Highly specific polyclonal antisera against estriol: cross-reactivity restriction following affinity chromatography.

An immunosorbent technique was developed to attenuate cross-reactivity of a polyclonal antiserum against a 4(2) (rho-carboxyphenylazo)-1,3,5[10]-estratrien-3,16 alpha,17 beta-triol-bovine serum albumin conjugate. The chromatographic separation of antiserum through stationary phases having either rho(carboxymethyl)phenylazo-phenol or rho(carboxymethyl)-phenylazo-2-naphthol side residues reduced the antiserum avidity, while increasing the apparent antiserum affinity and decreasing the residual cross-reactivities against heterologous ligands. The highly specific antiserum obtained allowed the development of a competitive binding assay over an extended analytical range, which opens up the possibility of direct measurement of estriol from the early pregnancy to delivery. The significance of the attenuation of antiserum cross-reactions after affinity chromatography is discussed with reference to epitope-paratope interaction in the case of small endogenous molecules like estrogens.

Synthesis of ring B unsaturated estriols. Confirming the structure of a diagnostic analyte for Smith-Lemli-Opitz syndrome.

[structure: see text] Brief partial syntheses are described for ring B unsaturated estriols, which are candidate metabolites diagnostic for Smith-Lemli-Opitz syndrome prenatally. These steroids are also likely metabolites of the Premarin preparation used in estrogen replacement therapy. Equilin (8) was converted in three steps to 7-dehydroestriol, which was isomerized to 8-dehydroestriol. The simplicity of the transformations belies the lability of these previously inaccessible metabolites and their synthetic precursors.

The synthesis of estriol 16- and 17-monoglucuronide from estriol.

An efficient and convenient procedure for the synthesis of estriol 16- and 17-monoglucuronides from estriol is described. This is achieved by the selective protection and deprotection of the hydroxy groups in estriol, Koenigs-Knorr reactions with methyl 1-bromo-1-deoxy-2,3,4-tri-O-acetyl-alpha-D-glucopyranuronate and subsequent hydrolysis. The products have been characterized by proton nuclear magnetic resonance (1H NMR), two-dimensional 1H homonuclear shift-correlated spectra (2D-COSY) and mass spectra. The selective Koenigs-Knorr reaction of the alcoholic hydroxyl group in the presence of a phenolic hydroxyl group is also reported.

Immunoassay of unconjugated estriol in serum of pregnant women monitored by chemiluminescence.

6-Oxoestriol-6-(O-carboxymethyl)oxime-aminobutylethyl- isoluminol conjugate was synthesized. This luminogenic estriol derivative enabled us to develop a solid phase immunoassay method for the determination of unconjugated estriol in serum of pregnant women by the measurement of the bound estriol-isoluminol conjugate upon oxidation with a hydrogen peroxide/microperoxidase system. The sensitivity of the assay was 700 pmol/l. Results obtained by radioimmunoassay and the described method showed good agreement (r = 0.95). The chemiluminescent method is applicable in the routine measurement of unconjugated estriol.

A short efficient synthesis of 16-oxygenated estratriene 3-sulfates.

A novel synthesis of sodium 17-oxo-16 alpha-hydroxy-1,3,5(10)-estratrien-3-yl sulfate (4), sodium 16 alpha, 16 beta-dihydroxy-1,3,5(10)-estratrien-3-yl sulfate (5) and sodium 16-oxo-17 beta-hydroxy-1,3,5(10)-estratrien-3-yl sulfate (6) is described. 16 alpha-Bromo-3-hydroxy-1,3,5(10)-estratrien-17-one (1) was efficiently synthesized in one step with 70-97% yield by bromination of 3-hydroxy-1,3,5(10)-estratrien-17-one with cupric bromide. 3,16 alpha-Dihydroxy-1,3,5(10)-estratrien-17-one (3) was quantitatively obtained by controlled stereospecific hydrolysis of the bromoketone 1 with sodium hydroxide in aqueous pyridine. The bromoketone 1 was converted to the 16 alpha-hydroxy-17-ketone 3-sulfate 4 by sulfation with chlorosulfonic acid in pyridine and a subsequent controlled hydrolysis in a high yield without formation of the other ketols. Treatment of the sulfate 4 with sodium borohydride have the triol sulfate 5. The sulfate 4 was also rearranged to the 17 beta-hydroxy-16-ketone 6 with sodium hydroxide in water in a quantitative yield.

Stereoselective halogenation of the 16-hydroxymethyl-3-methoxy-13alpha-estra-1,3,5(10)-trien-17-ols and their solvolytic investigation.

The primary hydroxy functions of 16alpha-hydroxymethyl-3-methoxy-13alpha-estra-1,3,5(10)-trien-17beta-ol (3a) and 16beta-hydroxymethyl-3-methoxy-13alpha-estra-1,3,5(10)-trien-17alpha-ol (4a) were stereoselectively transformed into good leaving groups. On alkaline methanolysis of the 16-halomethyl or 16-tolylsulfonyloxymethyl derivatives, a new D-seco-13alpha-estrone derivative was obtained in high yield.

