Stable and highly fluorescent europium(III) chelates for time-resolved immunoassays.

Derivatives of 4-[2-(4-isothiocyanatophenyl)ethynyl]-2,6,-bis{[N,N-bis(carboxymethyl)-amino]methyl}pyridine europium(III) (1) bearing one (6) or two (7) additional iminodiacetate coordinating arms have been synthesized. 6 and 7 were significantly more stable than 1 as evidenced by competition experiments with ethylenediaminetetraacetic acid (EDTA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). While the luminescence quantum yield of 1 remained modest, the other two complexes displayed substantial luminescence efficiency. The introduction of a supplementary iminodiacetate arm in 6 brought important improvements to both the stability and the luminescence properties of the Eu complex. In contrast, although 7 is more luminescent than 1, the introduction of a second iminodiacetate coordinating arm brings no further benefit on the photophysical properties. The most promising results were obtained with the nine-dentate chelate 6 and its Eu complex, which was conjugated to biotin and applied within the frame of a bioaffinity immunoassay of human C-reactive protein.

Reactions of 4-[Bis(2-chloroethyl)amino]benzenebutanoic acid (chlorambucil) with DNA.

4-[Bis(2-chloroethyl)amino]benzenebutanoic acid (=chlorambucil, 1; 2.5 mM) was allowed to react with single- and double-stranded calf thymus DNA at physiological pH (cacodylic acid, 50% base) at 37 degrees . The DNA-chlorambucil adducts were identified by analyzing the DNA hydrolysates by NMR, UV, HPLC, LC/ESI-MS/MS techniques as well as by spiking with authentic materials. ssDNA was more reactive than dsDNA, and the order of reactivity in ssDNA was Ade-N1>Gua-N7>Cyt-N3>Ade-N3. The most reactive site in dsDNA was Ade-N3. The Gua-N7 and Ade-N3 adducts were hydrolytically labile. Ade-N7 adduct could not be identified in the hydrolysates of ssDNA or dsDNA. The adduct Gua-N7,N7, which consists of two units of Gua bound together with a unit derived from chlorambucil, is a cross-linking adduct, and it was detected in the hydrolysates of ssDNA and dsDNA. Also several other adducts were detected which could be characterized by spiking with previously isolated authentic adducts or tentatively by MS. The role of chlorambucil-DNA adducts on the cytotoxicity and mutagenity of 1 is also discussed.

Derivatization of 11-alpha-hydroxyprogesterone using phosphoramidite chemistry.

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.

Bioconjugation with stable luminescent lanthanide(III) chelates comprising pyridine subunits.

The use of long-lifetime emitting lanthanide(III) chelate labels or probes together with time-resolved fluorometry in detection provides a method to generate sensitive bioaffinity assays. However, the use of stable chelates demands very complicated optimization of the chelate structure. A great number of chelates have been synthesized, but usually, only the most prominent structures were then converted to corresponding biomolecule labeling reactants. This review covers the syntheses of luminescent lanthanide chelates comprising a pyridine subunit that allow solution and solid-phase bioconjugation.

Genotyping of celiac disease-related-risk haplotypes using a closed-tube polymerase chain reaction analysis of dried blood and saliva disk samples.

Expansion of molecular diagnostics more widely into clinical routines requires simplified methods allowing automation. We developed a homogeneous, multilabel polymerase chain reaction (PCR) method based on time-resolved fluorometry, and studied the use of dried disk samples in PCR. Celiac disease-related HLA-DQA1*05, HLA-DQB1*02, and HLA-DQB1*0302 genotyping was used to verify the method with blood and saliva samples dried on S&S 903 and IsoCode sample collection papers. Three sample preparation procedures, including manufacturer's manual elution, an automated elution, and direct use of disk samples, were compared using dried disk samples. The three procedures gave successful amplification and correct genotyping results. Owing to the simplicity of the direct use of disk samples in PCR, this method was chosen for the subsequent homogeneous analysis of blood (n=194) and saliva (n=30) disk samples on S&S 903 paper. The results revealed that, in addition to DNA samples (n=29), both blood and saliva disk samples were successfully amplified and genotyped using the homogeneous PCR assays for HLA-DQA1 and HLA-DQB1. The homogeneous PCR assays developed provide a useful tool to genotype celiac disease-related HLA-DQA1*05, HLA-DQB1*02, and HLA-DQB1*0302 alleles. Furthermore, the method provides a direct way to perform a closed-tube PCR analysis of dried blood and saliva disk samples enabling simple automation.

