Chemical synthesis and biochemical characterization of a biotinylated derivative of 17beta-estradiol with a long side chain covalently attached to its C-7alpha position.
High-affinity biotinylated derivatives of 17beta-estradiol (E2) are of value for isolation of various estrogen-binding proteins (including estrogen receptors) and also for studying protein-protein interactions involving these proteins. In this study, we developed a simplified route for the chemical synthesis of a biotinylated derivative of E2 (compound 7) with a side chain attached to its C-7alpha position. Compound 7, i.e., 7alpha-{7-[8-(biotinamido)-octanamido]-heptyl}-estradiol, could be readily synthesized from 6-keto-estradiol-3,17beta-di-tetrahydropyranyl ether (compound 2, which can be prepared from E2), with a final yield of 36%. In vitro receptor-binding assay confirmed that the synthesized affinity ligand has a high binding affinity for both human estrogen receptor alpha and beta. When the affinity ligand (compound 7) was immobilized with avidin on an affinity column, it effectively bound human estrogen receptor alpha, and the receptor protein could be selectively eluted with a biotin-containing buffer. Using the same affinity ligand, prolyl 4-hydroxylase beta-subunit (also known as protein disulfide isomerase) was identified as one of the high-affinity E2-binding proteins in the whole cytosolic protein mixture prepared from MCF-7 human breast cancer cells. Computational molecular modeling analysis showed that compound 7 can bind to human estrogen receptor alpha in a similar manner as ICI-182,780 and raloxifene, and their binding energy values are also similar.
Jiang
Xiang-Rong
XR
Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA.
Wang
Pan
P
Fu
Xinmiao
X
Zhu
Bao Ting
BT
D001710:Biotin / Q000737:chemistry*; D004958:Estradiol / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D047628:Estrogen Receptor alpha / Q000378:metabolism; D047629:Estrogen Receptor beta / Q000378:metabolism; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular
D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D008024:Ligands; D004958:Estradiol; D001710:Biotin