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Luminescent Lanthanide Agents
Introduction

The unusual electronic properties of the trivalent lanthanide ions (Figure 1) make them well suited as luminescent reporter groups, with many currently developing applications in biotechnology. The visible emitting lanthanides (Sm, Eu, Tb and Dy) each have a characteristic luminescence color (Figure 2) allowing for easy multiplexing, for example in DNA hybridization assays.

Figure 1. Lanthanum and other Lanthanide elements (Z = 57-71) which correspond to successive filling of the 4f shell.
Figure 2. Visible emission (from left to right) of a Raymond Group 'IAM' type ligand, and the corresponding complexes with Tb(III), Eu(III), Dy(III) and Sm(III).

The unusual spectroscopic properties of the Ln(III) cations results from shielding of the 4f orbitals by the filled 5s2 and 5p6 sub-shells. For example, each of the elements have very characteristic and very narrow 'line-like' emission bands, mostly in the visible and near infrared range. As an example, the typical 'green' emission of Tb(III) is compared with fluorescein, a typical organic chromophore in Figure 3 below. Additionally, these f-f transitions are parity (and sometimes also spin) forbidden, resulting in very long lived excited states, with typical luminescence lifetimes on the micro- to millisecond timescale. These long lifetimes facilitate 'time-gated' emission experiments which result in drastic improvement in signal to noise ratios compared with more traditional steady-state measurements by removing short lived (eg. protein) emission and scattered excitation.

Figure 3. A comparison of characteristic Tb(III) emission compared to fluorescein, a typical organic emitter.

Unfortunately, as another consequence of the parity (Laporte) forbidden nature of the 4f transitions, the direct absorption of Ln(III) cations is only very weak, and they hence have very low molar absorption coefficients (typically less than 10 M-1cm-1) which limits their practical usage. In order to circumvent these low extinction coefficients, the luminescent metal ion can be chelated to a chromophore-containing group which functions as an 'antenna,' absorbing incident light then transferring this excitation to the metal ion, which can then deactivate by undergoing its typical luminescent emission (Figure 4).

Figure 4. An illustration of the 'antenna' effect, wherein incident excitation is first absorbed by a chelating organic chromophore and then transferred to the metal.

In addition to directing energy to the metal, chelation also serves to exclude solvent molecules from the first coordination sphere, which is essential to avoid quenching of the lanthanide luminescence through non-radiative decay via vibronic coupling to vibrational states of O-H and N-H bonds, and also to provide stable metal complexes.


Raymond Group Chromophores

In the Raymond group, one current interest is the efficient sensitization of the strongly luminescent visible emission from Tb(III) and Eu(III). To achieve a high overall quantum yield (Φ) of luminescence, we use either the 2-hydroxyisophthalamide 'IAM' or 1-hydroxypyridin-2-one '1,2-HOPO' chelate groups respectively (Figure 5).

Figure 5. Chemical structures of the 1-hydroxypyridin-2-one '1,2-HOPO' (left) and 2-hydroxisophthalamide 'IAM' (right) chromophore-containing organic chelates.

These two organic chelates are both anionic and rich in oxygen donors to satisfy the oxyphilic Ln(III) cations, and serve a dual purpose by strongly chelating the metal and also by acting as the antennae, to absorb incident light. The generic 'R' group can be a variety of differing ligand backbones such as alkyl or aryl amines chosen to control both the denticity of the ligand and also the geometry at the metal center for the ensuing Ln(III) complex. For example, both tetradentate and octadentate ligand topologies have been explored using both chromophores (eg. see Figure 6). The 2-hydroxyisophthalamide 'IAM' based ligands efficiently sensitize Tb(III) (Φ = 61%), Eu(III) (6%), Dy(III) (3%), and Sm(III) (1%) while 1-hydroxypyridin-2-one '1,2-HOPO' based ligands are more efficient for Eu(III) (Φ = 21.5%).

Figure 6. Chemical structures of octadentate ligands using 1,2-HOPO (left) and IAM (right) chromophores.
Figure 7. X-ray structures of two Eu(III) ML2 complexes with IAM (left) and 1,2-HOPO (right) based ligands.

We are also investigating the use of chiral, enantiopure ligands of IAM and 1,2-HOPO in order to be able to obtain "chiral" light emission (Figure 8). This phenomenon, called 'circularly polarized luminescence (CPL)', is the emission analogue of circular dichroism (CD) but probes the chirality of the excited state instead of the ground state. This cutting-edge technique is very promising for bioanalytical applications where highly specific reporters for conformational changes are needed (eg. for protein-protein interactions).

