Chemical synthesis and biochemical characterization of a biotinylated derivative of 17β-estradiol with a long side chain covalently attached to its C-7α position

Abstract

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.

Protein disulfide isomerase is a multifunctional regulator of estrogenic status in target cells

“…For isolation of E 2 -binding proteins, a novel biotinylated E 2 affinity ligand (recently synthesized in our laboratory [22]) was used. All of the following steps were performed at 4 • C. MCF-7 cells were harvested in 10 mM sodium phosphate buffer (pH 7.4) and sonicated in the presence of 1 mM EDTA and protease inhibitor cocktail (Sigma-Aldrich).…”

“…Using a newly developed biotinylated E 2 affinity ligand [22], we identified several potential E 2 -binding or ER-interacting proteins, including Hsp70, calmodulin and PDI, in the cytosol of MCF-7 human breast cancer cells (Fig. 1A and Fig.…”

Synthesis of Novel Estrogen Receptor Antagonists Using Metal-Catalyzed Coupling Reactions and Characterization of Their Biological Activity

“…The synthesis of compound 4 was described in our recent study. , For the synthesis of compound 5 , a 1.0 M solution of t -BuOK in THF (5.35 mL, 5.35 mmol) was added to a cooled solution (at 0 °C) of compound 4 (1.2 g, 2.67 mmol) in THF (20 mL). The reaction mixture was stirred at 0 °C for 40 min and then cooled to −78 °C.…”

“…The synthesis of compound 4 was described in our recent study. 21,22 For the synthesis of compound 5, a 1.0 M solution of t-BuOK in THF (5.35 mL, 5.35 mmol) was added to a cooled solution (at 0 °C) of compound 4 (1.2 g, 2.67 mmol) in THF (20 mL). The reaction mixture was stirred at 0 a The ligand−ERα interaction energy values (ΔE binding ) were calculated using the following equation:…”

Identifying the Cellular Targets of Bioactive Small Molecules with Activity-Based Probes

“…Biotinylated probes can be utilized in various ways in target identification strategies. They can be pre-complexed with a streptavidin resin to create a non-covalent affinity matrix [29][30][31][32][33][34][35][36], or incubated with live cells for desired time and then fished out of the solution with streptavidin resin after cell lysis [37][38][39][40]. The biotinylated probes and their attached proteins may be eluted from avidin resins by 8 M guanidineHCl, pH 1.5, or by boiling in a typical denaturing SDS-PAGE loading dye.…”