Synthesis and properties of a neutral derivative of diethylenetriaminepentaacetic acid (DTPA).

A neutral bifunctional derivative of diethylenetriaminepentaacetic acid europium(III) (11) was synthesized and its suitability to dissociation-enhanced lanthanide fluorescence immunoassay was investigated.

Synthesis of building blocks for solid-phase introduction of diethylenetriaminepentaacetic acid (DTPA) to oligonucleotides and oligopeptides.

Synthesis of building blocks that allow site-specific incorporation of diethylenetriaminepentaacetic acid (DTPA) to oligonucleotides and oligopeptides using phosphoramidite and Fmoc chemistries, respectively, is described.

Labeling of proteins and oligopeptides with luminescent lanthanide(III) chelates.

Synthesis of a building block that allows introduction of photoluminescent europium(III) and samarium(III) chelates to synthetic oligopeptides on solid phase using standard Fmoc chemistry is described. Upon completion of the oligopeptide synthesis, these conjugates were converted to the corresponding lanthanide(III) chelates by treatment with appropriate lanthanide(III) salt. Also synthesis of a new terpyridine-based europium(III) chelate designed for solution phase protein labeling is demonstrated.

Solid-phase synthesis of oligonucleotides labeled with luminescent lanthanide(III) chelates.

The synthesis of phosphoramidite building blocks that allow introduction of luminescent europium(III), terbium(III), dysprosium(III), and samarium(III) chelates to oligonucleotides on the solid phase is described. Several labeled oligonucleotides using these building blocks were prepared, and the photophysical properties of these bioconjugates were investigated.

Labeling of steroids on solid phase.

Up to four tetra-tert-butyl-1-[4-aminoacetamido)benzyl]diethylenetriaminetetrakis(acetato) derivatives of Fmoc glutamic acid (1) were attached to two steroids (17alpha-hydroxyprogesterone-3-O-carboxymethyloxime 2 and 1,3,5(10)-estratriene-3,16alpha,17beta-triol-6-one-6-O-carboxymethyloxime, 3)) on solid phase using an oligopeptide synthesizer. Upon deprotection and conversion to the corresponding europium(III) chelates, these steroid conjugates were used in DELFIA-based competitive fluoroimmunoassays. The more chelates conjugated to 17-alpha-hydroxyprogesterone, the more diluted antiserum could be used in an immunoassay for 17-alpha-hydroxyprogesterone, without any alteration of the measurement range. Hence, 17-alpha-hydroxyprogesterone tracers with several chelates are useful when a high serum dilution factor is desired i.e., when only a limited quantity of antiserum is available. The result demonstrates the suitability and usefulness of lanthanide(III) chelates as multilabels in bioaffinity assays.

Caspase multiplexing: simultaneous homogeneous time-resolved quenching assay (TruPoint) for caspases 1, 3, and 6.

Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparametric homogeneous assay capable of measuring activity of three different caspases in a single well of a microtiter plate is described. Different fluorescent europium, samarium, terbium, and dysprosium chelates were coupled to a caspase substrate peptide, their luminescence properties, were analyzed, and their function in a time-resolved fluorescence quenching-based caspase 3 assay was studied. Substrates for caspases 1, 2, 3, 6, and 8 and granzyme B were also synthesized and their specificities for different caspases were determined. By selecting suitable lanthanide chelates and substrates we developed a multiparametric homogeneous time-resolved fluorescence quenching-based assay for caspases 1, 3, and 6. The assay was capable of measuring the activity of both single caspases and a mixture of three caspases mixed in the same well.

Synthesis of nonluminescent lanthanide(III) chelates tethered to an aminooxy group and their applicability to biomolecule derivatization.

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.

Introduction of lanthanide(III) chelates to oligopeptides on solid phase.

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).