Study on luminescent ternary EuEDTA complexes with a set of substituted 4-phenylethynyl and 4-aryl pyridine-2,6-dicarboxylic acids Markus Räsänen a , Jaana Rosenberg b , Jukka Lukkari a , Keijo Haapakka a , Jouko Kankare a,n , Harri Takalo c,n a Department of Chemistry, University of Turku, FIN-20014 Turku, Finland b Department of Biochemistry/Biotechnology, University of Turku, FIN-20520 Turku, Finland c Radiometer Turku, Biolinja 12, FIN-20750 Turku, Finland article info Article history: Received 18 November 2016 Received in revised form 24 March 2017 Accepted 26 March 2017 Available online 28 March 2017 Keywords: Europium Dipicolinic acid EDTA Luminescence Lifetimes abstract A set of pyridine-2,6-dicarboxylic acids (dipicolinic acids, DPAD, Lx) were prepared to study the photo- physical properties of their ternary complexes with Eu(III) and ethylenediaminetetraacetic acid (EDTA or Y). The stability constant of the formation of EuY-L1 (L1 ¼ dipicolinic acid, measured log K ter ¼5.38) was determined and found to be comparable to the formation constant of Eu(L1) 3 (logK 3 ¼5.51). All the studied ternary complexes exhibit two predominant luminescence decay times. The origin of the shorter lifetime was shown to be due to the dissociation of carboxylate group(s) and their replacement by water molecules. The presence of the short-lived component decreases the quantum yields compared those reported for complexes of Eu(Lx) 3 . However, the use of ternary complexes of EuY-Lx offers a simple comparison method between different dipicolinic acids (Lx). Eight of the studied ligands Lx form brighter ternary complexes than the currently used ligand in the duplex PCR assay, with quantum yields over 20%, and the most luminescent one has three times higher signal level compared to the currently used ligand. & 2017 Elsevier B.V. All rights reserved. 1. Introduction Lanthanide chelates, especially the chelates of europium and terbium, are widely used as labels in various bioanalytical assays due to the long decay times of their luminescence. The unique properties of lanthanide ions place special requirements on the chelating compounds. The low absorption cross-section or ab- sorptivity of lanthanide ions means that the ligand should have both a high absorptivity and efficient energy transfer from the excited state of the “antenna” ligand to the proper energy level of the lanthanide ion. The lanthanide coordination occurs pre- dominantly via electrostatic interactions, meaning that the co- ordination has little or no directionality and the steric constraints have a strong influence on the stability of the complexes. Most trivalent lanthanide ions form octa- or nonacoordination com- plexes and at least six electronegative donors in the chelating li- gand are needed to guarantee the stability of the complex in aqueous solutions. The remaining free coordination sites of the lanthanide cation are filled with water molecules unless molecules of higher affinity for lanthanide ions are present. In this case a ternary complex or mixed-ligand complex is formed. Tendency of lanthanide cations to form ternary complexes has been utilized in various analytical techniques. For instance, a suitable complexing agent may be added just for displacing water from the inner coordination sphere of the lanthanide cation. Water is very effective in quenching luminescence by multiphonon deactivation and hence considerable enhancement of intensity is obtained. Other applications, for example, are molecular recognition and chirality sensing using luminescence and NMR techniques [1]. It is obvious that there are two essentially different ways to apply the ternary complexation. Either the lanthanide cation forms a relatively stable complex, (a) with an antenna-type ligand and the second molecule binds to the remaining free coordination sites, or (b) with a non- absorbing ligand and an antenna-type ligand binds to the remaining coordination sites. In the first case (a) the chelate is already lumi- nescent without the addition of the second ligand. The second ligand may change the luminescent properties and becomes characterized or recognized on this basis. In the case (b) the chelate becomes lu- minescent only after coordination of the second ligand. It is this case which is of our interest in this work. A ternary complex of terbium cation has been applied already about 25 years ago in a DNA-hybridization assay [2]. In this assay a specific nucleic acid sequence is identified by using two oligonu- cleotide sequences, one labelled by a Tb(III) complex of Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/jlumin Journal of Luminescence http://dx.doi.org/10.1016/j.jlumin.2017.03.061 0022-2313/& 2017 Elsevier B.V. All rights reserved. n Corresponding authors. E-mail addresses: kankare@utu.fi (J. Kankare), harri.takalo@radiometer.fi (H. Takalo). Journal of Luminescence 187 (2017) 471–478