Synthesis of 17 beta-hydroxyandrost-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 copper-catalyzed 1,6 Michael addition of a 6-(tertbutyldimethylsilyloxyhexyl) chain to 6-dehydrotestosterone 17 beta-acetate. After chromatographic 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 primary amine is easily biotinylated using biotinyl-N-hydroxysuccinimide 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 with Landsteiner's principle, which states that antibody specificity is directed primarily at that portion of the hapten furthest from the functional group linking it to the carrier protein. Thus, this negligible alteration in comparison to the structure of the respective testosterone hapten used to elicit antibodies offers the feasibility of applying the testosterone derivative as an optimal immunoadsorbent in affinity chromatography. The 7 alpha-biotinylated testosterone was used to obtain active antitestosterone antibodies from a specific antiserum by affinity chromatography. This was achieved by attaching the biotinylated testosterone to agarose-coupled streptavidin beads, Accordingly, a H-3-testosterone-binding test demonstrated a 20-fold increase in affinity of the purified antibody to the steroid compared to the original antiserum, and a recovery of >80% could be obtained. The antitestosterone 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 testosterone employed as tracer in combination with a streptavidin-linked reporter enzyme.