From antenna to assay: lessons learned in lanthanide luminescence.
ABSTRACT: Ligand-sensitized, luminescent lanthanide(III) complexes are of considerable importance because their unique photophysical properties (microsecond to millisecond lifetimes, characteristic and narrow emission bands, and large Stokes shifts) make them well suited as labels in fluorescence-based bioassays. The long-lived emission of lanthanide(III) cations can be temporally resolved from scattered light and background fluorescence to vastly enhance measurement sensitivity. One challenge in this field is the design of sensitizing ligands that provide highly emissive complexes with sufficient stability and aqueous solubility for practical applications. In this Account, we give an overview of some of the general properties of the trivalent lanthanides and follow with a summary of advances made in our laboratory in the development of highly luminescent Tb(III) and Eu(III) complexes for applications in biotechnology. A focus of our research has been the optimization of these compounds as potential commercial agents for use in homogeneous time-resolved fluorescence (HTRF) technology. Our approach involves developing high-stability octadentate Tb(III) and Eu(III) complexes that rely on all-oxygen donor atoms and using multichromophore chelates to increase molar absorptivity; earlier examples utilized a single pendant chromophore (that is, a single "antenna"). Ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield values up to approximately 60% that are stable at the nanomolar concentrations required for commercial assays. Through synthetic modification of the IAM chromophore and time-dependent density functional theory (TD-DFT) calculations, we have developed a method to predict absorption and emission properties of these chromophores as a tool to guide ligand design. Additionally, we have investigated chiral IAM ligands that yield Tb(III) complexes possessing both high quantum yield values and strong circularly polarized luminescence (CPL) activity. To efficiently sensitize Eu(III) emission, we have used the 1-hydroxypyridin-2-one (1,2-HOPO) chelate to create remarkable ligands that combine excellent photophysical properties and exceptional aqueous stabilities. A more complete understanding of this chromophore has been achieved by combining low-temperature phosphorescence measurements with the same TD-DFT approach used with the IAM system. Eu(III) complexes with strong CPL activity have also been obtained with chiral 1,2-HOPO ligands. We have also undertaken kinetic analysis of radiative and nonradiative decay pathways for a series of Eu(III) complexes; the importance of the metal ion symmetry on the ensuing photophysical properties is clear. Lastly, we describe a Tb(III)-IAM compound--now carried through to commercial availability--that offers improved performance in the common HTRF platform and has the potential to vastly improve sensitivity.
Project description:A series of octadentate ligands featuring the 2-hydroxyisophthalamide (IAM) antenna chromophore to sensitize Tb(III) and Eu(III) luminescence has been prepared and characterized. The length of the alkyl amine scaffold that links the four IAM moieties has been varied to investigate the effect of the ligand backbone on the stability and photophysical properties of the Ln(III) complexes. The amine backbones utilized in this study are N,N,N',N'-tetrakis-(2-aminoethyl)-ethane-1,2-diamine [H(2,2)-], N,N,N',N'-tetrakis-(2-aminoethyl)-propane-1,3-diamine [H(3,2)-], and N,N,N',N'-tetrakis-(2-aminoethyl)-butane-1,4-diamine [H(4,2)-]. These ligands also incorporate methoxyethylene [MOE] groups on each of the IAM chromophores to increase their water solubility. The aqueous ligand protonation constants and Tb(III) and Eu(III) formation constants were determined from solution thermodynamic studies. The resulting values indicate that at physiological pH the Eu(III) and Tb(III) complexes of H(2,2)-IAM-MOE and H(4,2)-IAM-MOE are sufficiently stable to prevent dissociation at nanomolar concentrations. The photophysical measurements for the Tb(III) complexes gave overall quantum yield values of 0.56, 0.39, and 0.52 respectively for the complexes with H(2,2)-IAM-MOE, H(3,2)-IAM-MOE, and H(4,2)-IAM-MOE, while the corresponding Eu(III) complexes displayed significantly weaker luminescence, with quantum yield values of 0.0014, 0.0015, and 0.0058, respectively. Analysis of the steady state Eu(III) emission spectra provides insight into the solution symmetries of the complexes. The combined solubility, stability, and photophysical performance of the Tb(III) complexes in particular make them well suited to serve as the luminescent reporter group in high sensitivity time-resolved fluoroimmunoassays.
