1600 Chem. Commun., 2013, 49, 1600--1602 This journal is c The Royal Society of Chemistry 2013 Cite this: Chem. Commun., 2013, 49, 1600 Very bright europium complexes that stain cellular mitochondria† James W. Walton, a Adrien Bourdolle, b Stephen J. Butler, a Marine Soulie, c Martina Delbianco, a Brian K. McMahon, a Robert Pal, a Horst Puschmann, a Jurriaan M. Zwier, c Laurent Lamarque, c Olivier Maury,* b Chantal Andraud b and David Parker* a The synthesis, structure and photophysical properties of a series of highly emissive europium complexes is reported. Certain complexes enter mammalian cells by macropinocytosis and stain the mitochondria selectively, allowing observation of the Eu emission in cellulo by time-gated spectral imaging. Emissive lanthanide complexes for use as tags in bioassays or as optical probes require both a high emission quantum yield and large molar absorptivity at an excitation wavelength in the range 337 to 405 nm to give high brightness, B, where B = ef. 1 Using sensitised emission, the incorporation of multiple chromo- phores into a polydentate ligand has been studied, allowing efficient energy transfer to a bound Eu(III) ion that is efficiently shielded from vibrational deactivation by solvent. 2 In aqueous media, no 1:1 [EuÁL] systems have been reported with a brightness (l exc 4 337 nm) exceeding 3000 M À1 cm À1 . Here, we report systems in which the brightness is an order of magnitude larger. Moreover, certain complexes are taken into mammalian cells, allowing their use in microscopic imaging. In designing these systems, we have combined the very effective shielding of the Eu(III) ion using nonadentate ligands based on triazacyclononane 3,4 with strongly absorbing p-substituted aryl–alkynyl groups, [EuL 1–4 ]. 5 Both carboxylate and phosphinate substituted systems have been prepared, 6 and the synthetic pathway allows the preparation of derivatives that can be conjugated to a vector. In the phosphinate systems, 3 the phosphorus substituents adopt a common configuration in the complex, and more effectively shield the excited Ln ion from intermolecular quenching processes. The ligands and their Eu complexes were prepared using established methods. 6 In the case of [EuÁL 2c ], the third substituent, bearing a remote protected amine group, was introduced last, following stepwise alkylation and de-protection of mono-BOC-triazacyclononane. 6 Crystals of [EuÁL 2a ] grew from aqueous methanol and the structure of [EuÁL 2a ] revealed that the Eu ion is encapsulated by the ligand in a tricapped trigonal prismatic array (Fig. 1). The nearest waters are over 6 Å from the metal ion, and the complex is slightly distorted from C 3 -symmetry. This distortion may be related to the presence of several disordered solvent molecules in the lattice. This complex, in common with the other triphosphinate systems, exists as a 50 : 50 mixture of D-(SSS) and L-(RRR) isomers. In solution, one 31 P NMR resonance is observed; the paramagnetically shifted 1 H NMR spectra of each Eu complex are consistent with average C 3 -symmetry. Each chromophore absorbs around 310 to 340 nm, with an overall extinction coefficient of 55–60 000 M À1 cm À1 (Table 1). Emission spectra in aqueous or methanol solutions were very similar for each Eu complex (Fig. 2). The Eu complexes of L 1a , L 2a ,L 3 and L 4 dissolved readily in MeOH, but were not soluble in water. The water solubility was higher with the complexes bearing PEG substituents, but did not allow greater than 2 mM solutions to be made up; for the PMe phosphinate complex, [EuÁL 2b ], the limiting solubility was about 20 mM. A very intense set of DJ = 2 transitions was observed around 610–620 nm, and the spectral form was consistent with C 3 -symmetry. Overall emission quantum yields ranged from 15–54%, with the lower values for the p-CF 3 substituted complex, [EuÁL 4 ] presumably reflecting a less efficient intramolecular energy transfer step. Following incubation of [EuÁL 2b ] (10 mM, 30 min) in the growth medium of NIH-3T3 (mouse skin fibroblasts), CHO (Chinese hamster ovarian) or PC-3 (human prostate cancer) cells, staining of the cell could be observed by confocal fluores- cence microscopy. The cell images (Fig. 3) revealed selective staining of the mitochondria, confirmed by co-localisation a Department of Chemistry, Durham University, South Road, Durham, DH1 3LE, UK. E-mail: david.parker@dur.ac.uk; Fax: +44 (0)191 3844737; Tel: +44 (0)191 3342033 b University of Lyon 1, ENS Lyon, CNRS - UMR 5182, 46 Alle ´e d’Italie, 69364 Lyon, France. E-mail: olivier.maury@ens-lyon.fr c Cisbio Bioassays, Parc Marcel Boiteux, BP 84175, 30200 Codolet, France † Electronic supplementary information (ESI) available: Details of complex synthesis and characterization, microscopy/spectroscopy instrumentation and the structural analysis of [EuÁL 2a ] are available. CCDC 857545. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/ c2cc35247h Received 20th July 2012, Accepted 19th December 2012 DOI: 10.1039/c2cc35247h www.rsc.org/chemcomm ChemComm COMMUNICATION Downloaded by Ecole Normale Superieure de Lyon on 31 January 2013 Published on 21 January 2013 on http://pubs.rsc.org | doi:10.1039/C2CC35247H View Article Online View Journal | View Issue