Concepts for the syntheses of biotinylated steroids. Part I: Testosterone derivatives as immunochemical probes

We describe synthetic strategies for the biotinylation of testosterone (T) at positions 3, 7 alpha, 17 alpha, and 19. These T probes are able to mimic ligand binding and may provide for a better understanding of the biospecif ic interaction with steroid-binding proteins such as the androgen receptor, anti-steroid antibodies, or steroid-binding serum globulins. For the 7 alp ha- and 17 alpha-derivatives, biotinyl-N-hydroxy-succinimide esters with di fferent types of spacer chains were used. The S-biotin hydrazone derivative was produced using N-(epsilon-biotinyl)-caproyl hydrazide, whereas for the 19-biotinylation, a biotinyl-1N-diamino-3,6-dioxaoctane-amide was applied. Key reaction for the biotinylation at position 3 is the oximation of the 3 -oxo function. The 17 alpha-position is accessible by the reaction of the 3 -protected 4-androsten-17-epoxide with oxygen in the beta-position, followe d by nucleophilic ring opening with cyanide which provides the 17 alpha-cya nomethyl derivative. The key step is the regioselective ketal protection of the 3-oxo function of androst-4-ene-3,17-dione using a stannoxane catalyst . An alternative pathway for the insertion of biotin at the 19-position was established by the synthesis of 17 beta-hydroxy-androst-4-en-3-one-19-yl c arboxymethyl ether. After activation by the carbodiimide method, the compou nd reacts with aminoterminal biotin derivatives. The copper(I)-catalyzed 1, 6 Michael addition of 17-acetoxy-6,7-dehydro-T leads to 7 alpha-derivatives by use of omega-silyl protected hydroxylalkyl-modified Grignard reagents. A functional group interconversion using the Staudinger reaction transforms the azide function into a primary omega-amino group. The absolute configur ations of the different biotinylated derivatives were investigated by H-1 N MR studies. For the 7 alpha-biotinylated T series, additionally, an X-ray a nalysis proved the axial position of the spacer group. This,results in a ve rtical orientation of the biotin moiety toward the alpha-face of the planar tetracyclic backbone. Thus, a negligible alteration of the original struct ure of the upper beta-face offers the feasibility of applying the 7 alpha-d erivatives as optimal immunochemical tracers in competitive immunoassays. B iotinylated T derivatives should be also suitable for ligand-binding studie s to the androgen receptor or to sex hormone-binding globulin.