Figure 8. An enantiopure Eu(III) complex for CPL applications.

Recently, we have obtained the first actinide CPL spectra with Cm (III) L1a and L1b complexes (S-) and (R+) (see below).  These spectra are mirror images of one another with opposite dissymmetry ratios (glum of + 0.0038 and -0.0040, respectively).

                                       cpl                        l1a
                                                                                                                                                                                                    L1a/b (a = S- / b = R+)
                                                          

Another interest is tuning the electronic structure of the chromophore via synthetic modification. The IAM chromophore has proven to be easily modifiable, allowing for the synthesis of a wide array of derivatives. Substituting the ring at the 5-position (Figure 9) has dramatic effects of the ligand triplet energies, and in turn, the efficiency of Tb(III) emission. By investigating a series of substituents (X = H, F, Cl, Br, Me, OMe, NO2, (C=O)NHMe, SO3-) we have been able to quantify the effect of the substituent on the ligand triplet level and Tb(III) quantum yield using the resonance Hammett parameter of the substituent. Currently we are exploring new substituents to further improve the quantum yield. We are also investigating substituents that can interact with species such as metal ions in order to transform our Ln(III) complexes into sensors.

Figure 9. A para-substitued IAM series has been prepared where X = H, F, Cl, Br, Me, OMe, NO2, (C=O)NHMe, SO3-.

Theoretical calculations are also being done to gain further insight into the electronic structure of the antenna ligands. TD-DFT calculations on the Na+ analogs of the Ln(III) complexes are consistent with the experimental results and in most cases calculate triplet energies within ~5% of experimental values (Figure 10). These calculations can be used to predict the energy levels of new chromophores and their derivatives.

Figure 10. TD-DFT output for Na+-IAM showing the calculated triplet energy (top) and the orbitals involved in the S0-T0 transitions (bottom).

Seitz, M.; Moore, E. G.; Ingram, A. J..; Muller, G.; Raymond, K. N. "Enantiopure Octadentate Ligands as Sensitizers for Europium and Terbium Circularly Polarized Luminescence in Aqueous Solution." J. Am. Chem. Soc. 2007, submitted.

Moore, E. G.; Jocher, C. J.; Xu, J.; Werner, E. J.; Raymond, K. N. "An Octadentate Luminescent Eu(III) 1,2-HOPO Chelate with Potent Aqueous Stability." Inorg. Chem. 2007, 46, 5468-5470.

Seitz, M.; Pluth, M. D.; Raymond, K. N., "1,2-HOIQO - A Highly Versatile 1,2-HOPO Analogue." Inorg. Chem. 2007, 46, 351-353.

Petoud, S.; Muller, G.; Moore, E. G.; Xu, J.; Sokolnicki, J.; Riehl, J. P.; Le, U.; Cohen, S. M.; Raymond, K. N. "Brilliant Sm, Eu, Tb and Dy Chiral Lanthanide Complexes with Strong Circularly Polarized Luminescence." J. Am. Chem. Soc. 2007, 129, 77-83.

Moore, E. G.; Xu, J.; Jocher, C. J.; Werner, E. J.; Raymond, K. N. "Cymothoe Sangaris: An Extremely Stable and Highly Luminescent 1,2 Hydroxy-pyridinonate Chelate of Eu(III)." J. Am. Chem. Soc. 2006, 128, 10648-10649.

Samuel, A. P. S.; Xu, J.; Raymond, K. N. "Adjusting 2-Hydroxyisophthalamide Energy Levels to Maximize Ln(III) Emission." 232nd ACS National Meeting, San Francisco, CA, USA, Sept. 10-14, 2006.

Johansson, M. K.; Cook, R. M.; Xu, J.; Raymond, K. N. "Time Gating Improves Sensitivity in Energy Transfer Assays with Terbium Chelate/Dark Quencher Oligonucleotide Probes." J. Am. Chem. Soc. 2004, 126, 16451-16455.

Petoud, S.; Cohen, S. M.; Bünzli, J.-C. G.; Raymond, K. N. "Stable Lanthanide Luminescence Agents Highly Emissive in Aqueous Solution: Multidentate 2-Hydroxyisophthalamide Complexes of Sm3+, Eu3+, Tb3+, Dy3+." J. Am. Chem. Soc. 2003, 125, 13324-13325.



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