Organometallic estrogens: synthesis, interaction with lamb uterine estrogen receptor, and detection by infrared spectroscopy

Abstract

As an integral part of the development of a new technique using organometallic markers for the detection of hormone receptors by FT-IR spectroscopy, a series of estradiol derivatives labeled with Cr(CO)3 or Cr(CO)2CS fragments on the A ring has been synthesized. The stereochemistry of one of these steroids, alpha-[3-(dimethyl-tert-butylsiloxy)-17 beta-estradiol]dicarbonyl(thiocarbonyl)chromium(0), has been established by X-ray diffraction. The organochromium-labeled steroids are stable in aqueous methanol solution, and their relative binding affinities to estrogen receptor have been determined; these values vary from 0.4 to 28%. The complex exhibiting the strongest affinity, [3-O-(3-hydroxypropyl)-17 beta-estradiol]-chromium tricarbonyl complex, has been prepared in a tritiated form with a high specific activity (4.1 Ci/mmol). This tritiated hormone binds reversibly to the estradiol receptor in lamb uterine cytosol with an affinity (Kd = 0.85 nM) and number of binding sites (n = 770 fmol/mg of protein) close to the values observed for estradiol itself. The level of nonspecific binding is low, and the hormone is not bound significantly to other nontarget tissues. The observation that the binding affinity of the steroid depends on which side of the steroidal A ring the organometallic label is bound demonstrates the nonequivalence of the two sides of the A ring with respect to the receptor site. The FT-IR spectra of the organochromium markers in the v(CO) region can be used for the detection of the estradiol receptor in lamb uterine cytosol.

Stabilization of Carbenium Ions Derived from Ethynylestradiol by Different Adjacent Organometallic Moieties. Implication in the Inactivation of the Estrogen Receptor

“…Melikyan et al resolved the structure of a Co 2 (CO) 6 -complexed propargyl cation showing that the C ϩ atom is no longer equidistant from Co atoms. [24] The same behavior was observed in 3 ϩ , where the organometallic moiety shifts below the estradiol D ring, enabling a Mo-C (17) ϩ interaction to be established. [6] In the case of 3 ϩ this interaction is weaker than that reported for 3a ϩ , and interestingly, this difference has been attributed to the conformational constraints imposed by the bioligand.…”

“…The quantification of the [ 3 H]estradiol specifically bound to the ER was then performed by protamine sulfate precipitation as previously described. [17] The residual radioactivity on the precipitate is related to the remaining estrogen binding activity: the higher the radioactivity, the lower the receptor inhibition (RI% ϭ 0 for estradiol itself). [18] Carbenium ions can be generated in presence of strong acids.…”

“…At the end of the incubation, separation of the bound fractions of the steroids was achieved by using protamine sulfate precipitation. [17] The bound fractions were collected on glass fiber paper (Whatman GF/C) under a light vacuum and washed extensively with phosphate buffer (pH ϭ 7.2). The filters were then transferred into a vial containing 10 mL of scintillation fluid (A.C.S., Amersham) and the radioactivity was measured in an LKB-1211 Rack-Beta scintillation counter.…”

Synthesis of cyclopentadienyltricarbonylrhenium substituted benzhydryl species and oestrogen receptor binding properties

“…• C) as previously described 26 was used as a source of cytosolic ERα. For purified ERβ, 10 µl of the solution containing 3500 pmol/mL were added to 16 ml of buffer B (10% glycerol, 50 mM bis-Tris-propane pH = 9, 400 mM KCl, 2 mM DTT, 1 mM EDTA, 0.1% BSA) in a silanized flask.…”

A variable-temperature 13C NMR study of (η5-C5H5)2Mo2(CO)4 complexes of chiral alkynes derived from a terpene or a hormonal steroid: interconversion of diastereomers via exchange of terminal and semi-bridging carbonyls

“…These molecules, in which the M2Cz tetrahedral moiety is situated on the a-face of the steroid, are known to bind to various steroidal receptor sites (7,8). Moreover, it has been demonstrated that one can take advantage of the intense metal carbonyl infrared stretching absorptions to assay the receptor sites without any of the inconveniences associated with radio-labels (9,10).…”