Synthesis of 3-Hydroxyestra-1,3,5(10)-trien-17-one and 3,17β-Dihydroxyestra-1,3,5(10)-triene 6α-N-(ϵ-Biotinyl)caproamide, Tracer Substances for Developing Immunoassays for Estrone and Estradiol

We describe the synthesis of 3-hydroxyestra-1,3,5(10)-trien-17-one 6α-2N-(ϵ-biotinyl)caproamide and 3,17β-dihydroxyestra-1,3,5(10)-triene 6α-N-(ϵ-biotinyl)caproamidefrom3-hydroxyestra-1,3,5(10)-trien-17-one and 3,17β-dihydroxyestra-1,3,5(10)-triene, via the 6-keto estrogenic derivatives. The reductive amination of these compounds is an effective step toward an epimeric mixture of the respective amines, which are easily biotinylated by use of N-(ϵ-biotinylcaproyl)-N-hydroxysuccinimide ester. The 6α-epimers could be isolated from the α/β-composition by application of isocratic HPLC, and overall yields were about 20% for the epimeric end products. The structures of the stereoisomers could clearly be assigned through ¹H NMR studies. The ratios of the respective isomers obtained from the reductive amination were found to be 3(α):2(β). The biotinylated estrogens can be used as tracers in a novel immunoassay concept for the determination of these analytes in human serum. Ring position 6 was selected for derivatization because of its distance from the functionalized positions 3 and 17 and, therefore, of a negligible alteration of the tracer's structure in comparison to underivatized estrone or estradiol.