Project description:The modular syntheses of three new octadentate, enantiopure ligands are reported, one with the bidentate chelating unit 2-hydroxyisophthalamide (IAM) and two with bidentate 1-hydroxy-2-pyridinone (1,2-HOPO) units. A new design principle is introduced for the chiral, non-racemic hexamines which constitute the central backbones for the presented class of ligands. The terbium(III) complex of the IAM ligand, as well as the europium(III) complexes of the 1,2-HOPO ligands, are synthesized and characterized by various techniques (NMR, UV, CD, luminescence spectroscopy). All species exhibit excellent stability and moderate to high luminescence efficiency (quantum yields Phi(Eu) = 0.05-0.08 and Phi(Tb) = 0.30-0.57) in aqueous solution at physiological pH. Special focus is put onto the properties of the complexes in regard to circularly polarized luminescence (CPL). The maximum luminescence dissymmetry factors (g(lum)) in aqueous solution are high with |g(lum)|(max) = 0.08-0.40. Together with the very favorable general properties (good stability, high quantum yields, long lifetimes), the presented lanthanide complexes can be considered as good candidates for analytical probes based on CPL in biologically relevant environments.
Project description:The synthesis, stability, and photophysical properties of several Eu(III) complexes featuring the 1-hydroxypyridin-2-one (1,2-HOPO) chelate group in tetradentate and octadentate ligands are reported. These complexes pair highly efficient emission with exceptional stabilities (pEu ? 20.7-21.8) in aqueous solution at pH 7.4. Further analysis of their solution behavior has shown the observed luminescence intensity is significantly diminished below about pH ? 6 because of an apparent quenching mechanism involving protonation of the amine backbones. Nonetheless, under biologically relevant conditions, these complexes are promising candidates for applications in Homogeneous Time-Resolved Fluorescence (HTRF) assays and synthetic methodology to prepare derivatives with either a terminal amine or a carboxylate group suitable for bioconjugation has been developed. Lastly, we have demonstrated the use of these compounds as the energy donor in a Luminescence Resonance Energy Transfer (LRET) biological assay format.
Project description:The synthesis, characterization, and photophysical properties of two novel ligands, 5LINMe-1,2-HOPO (1) and H(2,2)-1,2-HOPO (2), which utilize the 1,2-HOPO chelate as a sensitizer for Eu(III) are reported. In addition, the former ligand was structurally characterized as the Eu(III) complex by X-ray crystallography. The [Eu(1)2]- complex of the tetradentate ligand (1) is stable in aqueous solution, to a limiting concentration of ca. 7x10(-9) M, and retains the superior photophysical performance noted for the 1,2-HOPO sensitizer. By contrast, the octadentate ligand (2) has vastly improved stability as the [Eu(2)]- complex upon further dilution, to a limiting concentration of ca. 5x10(-17) M, which is beyond the minimum detectable concentration of most fluorimeters. The presence of a single coordinated water molecule for the latter complex reduces the overall metal-centered luminescence.
Project description:The synthesis of a series of octadentate ligands containing the 1-hydroxypyridin-2-one (1,2-HOPO) group in complex with europium(III) is reported. Within this series, the central bridge connecting two diethylenetriamine units linked to two 1,2-HOPO chromophores at the extremities (5-LIN-1,2-HOPO) is varied from a short ethylene chain (H(2,2)-1,2-HOPO) to a long pentaethylene oxide chain (H(17O5,2)-1,2-HOPO). The thermodynamic stability of the europium complexes has been studied and reveals these complexes may be effective for biological measurements. Extension of the central bridge results in exclusion of the inner-sphere water molecule observed for [Eu(H(2,2)-1,2-HOPO)](-) going from a nonacoordinated to an octacoordinated Eu(III) ion. With the longer chain length ligands, the complexes display increased luminescence properties in aqueous medium with an optimum of 20% luminescence quantum yield for the [Eu(H(17O5,2)-1,2-HOPO)](-) complex. The luminescence properties for [Eu(H(14O4,2)-1,2-HOPO)](-) and [Eu(H(17O5,2)-1,2-HOPO)](-) are better than that of the model bis-tetradentate [Eu(5LIN(Me)-1,2-HOPO)2](-) complex, suggesting a different geometry around the metal center despite the geometric freedom allowed by the longer central chain in the H(mOn,2) scaffold. These differences are also evidenced by examining the luminescence spectra at room temperature and at 77 K and by calculating the luminescence kinetic parameters of the europium complexes.
Project description:Although widely used in bioassays, the spectrofluorimetric method described here uses the antenna effect as a tool to probe the thermodynamic parameters of ligands that sensitize lanthanide luminescence. The Eu(3+) coordination chemistry, solution thermodynamic stability, and photophysical properties of the spermine-based hydroxypyridonate octadentate chelator 3,4,3-LI(1,2-HOPO) are reported. The complex [Eu(III)(3,4,3-LI(1,2-HOPO))](-) luminesces with a long lifetime (805 mus) and a quantum yield of 7.0% in aqueous solution, at pH 7.4. These remarkable optical properties were exploited to determine the high (and proton-independent) stability of the complex (log beta(110) = 20.2(2)) and to define the influence of the ligand scaffold on the stability and photophysical properties.
