Ohno Akiko, Shimizu Masato, Yamada Sachiko
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Japan.
Chem Pharm Bull (Tokyo). 2002 Apr;50(4):475-83. doi: 10.1248/cpb.50.475.
To investigate vitamin D conformation, specifically the A-ring and seco-B ring parts, in its complex with the vitamin D receptor (VDR), we have previously suggested the use of 19F-NMR spectroscopy and have synthesized three fluorovitamin D derivatives to be used for the study, 4,4-difluoro-1alpha,25-dihydroxyvitamin D3 [4,4-F2-1,25-(OH)2D3, 2a], and (10Z)- and (10E)-19-fluoro-1alpha,25-dihydroxyvitamin D3 [19-F-1,25-(OH)2D3, 3a, 4a]. In this paper, we examined the 19F-NMR spectra of these and related fluorovitamin D compounds in detail. In the low temperature 19F-NMR spectra, we observed two well-separated rigid conformations of 2a (51:49) and 4a (84:16), while only one conformation was detected with 3a. The two observed conformers of 2a and 4a were respectively assigned to the known alpha- and beta-conformers formed by the flipping of the A-ring where the C(19) exocyclic methylene points to the alpha- and beta-faces. The single conformation observed for 3a was assigned to the alpha-conformer. We detected no other conformation in the 19F-NMR of all vitamin D compounds examined. The effect of solvents on the 19F chemical shifts of fluorovitamin D compounds was found to be small (less than 6.3 ppm). This was much smaller than the difference between the two A-ring conformers (13-30 ppm). Using the dynamic 1H-NMR studies of fluorovitamin D compounds, we determined the free energy of activation for the interconversion between the two conformations of 2a (9.9 kcal/mol) and 4a (10.8, 11.5 kcal/mol). Introduction of the 1alpha-hydroxyl group raised the activation energy about 1 kcal/mol. The affinity for VDR was evaluated, and the relative potency of 2a, 3a and 4a was found to be 1%, 8% and 9%, respectively, of that of 1,25-(OH)2D3 (1). Though the affinity for VDR was considerably reduced in these compounds, the ability to activate gene transcription was similar and remained in the range 30-50% of the effect of 1. This biological information in combination with the NMR properties indicates that 2a and 4a are promising probes for studying the VDR-bound A-ring conformation of vitamin D.
为了研究维生素D与维生素D受体（VDR）形成复合物时的构象，特别是A环和开环B环部分，我们之前建议使用19F-NMR光谱法，并合成了三种用于该研究的氟代维生素D衍生物，即4,4-二氟-1α,25-二羟基维生素D3 [4,4-F2-1,25-(OH)2D3, 2a]，以及(10Z)-和(10E)-19-氟-1α,25-二羟基维生素D3 [19-F-1,25-(OH)2D3, 3a, 4a]。在本文中，我们详细研究了这些以及相关氟代维生素D化合物的19F-NMR光谱。在低温19F-NMR光谱中，我们观察到2a（51:49）和4a（84:16）有两种明显分开的刚性构象，而3a仅检测到一种构象。观察到的2a和4a的两种构象分别对应于已知的由A环翻转形成的α-和β-构象，其中C(19)环外亚甲基指向α-面和β-面。观察到的3a的单一构象被归为α-构象。在所有检测的维生素D化合物的19F-NMR中，我们未检测到其他构象。发现溶剂对氟代维生素D化合物的19F化学位移的影响很小（小于6.3 ppm）。这比两个A环构象之间的差异（13 - 30 ppm）小得多。通过对氟代维生素D化合物的动态1H-NMR研究，我们确定了2a（9.9 kcal/mol）和4a（10.8, 11.5 kcal/mol）两种构象之间相互转化的活化自由能。引入1α-羟基使活化能提高了约1 kcal/mol。评估了它们对VDR的亲和力，发现2a、3a和4a的相对效力分别为1,25-(OH)2D3（1）的1%、8%和9%。尽管这些化合物对VDR的亲和力大幅降低，但激活基因转录的能力相似，仍保持在1的作用效果的30 - 50%范围内。这些生物学信息与NMR性质相结合表明，2a和4a是研究维生素D与VDR结合时A环构象的有前途的探针。
