Biomedical subjects
Publications and source records attributed to Jari Hovinen.
A neutral bifunctional derivative of diethylenetriaminepentaacetic acid europium(III) (11) was synthesized and its suitability to dissociation-enhanced lanthanide fluorescence immunoassay was investigated.
The syntheses of three new aminooxy-tethered lanthanide(III) chelates, compounds 1-3, incorporating DOTA (= 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DTPA (= diethylenetriaminepentaacetic acid), or a substituted terpyridine (2,2',2'',2'''-[2,2': 6',2''-terpyridine-6,6''-diylbis(methylenenitrilo)]tetraacetic acid), respectively, are described. Reagents 1-3 can be used for carbonyl 'labeling', as shown by the formation of the corresponding oxime-ether bioconjugates of naltrexone (16) and 2-deoxy-beta-D-glucose (17) (Scheme 4).
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.
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.
N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil, 1; 2.5 mM) was allowed to react with 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, and thymidine (16.1 mM) at physiological pH (cacodylic acid, 50% base), and the reactions were followed by HPLC and HPLC-MS technique. Although the predominant reaction observed was chlorambucil hydrolysis, 1 reacted with various heteroatoms of the nucleosides. The principal site of alkylation with all pyrimidine nucleosides was N3, as judged by 1H NMR and HPLC-MS analyses. Also, several other adducts were detected, which could be tentatively characterized by means of HPLC-MS and MS/MS. As expected, thymidine was the least reactive pyrimidine nucleoside studied, and in addition of the N3 derivative, it reacted only at the carbohydrate moiety. Overall reactivity of cytosine nucleosides with 1 was considerably higher. The N3 adducts of dCyd and 5-Me-dCyd partially deaminated under the reaction conditions employed, but the reaction was not catalyzed by the participation of the omega-hydroxy function of the alkyl substituent but presumably by the nitrogen atom of the chlorambucil moiety. In the case of cytosine nucleosides, the O2 derivatives were the second most abundant species. 5-Me-dCyd reacted more readily at O2 than dCyd. These O2 adducts were labile under acidic, neutral, and basic conditions. No N4 derivatives or cross-links were detected, but dCyd reacted also at C5, although the yield of this derivative was very low. The role of chlorambucil-pyrimidine 2'-deoxyribonucleoside adducts on the cytotoxicity and mutagenity of 1 is also discussed.
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N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil, 1; 0.6 mM) was allowed to react with 2'-deoxyadenosine (16.1 mM) at physiological pH (cacodylic acid, 50% base), and the reactions were followed by HPLC-MS and HPLC-MS/MS techniques. Although the predominant reaction observed was chlorambucil hydrolysis, ca. 7% of 1 reacted with various heteroatoms of the nucleoside. The principal site of alkylation was N1. Several other adducts were also detected. The N1, N6, N3, and N7 derivatives were characterized by means of MS/MS, UV, and (1)H NMR. The N6 adduct is derived directly from alkylation of N6 of 2'-dAdo. Dimroth rearrangement of the N1 adduct to the N6 adduct was very slow under the reaction conditions employed. Minor adducts such as a carbohydrate derivative were tentatively characterized by MS/MS. No cross-links were detected. The role of chlorambucil-2'-deoxyadenosine adducts in the cytotoxicity and mutagenicity of 1 is also discussed.
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).
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.
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.
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.
Synthesis of a nucleosidic phosphoramidite building block which allows solid-phase introduction of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to synthetic oligonucleotides using a standard oligonucleotide synthesizer is described.
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