Synthesis of 17 beta-hydroxy esters of 4-estren-17 beta-ol-3-one and carbenicillin, ticarcillin, or functionalized oxacillin: potentially useful conjugates for beta-lactamase-based homogeneous immunoassays.
On the basis of the large range of kinetic constants of their substrates, beta-lactamases seem to be interesting enzymes for the development of homogeneous immunoassays. For this purpose, hapten-penicillin or -cephalosporin conjugates have to be prepared. The aim of this work is to couple the anabolizing steroid nandrolone to several penicillins characterized by extremely low K(m) and kcat values: ticarcillin, carbenicillin, and oxacillin. The easy decarboxylation of derivatives of phenylmalonic acid (carbenicillin) and thienylmalonic acid (ticarcillin) imposes the choice of very mild procedures which have been specifically adapted to each substance investigated. 4-Estren-17 beta-ol-3-one hemiphenylmalonate is conjugated to 6-aminopenicillanic acid after 1,1'-carbonyldiimidazole activation, while 4-estren-17 beta-ol-3-one hemi(3-thiophene)malonate is coupled to 6-aminopenicillanic acid after activation using methanesulfonyl chloride. Before conjugation of oxacillin, a carboxylated analogue of its side chain has been prepared. A procedure resorting to tert-butyl ester protection of the carboxyl group present on the isoxazole ring allows the binding of nandrolone to the remaining carboxyl followed, after specific deprotection, by the conjugation to 6-aminopenicillanic acid giving the oxacillin derivative. In this way, conjugates retaining immunological properties of nandrolone and high inhibiting power of beta-lactamases should be obtained.