Complexes luminescents de lanthanides avec des dérivés du cyclène comme briques pour l'ingénierie de sondes analytiques biomédicales

This work deals with the engineering of a new luminescent complex of lanthanide, able to be specifically grafted onto biological materials and to emit a characteristic light useful in quantifying the concentration of the target molecules. We have based our molecular design on TbL1, wich possesses a significant quantum yield in water pointing to an efficient intramolecular energy transfer. We have elaborated the synthesis of a ligand derived from L1 and named L3, by functionalizing one of the chromophoric groups into a moiety able to couple the luminescent sensor to biological materials. The synthesis of the complexes was then realized. The thermodynamic study of the ligands L1 and L3, and their terbium complexes in water was made to establish the distribution diagrams and to compare their behaviour, especially at neutral pH. In addition, EuL1 was also investigated and the complexation constants of TbL1, EuL1 and TbL3 in water showed that these molecules are thermodynamically stable in aqueous media. Characterization of EuL3 in the solid state by high resolution luminescence allowed us to determine the geometry of the coordination sphere of the Eu(III) ion, which possesses a distorded C4 symmetry. The detailed study of the Eu(III) ion transitions evidenced two different sites, possibly arising from an equilibrium induced by the competition between a water molecule and the carboxylate function. This interpretation is in line with the measured lifetimes of the Tb(III) and Eu(III) excited states and the calculation of the hydration number of the complexes in aqueous solution. The determination of the energy of the singlet state and triplet states of the ligand, as well as of the emitting levels of the Ln(III) ions in solution showed that their relative values are favorable for a good intramolecular energy transfer. The quantum yields of TbL3 and EuL3, which amount to 2.6 % and 1.4 %, respectively, are lower than those of the complexes with L1, denoting that the presence of the acid function increases losses in the energy transfer. However, these values remain sizeable enough for potential biomedical applications. The study of the intermolecular energy transfer from EuL1, EuL3 and TbL3 to the acceptor Cy5 has proved promising, and the resulting yields were 90 % for EuL1 and 80 % for the complexes with L3.

Non-Cytotoxic, Bifunctional EuIII and TbIII Luminescent Macrocyclic Complexes for Luminescence Resonant Energy- Transfer Experiments

Jean-Claude Bünzli, Anne-Sophie Chauvin, Caroline Vandevyver

A new macrocyclic ligand,

L^3

, has been synthesised, based on the cyclen framework grafted with three phenacyl light-harvesting groups and a C5-alkyl chain bearing a carboxylic acid function as a potential linker for biological material. Acidity constants are determined by spectrophotometric titrations, as well as conditional stability constants for the resulting 1:1 complexes with trivalent lanthanide ions. The complexes have stabilities comparable to 1,4,7,10-tetrakis(carbamoylmethyl)- 1,4,7,10- tetraazacyclododecane (dtma) complexes, with

pLn \approx 12-13

. Photophysical properties of the ligand and of the

EuL^3

and

TbL^3

complexes have been determined for both microcrystalline samples and solutions in water and acetonitrile. They point to the metal ion being present in an environment with axial symmetry derived from the

C_4

point group. The hydration number determined for

TbL^3

decreases with increasing pH value and becomes fractional at pH 7.5, which points to an equilibrium between two differently solvated species and probably to the participation of the deprotonated carboxylic acid chain in the complexation. The quantum yields in water (1.9% for

Eu^{III}

, 3.4% for

Tb^{III}

) are smaller than those for complexes with the symmetrically substituted parent macrocycle, but efficient luminescence resonant energy transfer (LRET) was observed when Cy5 dye was added to the solutions. Finally, the influence of the

TbL^3

complex on cell viability is tested on both malignant (5D10 mouse hybridoma, Jurkat human T leukaemia, MCF-7 human breast carcinoma) and non-malignant (Hacat human keratinocyte) cell lines. Cell viability after 24 h incubation at

37^ {\circ}C

with

500\mu m

TbL^3

was >90% for all cell lines, except Jurkat (>70%). All of these properties make

LnL^3

complexes interesting potential probes for bioanalyses.

2007

Luminescent lanthanides bioprobes emitting in the visible and/or near-infrared ranges

