SYNTHESIS OF 3-HYDROXYESTRA-1,3,5(10)-TRIEN-17-ONE AND 3,17-BETA-DIHYDROXYESTRA-1,3,5(10)-TRIENE 6-ALPHA-N-(EPSILON-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 6alpha-N-(epsilon-biotinyl)caproamide and 3,17beta-dihydroxyestra-1,3,5(10)-triene 6alpha-N-(epsilon-biotinyl)caproamide from 3-hydroxyestra-1,3,5(10)-trien-17-one and 3,17 beta-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-(epsilon-biotinylcaproyl)-N-hydroxysuccinimide ester. The 6alpha-epimers could be isolated from the alpha/beta-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 H-1 NMR studies. The ratios of the respective isomers obtained from the reductive amination were found to be 3(alpha):2(beta). 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.