Synthesis of 17β-Hydroxyandrost-4-en-3-one−7α-(Biotinyl-6-N-hexylamide), a Conjugate Useful for Affinity Chromatography and for Testosterone Immunoassays

Abstract

We describe the synthesis of 17 beta-hydroxyandrost-4-en-3-one-7 alpha-(biotinyl-6-N-hexylamide) 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 1H 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 3H-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.

Concepts for the Syntheses of Biotinylated Steroids. Part I: Testosterone Derivatives as Immunochemical Probes

“…This presentation describes synthetic approaches for the biotinylation of T at the following positions: 3, 7R, 17R, and 19. Besides the synthetic pathways for the aliphatic 7R-biotinylated derivatives using the 1,6 Michael addition of hydroxylalkyl-modified Grignard compounds to 17-acetoxy-6,7dehydro-T [already described by our group (Luppa et al, 1996)], which represents a nonstereoselective reaction, the 1,6-addition of the 9-t-butyldimethyl-silyloxy-5-oxanonyl-and 13-t-butyldimethylsilyoxy-7-oxa-tridecyl-magnesium bromides provides ether chain-linked 7R-derivatives with high R-stereoselectivity. 3-Biotinylated T derivatives are available by the well-known hydrazone formation procedures at the 3-oxo position in an one pot reaction.…”

“…High specificity is achievable only if the tracer resembles in structure to the native steroid (Chames and Baty, 1998). Recently, we showed that the introduction of a biotin label to E1 at the ring position 6R and to T at the position 7R leads to negligible alterations in comparison to the structure of the respective steroidal hapten (Luppa et al, 1994(Luppa et al, , 1996. These characteristics allow sensitive and reproducible determinations of E1 and T over large dynamic ranges (Luppa et al, 1995(Luppa et al, , 1997.…”

“…The resulting clear solution was diluted with further solvent and after 1 h cooled to -40 to -30 °C in an 2-propanol/dry ice bath. A total of 2 equiv of freshly prepared CuI was added, 15 min later, 1 equiv of 17Rhydroxyandrost-4,6-dien-3-one 17β-acetate [6-dehydro-T-17β-acetate 3, preparation according to the prescription given in (Luppa et al, 1996)] in THF was dripped to the solution within 50 min at -45 to -35 °C, giving a reddish suspension. After being stirred for further 40 min, the reaction mixture was quenched with glacial acetic acid at -30 °C, then subsequently basified with a saturated NH 4 Cl solution.…”

“…17β-Acetoxy-7R/β-(6′-dimethyl-t-butylsilyloxy-hexyl)androst-4-en-3-one, 5b. The synthesis is already described in Luppa et al, (1996).…”

Synthesis of 15?-hydroxyalkyl-substituted (17Z)-pregn-17-enes, their ethers and esters

“…The introduction of an alkenyl chain was carried out by adding to unsaturated ketone II the Grignard reagent prepared from 5-bromopentene [9] under catalysis with copper bromide complex with dimethyl sulfide [7,10,11]. Adding to the reaction mixture the trimethylsilyl chloride (TMSCl) and the hexamethylphosphoramide (HMPA) [12] we raised the yield of adduct IV from 85 to 95%.…”