No abstract

We have examined the binding behavior and fluorescence characteristics of a series of novel ligands for the estrogen receptor (ER). These ligands are derivatives of 5,6,11,12-tetrahydrochrysene (THC), a structure that embodies a stilbene chromophore, found in many nonsteroidal estrogens, within a rigid tetracyclic system where it cannot easily be distorted from planarity, thus providing the conjugation and rigidity required for efficient fluorescence. Additional steric bulk, as trans-disposed ethyl substituents at the internal C-5 and C-11 positions, is required for the highest relative binding affinity (RBA), and the trans-5,11-diethyl-2,8-dihydroxy-THC derivative binds to ER with an affinity greater than that of estradiol. The replacement of one of the phenolic hydroxyl groups of this THC derivative with an electron-withdrawing group (COMe, COOMe, CONH2, CN, or NO2) yields unsymmetrical THCs with binding affinities 15-40% that of estradiol (E2). The fluorescence emission shifts from about 380 nm for the dihydroxy THC to 475-688 nm for the donor-acceptor THCs. The emission of these donor-acceptor THCs is highly solvatochromic and shifts to longer wavelengths as the solvent polarity increases. In ethanol, the fluorescence quantum yield of the first four of these compounds is high (phi f = 0.43-0.69), but the fifth compound, the nitro-THC, is almost nonemissive in protic solvents. When they are incubated with protein solutions containing ER (approximately 10(-9) M), the emission from the donor-acceptor THCs bound specifically to ER is in the 500-570-nm range, whereas fluorescence from non-receptor-bound fluorophores is in the 425-460-nm range. Thus, fluorescence from these probes bound specifically to ER could be measured under equilibrium conditions as well as after the removal of free and non-receptor-bound material by treatment with charcoal-dextran. This is one of the first demonstrations of ligands whose fluorescence is distinctly different when free, when bound to ER, or when bound to non-receptor proteins. It is also the first demonstration of ER assay by fluorescence under equilibrium conditions.

We have analyzed four fluorescent nonsteroidal estrogens for their potential to serve as vital cytological stains to visualize the estrogen receptor (ER) in a model receptor expression system. The novel estrogen fluorophores are based on the rigidified stilbene-like structure of 5,6,11,12-tetrahydrochrysene (THC), and they embody electron-donor (hydroxyl) and electron-acceptor groups (nitrile, amide, ester, or ketone) that afford efficient, long wavelength, and environment-sensitive fluorescence. These probes bind with high affinity to human ER (relative binding affinity, 22-85 vs. estradiol, 100), and they stimulate the transcriptional activity of this receptor. The strong fluorescence of the estrogenic THCs permits visualization, using conventional epifluorescence microscopy, of ER in transfected Cos-7 cells that express elevated levels of receptor. Cell staining by the donor-acceptor THCs characteristically displays a nonuniform pattern of nuclear fluorescence that can be fully inhibited by nonfluorescent estrogens such as estradiol or diethylstilbestrol. Additionally, this staining appears to be specific for ER, since it coincides with the distribution of receptor as determined by indirect immunofluorescence analysis using an ER-specific monoclonal antibody. Using these probes, we have analyzed the intracellular distribution of ER mutants containing a variety of deletions. Evidence is presented to show that removal of amino-terminal sequences within the ER polypeptide results in an altered pattern of intranuclear distribution with preferential accumulation of receptor protein within the nucleolus. These THC fluorophores therefore represent excellent probes for cytological studies involving ER expressed in cultured cells and represent an important advance toward the goal of exploiting fluorescence technology to analyze the expression and distribution of ER within tissue samples.

Crystals of the title compound, CaaHxxO2N, are orthorhombic, space group Fdd2, Z= 8, a=27.289 + 0.004, b = 13.108 + 0.005, c = 9-165 + 0.002/~, Dx = 1.362 g.cm -3. Data for 454 reflections were collected at room temperature on a Picker automated diffractometer. The structure was solved by direct methods, using a computerized multiple-solution procedure, and it was refined by full-matrix least-squares methods to a final R on F of 0"046, with 377 observed reflections. The average e.s.d.'s of the bond lengths and angles not involving hydrogen are about 0.008 A and 0.5 °, respectively. Bond lengths and angles were corrected for thermal motion. Some C-C single bonds are found to be significantly shorter than the normal C-C single bond length of 1.54 A. The shortening of the C-C bonds is explained by the conjugation of the C=O bond and C=C bond with the three-membered ring.