Synthesis of 15 alpha-hydroxyestrogen 15-N-acetylglucosaminides.

The synthesis of 15-N-acetylglucosaminides of 15 alpha-hydroxyesterone, 15 alpha-hydroxyestradiol, and 15 alpha-hydroxyestriol (estetrol) is described. The latter two were prepared by condensation of 2-acetamido-1 alpha-chloro-1,2-dideoxy-3,4,6-trio-O-acetyl-D-glucopyranose with appropriately protected 15 alpha-hydroxyestrogens by the Koenigs-Knorr reaction employing cadmium carbonate as a catalyst. Subsequent removal of protecting groups with methanolic potassium hydroxide provided the desired conjugates. 15 alpha-Hydroxyestrone 15-N-acetylglucosaminide was synthesized from the corresponding 15 alpha-hydroxyestradiol derivative by Jones oxidation followed by brief alkaline hydrolysis. These conjugates underwent enzymatic hydrolysis with beta-N-acetylglucosaminidase from Jack beans to produce 15 alpha-hydroxyestrogens.

Ent-16 alpha-methylamino-3-methoxy-1,3,5(10)-estratrien-17 beta-ol hydrochloride, a selective anti-arrhytmic enantiosteroid.

The title compound and its antipode with natural steroid configuration were synthesized and tested. Both compounds showed equal potency as anti-arrhythmic compounds. An additional effect found for the steroid with natural configuration was its plasma cholesterol lowering activity in the rat. This suggests that enantiosteroids may have a more selective action than the steroids with natural configuration.

Synthesis of 2-hydroxyestriol monoglucuronides and monosulfates.

The ring A monoglucuronides and monosulfates of 2-hydroxyestriol were synthesized from 2-hydroxyestriol 16,17-diacetate by means of the Koenigs-Knorr reaction with methyl alpha-acetobromoglucuronate and sulfation with sulfur trioxide-pyridine complex, respectively. The conjugated positions of these compounds were definitely established by conversion to 2-hydroxyestriol monomethyl ethers by methylation, then enzymatic hydrolysis. The ring D monoglucuronides and monosulfates of 2-hydroxyestriol were also prepared from 2-hydroxyestriol 2,3-dibenzyl ether by glucuronidation and sulfation in a similar fashion followed by debenzylation, respectively. The positions of conjugation were established on the basis of their 1H-nuclear magnetic resonance spectral data.

Preparation of specific antisera to estrone-3-glucuronide and estriol-3-glucuronide.

The preparation and antigenic properties of estrone-3-glucuronide- and estriol-3-glucuronide-bovine serum albumin conjugates in which the hapten is linked to the carrier protein through an (O-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Antibodies raised against the two immunogens in the rabbit possessed high specificity to estrone-3-glucuronide and estriol-3-glucuronide, respectively, exhibiting little cross-reactivities with other estrogen conjugates and no cross-reactions with related steroids except for free estrogens, their 3-methyl ethers and 3-sulfates. The cross-reactive antibodies were eliminated by partial immunoadsorption on affinity chromatographic media using the estrone-3-methyl ether 17-(O-carboxymethyl)oxime- and estriol-3-methyl ether 16 (or 17)-hemisuccinate-aminohexyl Sepharose conjugates, respectively. The purified antisera exhibited no cross-reactivities with free estrogens and ring A conjugates of estrone and estriol.

The use of disulfonatonaphthalimide fluorescent dyes for the fluorescence polarization immunoassay of steroids.

A series of fluorescent disulfonatonaphthalimide derivatives of testosterone and estriol have been synthesized and their fluorescent properties investigated. The fluorescence lifetimes of these derivatives were higher than that of the unreacted fluorescent dye while the quantum yields were of the same order. The compounds were therefore compared in terms of their utilizability in steroid fluorescence polarization immunoassays. The assay sensitivity and precision with each compound is discussed in terms of the position, type, and length of the chemical "bridge" linking the steroid to the fluorescent dye. It is proposed that these fluorescent labels are highly appropriate to this type of immunoassay.

Syntheses of (14 beta,15 beta,16 beta,17 alpha)- and (14 beta,15 alpha,16 alpha,17 alpha)-1,3,5(10)-estratriene-2,3,14,15,16,17- hexaols, possible candidates for the Inagami--Tamura endogenous digitalis-like factor, and their activity.

Structures are proposed for the Inagami-Tamura endogenous digitalis-like factor (EDLF), and two possible candidates, (14 beta,15 beta,16 beta,17 alpha)- and (14 beta,15 alpha,16 alpha,17 alpha)-2,3,14,15,16,17-hexahydroxy-1,3,5(10)-estratrienes, were synthesized. Both compounds were potent in inducing a contractile response in isolated rat aorta and guinea pig left atrium.