Minisequencing with acyclonucleoside triphosphates tethered to lanthanide(III) chelates.

Four acyclic nucleoside triphosphates (derivatives of cytosine, thymine, 7-deazaadenine, and 7-deazaguanine) labeled with nonluminescent europium, terbium, dysprosium, and samarium chelates of 2,2',2'',2'''-[[4-(4-isothiocyanatophenyl)ethyl]pyridine-2,6-diyl]bis(methylenenitrilo)]tetrakis(acetic acid) were applied to minisequencing using two mutations (Delta F 508 and 1717-1 G to A) of cystic fibrosis as a model system. When synthetic targets were used, all four alleles involved could be analyzed in a single reaction using four terminating substrates labeled with four different lanthanide(III) chelates and DELFIA technology for detection. Blood spot samples without DNA isolations were used for PCR amplification and genotyping the target mutations by minisequencing. The single- and dual-labeled minisequencing assays were robust, while the four-label assay still requires further optimization of the multiplexed PCR amplification.

Labeling of oligonucleotides with DTPA and DOTA on solid phase.

Oligonucleotide conjugates labeled with metal chelates of diethylenetriaminepentaacetic acid (DTPA) and tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were synthesized on solid phase using appropriate nucleosidic phosphoramidite building blocks (3, 4) and a modified deprotection-metal chelation protocol. The major differences on the properties of the oligonucleotide conjugates also are discussed.

Convenient synthesis of maleimido-derivatized lanthanide(III) chelates and their use in mercapto group conjugation.

Simple synthesis of luminescent europium(III) and terbium(III) chelates tethered to a maleimido function (7, 8) is described. The method is based on the following: (i) synthesis of protected ligands tethered to a maleimido function and their purification on silica gel; (ii) deprotection by acidolysis; (iii) conversion of the deprotected ligands to the corresponding lanthanide(III) chelates by passing them through a column of strong cation exchange resin loaded with the appropriate lanthanide(III) ions. According to this procedure, large quantities of mercapto-selective biomolecule-labeling reactants of high purity can be prepared.

Reactions of {4-[bis(2-chloroethyl)amino]phenyl}acetic acid (phenylacetic acid mustard) with 2'-deoxyribonucleosides.

Phenylacetic acid mustard (PAM; 2), a major metabolite of the anticancer agent chlorambucil (CLB; 1), was allowed to react with 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG), 2'-deoxycytidine (dC), 2'-deoxy-5-methylcytidine (dMeC), and thymidine (T) at physiological pH (cacodylic acid, 50% base). The reactions were followed by HPLC and analyzed by HPLC/MS and/or (1)H-NMR techniques. Although the predominant reaction observed was hydrolysis of PAM, 2 also reacted with various heteroatoms of the nucleosides to give a series of products: compounds 5-31. PAM (2) was found to be hydrolytically slightly more stable than CLB (1). The principal reaction sites of 2 with dA, dG, and with all pyrimidine nucleosides were N(1), N(7), and N(3), resp. Also, several other adducts were detected and characterized. There was no significant difference in the reactivity of 1 and 2 with dG, dA or T, but the N(3) dC-PAM adduct was deaminated easier than the corresponding CLB derivative. The role of PAM-2'-deoxyribonucleoside adducts on the cytotoxic and mutagenic properties of CLB (1) is discussed.

Solid-phase oligonucleotide labeling with DOTA.

This unit describes a method to construct oligonucleotide conjugates labeled with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) on a solid support. A nucleosidic phosphoramidite that contains a protected DOTA ligand compatible with normal chain assembly is prepared first. As the chain assembly is completed, the oligonucleotide is deprotected and converted to the corresponding gadolinium(III) chelate, resulting in an oligonucleotide conjugate containing the chelate at the 5'-terminus.

Synthesis of aminooxy-functionalized lanthanide(III) chelates for carbonyl-group conjugation.

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).

Simple synthesis of a building block for solid-phase labeling of oligonucleotides with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

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.

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.

Reactions of N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, and thymidine.

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.