Project description:We report the preparation and new insight into photophysical properties of luminescent hydroxypyridonate complexes [M(III)L](-) (M = Eu or Sm) of the versatile 3,4,3-LI(1,2-HOPO) ligand (L). We report the crystal structure of this ligand with Eu(III) as well as insights into the coordination behavior and geometry in solution by using magnetic circular dichroism. In addition TD-DFT calculations were used to examine the excited states of the two different chromophores present in the 3,4,3-LI(1,2-HOPO) ligand. We find that the Eu(III) and Sm(III) complexes of this ligand undergo a transformation after in situ preparation to yield complexes with higher quantum yield (QY) over time. It is proposed that the lower QY in the in situ complexes is not only due to water quenching but could also be due to a lower degree of f-orbital overlap (in a kinetic isomer) as indicated by magnetic circular dichroism measurements.
Project description:The coordination compounds of the trivalent lanthanide ions (Ln(III)) have unique photophysical properties. Ln(III) excitation is usually performed through a light-harvesting antenna. To enable Ln(III)-based emitters to reach their full potential, an understanding of how complex structure affects sensitization and quenching processes is necessary. Here, the role of the linker between the antenna and the metal binding fragment was studied. Four macrocyclic ligands carrying coumarin 2 or 4-methoxymethylcarbostyril sensitizing antennae linked to an octadentate macrocyclic ligand binding site were synthesized. Complexation with Ln(III) (Ln = La, Sm, Eu, Gd, Tb, Yb and Lu) yielded species with overall -1, 0, or +2 and +3-charge. Paramagnetic <sup>1</sup>H NMR spectroscopy indicated subtle differences between the coumarin- and carbostyril-carrying Eu(III) and Yb(III) complexes. Cyclic voltammetry showed that the effect of the linker on the Eu(III)/Eu(II) apparent reduction potential was dependent on the electronic properties of the N-substituent. The Eu(III), Tb(III) and Sm(III) complexes were all luminescent. Coumarin-sensitized complexes were poorly emissive; photoinduced electron transfer was not a major quenching pathway in these species. These results show that seemingly similar emitters can undergo very different photophysical processes, and highlight the crucial role the linker can play.
Project description:Studies concerning synthesis, structure and luminescence of eight-coordinate Eu, Tb, Sm and Dy complexes of the type [Ln(acac)2(L)]Cl (Hacac = pentanedione-2,4 and L = bis(5-(pyridine-2-yl)-1,2,4-triazol-3-yl)propane) are reported in detail. The obtained complexes were investigated by various means including elemental- and thermogravimetric analysis, IR- and electron transition spectroscopy. The structure of the Tb complex was determined by single-crystal X-ray crystallography: Tb is eight-coordinate, and L acting only as a tetradentate chelate together with two bidentate acac ligands. Photophysical studies of the complexes were carried out. The Tb(III) and Eu(III) complexes show strong emissions both in solid state and solution. The intensity of the luminescence of Dy(III) and Sm(III) are relatively weak. The factors determining the intensity of the photoluminescence are discussed.
Project description:The synthesis, structure, and photophysical properties of several Tb(III) complexes with octadentate, macrotricyclic ligands that feature a bicapped topology and 2-hydroxyisophthalamide (IAM) chelating units are reported. These Tb(III) complexes exhibit highly efficient emission (?(total) ? 50%), large extinction coefficients (?(max) ? 20,000 M(-1) cm(-1)), and long luminescence lifetimes (?(H(2)O) ? 2.45 ms) at dilute concentrations in standard biological buffers. The structure of the methyl-protected ligand was determined by single-crystal X-ray diffraction and confirms the macrotricyclic structure of the parent ligand; the amide groups of the methyl-protected cage compound generate an anion binding cavity that complexes a chloride anion. Once the ligand is deprotected, a conformational change generates a similar cavity, formed by the phenolate and ortho amide oxygen groups that strongly bind lanthanide ions. The Tb(III) complexes thus formed display long-term stability, with little if any change in their spectral properties (including lifetime, quantum yield, and emission spectrum) over time or in different chemical environments. Procedures to prepare functionalized derivatives with terminal amine, carboxylate, and N-hydroxysuccinimide groups suitable for derivatization and protein bioconjugation have also been developed. These bifunctional ligands have been covalently attached to a number of different proteins, and the terbium complexes' exceptional photophysical properties are retained. These compounds establish a new aqueous stability and quantum yield standard for long-lifetime lanthanide reporters.