title: Concepts for the syntheses of biotinylated steroids. Part I: testosterone derivatives as immunochemical probes.
abstract: We describe synthetic strategies for the biotinylation of testosterone (T) at positions 3, 7alpha, 17alpha, and 19. These T probes are able to mimic ligand binding and may provide for a better understanding of the biospecific interaction with steroid-binding proteins such as the androgen receptor, anti-steroid antibodies, or steroid-binding serum globulins. For the 7alpha- and 17alpha-derivatives, biotinyl-N-hydroxy-succinimide esters with different types of spacer chains were used. The 3-biotin hydrazone derivative was produced using N-(epsilon-biotinyl)-caproyl hydrazide, whereas for the 19-biotinylation, a biotinyl-1-N-diamino-3, 6-dioxaoctane-amide was applied. Key reaction for the biotinylation at position 3 is the oximation of the 3-oxo function. The 17alpha-position is accessible by the reaction of the 3-protected 4-androsten-17-epoxide with oxygen in the beta-position, followed by nucleophilic ring opening with cyanide which provides the 17alpha-cyanomethyl 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 17beta-hydroxy-androst-4-en-3-one-19-yl carboxymethyl ether. After activation by the carbodiimide method, the compound reacts with aminoterminal biotin derivatives. The copper(I)-catalyzed 1,6 Michael addition of 17-acetoxy-6,7-dehydro-T leads to 7alpha-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 configurations of the different biotinylated derivatives were investigated by (1)H NMR studies. For the 7alpha-biotinylated T series, additionally, an X-ray analysis proved the axial position of the spacer group. This results in a vertical orientation of the biotin moiety toward the alpha-face of the planar tetracyclic backbone. Thus, a negligible alteration of the original structure of the upper beta-face offers the feasibility of applying the 7alpha-derivatives as optimal immunochemical tracers in competitive immunoassays. Biotinylated T derivatives should be also suitable for ligand-binding studies to the androgen receptor or to sex hormone-binding globulin.
authors.affiliation: Institute for Organic Chemistry, Universität Regensburg, D-93053 Regensburg, Germany, Institute for Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar, Technische Universität München, D-81675 München, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Hauptmann
authors.forename: B
authors.initials: B
authors.lastname: Paulus
authors.forename: T
authors.initials: T
authors.lastname: Kaiser
authors.forename: E
authors.initials: E
authors.lastname: Herdtweck
authors.forename: E
authors.initials: E
authors.lastname: Huber
authors.forename: P B
authors.initials: PB
authors.lastname: Luppa
mesh_terms: D019921:Biotinylation / Q000379:methods; D002942:Circular Dichroism; D007120:Immunochemistry / Q000379:methods; D007536:Isomerism; D013058:Mass Spectrometry; D008958:Models, Molecular; D015335:Molecular Probes / Q000138:chemical synthesis*; D019906:Nuclear Magnetic Resonance, Biomolecular; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet; D013256:Steroids / Q000737:chemistry*; D013739:Testosterone / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D015335:Molecular Probes; D013256:Steroids; D013739:Testosterone