Preparation of 6α- and 6β-carboxymethyl steroid conjugates and their use in radioimmunoassay for progesterone

Abstract

A method was devised to utilize the Δ 4-3-ketone structure of steroids to obtain a functional group at the 6 position which could be conjugated to proteins. The bis ethylene ketal of progesterone was formed and the resulting Δ 5 double bond was subjected to epoxidation. The 5α,6α-epoxide formed was opened with allyl magnesium bromide after which the terminal carbon-carbon double bond was oxidized to provide a carboxyl group which could be coupled to the free amino groups of proteins. The 6α and 6β carboxymethyl progesterone derivatives formed by this method were coupled to bovine serum albumin (BSA) using tributyl amine and i -butyl chlorocarbonate. The 6α conjugate averaged 19 residues per molecule of BSA and the 6β, 24 residues per BSA. Antisera to the conjugates produced in rabbits showed very little cross-reaction with steroids which differed from progesterone at positions other than C-5 or C-6. There appeared to be very little difference between the specificities of the 6α and 6β antisera.