生素D类似物在可变温度下的1HF-NMR光谱。这些化合物中的每一个在低于-60 ° C时都给出了单独的信号，为towing A构象。大鼠维生素D受体配体结合结构域（VDR-LBD）在细菌中表达为融合蛋白。VDR-LBD<19>-氟维生素D复合物的1H-NMR光谱代表来自结合配体的额外信号。我们已经开始了一个系统的调查分配的配体构象在结合状态。

The active vitamin D induces biological responses via genomic michanism by binding to the vitamin receptor (VDR) which mediates gene expression. Formation of vitamin D-VDR complex is the first step for expressing of biological actions and the three-demensional structure of the active vitamin D at the time of interacting with VDR has not been determined up to now. To directly examine a strereostructure of vitamin D in the VDR-ligand complex by ^<19>F-NMR spectroscopy, we designed the fluorinated vitamin D analogs as probe compounds.We successfully synthesized 4,4-difluorovitamin D and 19-fluorovitamin D analogs as probes to monitor the A-ring and the conjugated triene conformations of vitamin D,respectively. Binding affinity of 4,4-difluorovitamin D and 19-fluorivitamin D analog to bovine thymus VDR was about 1/100 and 1/10, respectively, as effective as the active vitamin D.To analyze the A-ring and the conjugated triene conformational equilibria, we investigated the ^<19>F-NMR spectra of the fluorovitamin D analogs at a variable temperature. Each of these compounds gave separate signals at below-60ﾟC,for the tworing A conformations. These analogs may serve as excellent probes for the A-ring conformational analysis.The rat vitamin D receptor ligand binding domain (VDR-LBD) was expressed as fusion proteins in bacteria. The ^<19>F-NMR spectrum of VDR-LBD-fluorovitamin D complex represented additional signals from bound ligand. We have begun a systematic investigation of the assignment of the ligand conformation in the bound state.