Synthesis of 3-Hydroxyestra-1,3,5(10)-trien-17-one and Estra-1,3,5(10)-triene-3,17β-diol 6α-N-(iε-Biotinyl)caproamide, Tracer Substances for Developing Immunoassays for Estrone and Estradiol

Abstract

We describe the synthesis of 3-hydroxyestra-1,3,5(10)-trien-17-one 6 alpha-N-(epsilon-biotinyl)caproamide and 3,17 beta-dihydroxyestra-1,3,5(10)-triene 6 alpha-N-(epsilon-biotinyl) caproamide from 3-hydroxyestra-1,3,5(10)-trien-17-one and 3,17 beta-dihydroxyestra-1,3,5(10)-triene, via the 6-keto estrogenic derivatives. The reductive amination of these compounds is an effective step toward an epimeric mixture of the respective amines, which are easily biotinylated by use of N-(epsilon-biotinylcaproyl)-N- hydroxysuccinimide ester. The 6 alpha-epimers could be isolated from the alpha/beta-composition by application of isocratic HPLC, and overall yields were about 20% for the epimeric end products. The structures of the stereoisomers could clearly be assigned through 1H NMR studies. The ratios of the respective isomers obtained from the reductive amination were found to be 3(alpha):2(beta). The biotinylated estrogens can be used as tracers in a novel immunoassay concept for the determination of these analytes in human serum. Ring position 6 was selected for derivatization because of its distance from the functionalized positions 3 and 17 and, therefore, of a negligible alteration of the tracer's structure in comparison to underivatized estrone or estradiol.

Specific Photoaffinity Labeling of Tyr-49 on the Light Chain in the Steroid-Combining Site of a Mouse Monoclonal Anti-Estradiol Antibody Using Two Epimeric 6α- and 6β-(5-Azido-2-nitrobenzoyl)amidoestradiol Photoreagents,

“…Synthesis of RadioactiVe Photoaffinity Labeling Reagents. A mixture of 6R-and 6β-aminoestrone epimers (1.5 mg or 5.3 µmol in 100 µL of ethanol containing 10 µL of pyridine), prepared by chromic oxidation (Collins et al, 1968) of the 17-hydroxy group of unseparated 6-aminoestradiol epimers (Luppa et al, 1994), was reduced with a solution of [ 3 H]-NaBH 4 in ethanol (30 µL, 15 mCi) for 48 h at 4 °C. The reaction mixture was purified by TLC on silica gel (Macherey-Nagel, Alugram SIL G/UV 254 , 100:20:5 chloroform-CH 3 OH-NH 4 OH) to give an unseparated mixture of 6Rand 6β-amino[17R-3 H]estradiol epimers (specific activity of ∼1.45 Ci/mmol, estimated as 1 / 4 of that of the commercial [ 3 H]NaBH 4 ).…”

Synthesis of 6β-[(2‘-Aminoethyl)carboxamidomethyl]estradiol and Preparation of Estradiol Probes

“…After the mixture was stirred for 16.5 h, the solvent was removed on a rotary evaporator and dried on a vacuum pump. Purification of the crude product by preparative RP HPLC [Waters µBondpak C18 10 µm column (40 mm × 10 mm) with 25:75 MeCN/0.1% aqueous trifluoroacetic acid, 45 mL/min, 225 nm] and lyophilization afforded 0.011 g of the biotinylated probe (14)…”

“…Introduction of an auxiliary functionality at the C6 or C7 of the estradiol molecule can address this drawback. Among the many available labels, fluorescein and biotin have often been chosen for the construction of probes at the C6 (11)(12)(13)(14)(15), C7 (16)(17)(18), and C17 positions (19)(20)(21) of estradiol (1). Increasingly, chemiluminescent isoluminol (9) conjugates at the C3, C6, and C17 positions and acridinium phenyl esters at the C3 position (8-10) have been employed.…”

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

“…17β-Hydroxyandrost-4-en-3-one 17β-Acetate-7R-(6-Hexylamine) (5a). As described previously by Luppa et al (7), 2 mL of a buffer solution, prepared by dissolving 4.05 g of NH 4 OAc in 15 mL of dry MeOH, was added to 325 mg (0.760 mmol) of 4a and 287 mg (4.560 mmol) of NaCNBH 3 under N 2 . The reaction was worked up after 2 h by removing the solvent and extracting the residue with aqueous NaHCO 3 and Et 2 O.…”

“…This leads one to expect high specificity due to a greater conformational similarity to the native steroid. As previously shown for the estrogen estrone (E1) (7), we were able to introduce a biotin residue as the label in ring B position 6, according to Landsteiner's principle (8) that antibody specificity is directed primarily at that portion of the hapten furthest from the group linking it to the carrier protein. We applied an anti-E1 antibody raised against the hapten E1 conjugated to a carrier protein via its benzylic 6 position using the (O-carboxymethyl)oximation method to reach a nearly identical recognition site between the immunogen and the tracer.…”