Jari Hovinen

Novel luminescent samarium(III) chelates

Abstract A series of new stable, luminescent samarium(III) chelates were synthesized and their photophysical properties were measured. The iodoacetamido activated chelate coupled to a peptide was used in a caspase-3 assay.

Synthesis and properties of nanospheres copolymerised with luminescent europium(III) chelates

Europium(III) chelates based on β-diketone and thienylpyridine subunits tethered to an acrylamide function were synthesized. They were polymerised with styrene and acrylic acid in the presence or absence of trioctylphosphine. The photophysical properties of the nanobeads obtained are also discussed.

Oligonucleotide conjugates based on acyclonucleosides and their use in DNA hybridization assays

Synthesis of two oligonucleotide building blocks based on acyclonucleosides (10, 11) which enable the introduction of several nonluminescent and luminescent lanthanide(III) chelates to the oligonucleotide structure is described. They were used in an instrument-assisted DNA synthesis in a standard manner. A modified deprotection procedure was used to ensure metal complexation. Also the applicability of these oligonucleotide conjugates to DNA hybridization assays is demonstrated.

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°. 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.

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.

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 manufacturers 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.

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.