Steve Comby

This Ph.D. work deals with the development of lanthanide-based bioprobes for biomedical analyses and imaging. For this purpose, two different approaches have been followed, one focusing on visible-emitting complexes, while the second addresses the sensitization of NIR-emitting LnIII ions. The homoditopic ligands H2LC2 and H2LC3 have been tailored to self-assemble with lanthanide ions (LnIII) into neutral triple-stranded bimetallic helicates of overall composition [Ln2(L)3] and also to provide a versatile platform for further derivatization. The grafting of polyoxyethylene groups onto the pyridine (H2LC2) or the benzimidazole (H2LC3) units ensures water solubility, while maintaining large thermodynamic stability and adequate antenna effect for the excitation of LnIII ions. The conditional stability constants are large at physiological pH, with logβ23 reaching values in the range 22-25 for helicates with H2LC2 and H2LC3. The ligands triplet states feature adequate energy to efficiently sensitize the EuIII luminescence (QEu L = 21 and 11 % for L = H2LC2 and H2LC3, respectively) in aerated water at pH 7.4. Conversely, energy back transfer occurring in [Tb2(LC3)3] precludes a good sensitization of the TbIII luminescence, and only the TbIII helicate with H2LC2 displays sizeable quantum yield (QTbL = 11 %). Furthermore, it has been demonstrated that ligand H2LC2 sensitizes the emission of other visible- and NIR-emitting LnIII ions, such as SmIII or YbIII, for which in cellulo luminescence is evidenced, a rare observation. The EuIII emission spectra of both helicates arise from a main species with pseudo-D3 symmetry and without coordinated water. Cytotoxicity experiments reveal no significant effect of the helicates on the viability of several cancerous cell lines and cell imaging properties of the EuIII helicates are demonstrated for the HeLa cell line by luminescence microscopy. Bright EuIII emission is seen after relatively short time (15-30 min) and low helicate incubation concentrations (10 µM). Similar images were obtained for [Sm2(LC2)3] and [Tb2(LC2)3] using slightly higher loading concentration. The helicates stain the cytoplasm and the permeation mechanism is likely to be endocytosis. The described luminescent helical stains are robust chemical species which remain mostly undissociated in the cell medium and which resist to the presence of other complexing agents, as demonstrated in the case of H2LC2. All these properties make these helicates potential bioprobes for interacting with biological material and for cell imaging applications. As near-infrared (NIR) light, in addition to be less energetic than visible light, displays better penetrability into the human body, the development of bioprobes emitting in the near-infrared range is of prime importance for deeper tissue imaging, especially for early diagnosis of tumors. We therefore investigate the ability of new polydentate podands to sensitize the NIR luminescence of NdIII and YbIII ions. These ligands have been designed in a way to take advantage of the chelating effect of bidentate (Tsox, TsoxMe) or tridentate 8-hydroxyquinolinate (8-HQ) subunits (T2soxMe). The interaction between the ligands and LnIII ions in aqueous solution leads to the formation of stable 1:1 chelates (pLn in the range of 12-16), all the chromophoric 8-HQ units being coordinated to the metal center, exploiting the entropic effect generated by the anchor. At physiological pH, one major species is present for all the ligands regardless of the LnIII ion used. The low energy triplet states of the ligands are particularly well-suited for an efficient sensitization of the NdIII- or YbIII-centered luminescence (QNdL = 0.02-0.04 % and QYbL = 0.13-0.37 %) in buffered aqueous solution. ErIII NIR luminescence is also detected in water for the chelates with Tsox and TsoxMe. Lifetime determinations point in all cases to the absence of coordinated water molecules in the inner coordination sphere of the LnIII ion. It has also been demonstrated that methylation of the amide functions of Tsox removes the quenching mechanism induced by the proximate N-H groups and increases both the lifetimes and quantum yields of the TsoxMe chelates, with the YbIII complex being one of the most NIR luminescent lanthanide-based edifices described so far. Ligand TsoxMe thus appears to be the best candidate of the series regarding to the development of NIR probes for bioanalyses. Furthermore, other applications, for instance in telecommunications, are still conceivable since the incorporation of the Yb-TsoxMe chelate into sol-gel silica films has proven to be successful, the chelate withstanding the sol-gel process, while providing a slight enhancement of photophysical properties compared to the same chelate in aqueous solution.

EPFL2008

Luminescent Lanthanide Helicates Self-Assembled from Ditopic Ligands Bearing Phosphonic Acid or Phosphoester Units

Matthias Baud, Jean-Claude Bünzli, Anne-Sophie Chauvin, Steve Comby, Cyntia De Piano, Céline Duhot

A series of hexadentate ditopic receptors incorporating benzimidazole moieties have been designed, which are fitted with phosphonic acid or phosphoethylester coordinating units. In addition, poly(oxyethylene) pendants have been introduced on the benzimidazole backbone of two ligands to increase water solubility. The ligands self-assemble with lanthanide ions under stoichiometric conditions, yielding triple-stranded homobimetallic helicates, as ascertained by mass spectrometry and UV-visible titrations. The helicates display large thermodynamic stability, for example, log β23 ≈ 21-24 for all the EuIII complexes. Photophysical measurements reveal sensitization of the metal-centered luminescence in the europium and terbium complexes, which is modulated by the nature of the ligand. Hydration numbers determined by the lifetime method are essentially zero. The Eu(5D0) lifetimes are long and reach values up to 3.2 ms, while quantum yields as high as 25% are obtained in water at pH 7.4. Back transfer limits the sensitization efficiency for TbIII luminescence, and both lifetimes and quantum yields are much smaller. The properties of the helicates are discussed with respect to those self-assembled from ligands bearing carboxylate coordinating units.

2009