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The well-known phosphoramidite chemistry routinely used in solid-phase oligonucleotide synthesis is exploited here in the preparation of steroid conjugates in solution. The applicability of the method is tested by conjugating one nucleosidic and one non-nucleosidic phosphoramidite building block to 11-alpha-hydroxyprogesterone. The suitability of one of the conjugates synthesized (5) for a competitive binding assay is also demonstrated.

We have developed assay technologies to measure hydrolyzing enzymes based on homogeneous time-resolved fluorescence quenching (TruPoint). High sensitivity was obtained using fluorescent europium chelates as labels, internally quenched by suitable quenchers and released upon enzymatic reaction. This approach allows robust and sensitive monitoring of low enzyme activities. The assay technology and the choice of donor-acceptor pairs were evaluated in three different enzymatic assays, a protease related to apoptosis, helicase involved in DNA unwinding, and phosphatase having an important role in cellular signaling cascades. All the assays produced an increasing signal, were sensitive, and had a good dynamic range. There were significant differences in optimized quenchers for each of the assays depending on the size, flexibility, and rigidity of the substrates. Also, clear differences in the energy-transfer reactions, their requirements for spectral overlapping, ionic interactions, and energy-transfer distances were found. Each of the enzymatic assays was briefly tested in a high-throughput screening environment by analyzing signal dynamics and statistical relevance as Z' factors.

Synthesis of nonluminescent lanthanide(III) chelates tethered to an aminooxy group (i.e., 1-[4-(6-aminooxyhexamido)benzyl]diethylenetriaminetetraacetic acid lanthanides(III), 6a-d, where Ln(3+) is Eu, Dy, Sm, and Tb) is described. Their applicability to biomolecule derivatization is demonstrated by allowing them to react with a synthetic oligopeptide, a protein, two synthetic drugs, and a steroid. The oligopeptide and protein were linked to 6 after preoxidation of their N-terminal serine residues, while the drugs and the steroid reacted via their ketone functionality. Also some application data is included.

The synthesis of oligopeptide building blocks for the introduction of nonluminescent and luminescent lanthanide(III) chelates to the oligopeptide structure on the solid phase is described. The oligopeptide conjugates synthesized were used in DELFIA-based receptor binding assay (motilin) as well as in LANCE time-resolved fluorescence quenching assay (caspase-3).

A chemical method for labeling of oligonucleotide probes with europium chelates is presented. A modified deoxycytidine phosphoramidite is used to introduce multiple reactive amino groups to the oligonucleotide during the synthesis phase. Upon deprotection and purification of the modified oligonucleotide, an isothiocyanate derivative of a stable Eu chelate is reacted with the primary amino groups. The labeling technology enables the coupling of a high number of Eu chelates to a single probe. The melting temperatures and hybridization efficiencies of the oligonucleotides are not significantly altered by the labeling process. However, hybridization kinetics of the oligonucleotides are affected by the introduction of multiple modified deoxycytidine residues. In a solid-phase hybridization assay up to 10(7) target molecules can be detected.

Pseudo-first-order rate constants for the bisulfite ion-catalyzed transamination of cytidine with 1,2-ethanediamine, 1,3-propanediamine, 1,4-butanediamine, and 1,6-hexanediamine have been determined. Hydrolytic deamination has been shown to compete with transamination under acidic conditions, but is of minor importance at pH > 5.3 when the total concentration of diamine is greater than 0.2 mol dm-3. The dependence of the transamination rate on pH and the concentration of diamine and bisulfite ion indicates that the major reaction involves nucleophilic attack of the diamine monocation on the N3 protonated bisulfite adduct of cytidine. The effect of the chain length of the diamine on the rate of transamination is discussed, and the results are compared with those obtained by reacting single-stranded DNA with the same diamines and labeling the transaminated product with a europium chelate.