SYNTHESIS OF ROST-4-EN-3-ONE-7-ALPHA-(BIOTINYL-6-N-HEXYLAMIDE), A CONJUGATE USEFUL FOR AFFINITY-CHROMATOGRAPHY AND FOR TESTOSTERONE IMMUNOASSAYS

We describe the synthesis of 17 beta-hydroxyandrost-4-en-3-one-7 alpha -(biotin from 17 beta-hydroxyandrost-4-en-3-one (testosterone) via cop per-catalyzed 1,6 Michael addition of a 6-(tertbutyldimethylsilyloxyhe xyl) chain to 6-dehydrotestosterone 17 beta-acetate. After chromatogra phic separation of the 7 alpha-isomer from the alpha/beta mixture and cleavage of the silyl ether, the alcohol was oxidized to the 6-hexanal side chain and then subjected to reductive amination. The resulting p rimary amine is easily biotinylated using biotinyl-N-hydroxysuccinimid e ester. The overall yield for the epimeric 7 alpha-end product was 30 %. The absolute configurations of the epimers were investigated by H-1 NMR studies by the nuclear Overhauser effect. We introduced a biotin label to the testosterone molecule at ring position 7 in compliance wi th Landsteiner's principle, which states that antibody specificity is directed primarily at that portion of the hapten furthest from the fun ctional group linking it to the carrier protein. Thus, this negligible alteration in comparison to the structure of the respective testoster one hapten used to elicit antibodies offers the feasibility of applyin g the testosterone derivative as an optimal immunoadsorbent in affinit y chromatography. The 7 alpha-biotinylated testosterone was used to ob tain active antitestosterone antibodies from a specific antiserum by a ffinity chromatography. This was achieved by attaching the biotinylate d testosterone to agarose-coupled streptavidin beads, Accordingly, a H -3-testosterone-binding test demonstrated a 20-fold increase in affini ty of the purified antibody to the steroid compared to the original an tiserum, and a recovery of >80% could be obtained. The antitestosteron e antibody, obtained by that method, is an effective component for use in a competitive immunoassay for testosterone in human sera. An assay configuration is conceivable with the same 7 alpha-biotinylated testo sterone employed as tracer in combination with a streptavidin-linked r eporter enzyme.