Concepts for the Syntheses of Biotinylated Steroids. Part II:  17β-Estradiol Derivatives as Immunochemical Probes

Abstract

Biotinylated 17beta-estradiol (E2) derivatives are helpful probes for a better understanding of biospecific E2 interactions with steroid-binding proteins such as the estrogen receptor and anti-steroid antibodies. We describe synthetic strategies for the biotinylation of E2 toward the 3, 6alpha, and 7alpha positions using biotinyl-N-hydroxysuccinimide esters with different spacers, varying in structure and chain length. Key reaction for biotinylation at the 3 position is the regioselective ether formation of the phenolate E2 anion with a linker mesylate without protecting the 17beta-hydroxyl group. The 6alpha position is accessible via a 3,17beta protected 6alpha-hydroxy E2, prepared by stereospecific sodium borohydride reduction of 6-oxo E2. Direct cyanoethylation of the alcohol followed by reduction to the amine allows the biotinylation to 6alpha-O-coupled cyanoethyloxy linker E2 derivatives. Alternatively, 6alpha-O-coupled cyanoalkyloxy polyether linker E2 probes are obtained by a Williamson ether synthesis of the alcohol precursor with omega-t-butyl-dimethylsilyloxy-5-oxa-nonylmesylate. Cyanoethylation of the desilylated compound and further reduction of the nitrile led to the terminal amine. Reductive amination of the 3, 17beta acetylated 6-oxo E2 compound with 6-cyanoethyloxyhexyl ammonium acetate yields in a mixture of 6alpha/beta-N-alkylated E2 nitriles. The epimers are separated by reversed-phase HPLC and the 6alpha-compound subsequently reduced to the terminal amine. The 7alpha-biotinylated E2 compound is derived from 7alpha-(11'-undecyl-N-methyl-N-butylamide) E2, which is already known from literature. Subsequently, the 3 and 17beta positions are protected, and the amide is reduced to the 7alpha-(11'-undecanol) compound. Further cyanoethylation and reduction led to the 11'-amino-ethyloxyundecyl E2. Using (1)H NMR analysis, it could be shown that the biotin moiety of the biotinylated 6alpha- and 7alpha-E2 derivatives has an axial position which results in a vertical orientation of the substituent 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 6alpha- and 7alpha-derivatives as optimal tracers in competitive immunoassays.

Biotinylated Steroid Derivatives as Ligands for Biospecific Interaction Analysis with Monoclonal Antibodies Using Immunosensor Devices

“…The following biotinylated T and E2 derivatives were used: 17␤-hydroxyandrost-4-en-3-(carboxymethyloxime-(biotinyl-1-N-diamino-3,6-dioxaoctylamine) (bio-t-3); 17␤-hydroxy-7␣-(9Ј-biotinylaminocaproyl-5Ј-oxanonylamine)-androst-4-en-3-one) (bio-t-7); 17␤-hydroxyandrost-4-en-3-one-19-yl-oxymethylenecarbonyl-(biotinyl-1-N-diamino-3,6-dioxaoctylamine) (bio-t-19); 17␤-hydroxy-3-(13Ј-biotinylaminocaproyl-5Ј,10Ј-dioxatridecylaminooxy)-estra-1,3,5(10)-triene (bio-e-3), and 3,17␤-dihydroxy-6␣-(10Ј-biotinylaminocaproyl-5Ј-oxadecylamine)-estra-1,3,5(10)-triene (bio-e-6). The syntheses of the steroid derivatives are described in detail elsewhere (14,15).…”

“…For the investigation of the kinetics and the position specificity of various testosterone (T) and 17␤-estradiol (E2) mAbs in the course of the binding process to the respective different steroidal derivatives, several biotinylated T and E2 compounds (bio-s) were synthesized with sterically defined structures (14,15) and bound to tetravalent SAv molecules that were coated to the sensor surfaces. To guarantee high affinities to the ligandbinding domains of the respective antibody, the site of substitution, structure, stereochemistry and functionality of the modification are of paramount importance (14,15).…”

“…To guarantee high affinities to the ligandbinding domains of the respective antibody, the site of substitution, structure, stereochemistry and functionality of the modification are of paramount importance (14,15). Therefore, biotin derivatization was performed at the 3, 7␣, and 19 positions for T and at the 3 and 6␣ positions for E2 in order to mimic the epitope structure of the respective hapten that was used for the immunization process.…”

Novel Synthetic Antiestrogens That Block Nuclear Estrogen Receptor Function Through Plasma Membrane Localization

“…The synthesis of compounds 5-7 required cholesterolderived electrophiles 12-14. The acid chloride 12 was prepared from cholesterol (9) in three steps as shown in Scheme 1. As shown in Figure 4, cholesteryl chloroformate (13) was commercially available, and the protected cholesterylamine (14) was prepared as previously described."…”

Synthesis, Photophysical and Electrochemical Properties, and Protein‐Binding Studies of Luminescent Cyclometalated Iridium(III) Bipyridine Estradiol Conjugates

“…However, the potential health hazards of these reagents and the long experimental time required have prompted the search for nonisotopic reagents. Different estradiol derivatives equipped with reporters such as organic fluorophores, [7][8][9][10] biotin, [10][11][12] transition-metal complexes, [13,14] and organometallic compounds [15] have been reported. In particular, a wide range of estradiol derivatives containing chromium, rhenium, technetium, cobalt, iron, and manganese carbonyl complexes have been investigated.…”