From Antenna to Assay: Lessons Learned in

Lanthanide Luminescence
EVAN G. MOORE, AMANDA P. S. SAMUEL, AND
KENNETH N. RAYMOND*
Department of Chemistry, University of California, Berkeley, California 94720
and Chemical Sciences Division, Lawrence Berkeley National Laboratories,
Berkeley, California 94720
RECEIVED ON SEPTEMBER 25, 2008

CON SPECTUS

L igand-sensitized, luminescent lanthanide(III) complexes are

of considerable importance because their unique photophysi-
cal properties (microsecond to millisecond lifetimes, character-
istic 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 emis-
sive complexes with sufficient stability and aqueous solubility for
practical applications.
In this Account, we give an overview of some of the gen-
eral properties of the trivalent lanthanides and follow with a
summary of advances made in our laboratory in the develop-
ment 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) tech-
nology. 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 chro-
mophore (that is, a single “antenna”).
Ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield val-
ues up to ∼60% that are stable at the nanomolar concentrations required for commercial assays. Through synthetic modifica-
tion 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 inves-
tigated 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 chro-
mophore 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 compoundsnow car-
ried through to commercial availabilitysthat offers improved performance in the common HTRF platform and has the potential
to vastly improve sensitivity.

Introduction relatively abundant: yttrium, for example, is one of

the 30 most abundant elements and even the
The “rare earths” consist of elements Z ) 21 (Sc), “rarest” of these elements, thulium (Z ) 69) and
Z ) 39 (Y), and Z ) 57-71 (La-Lu).1 Despite this lutetium (Z ) 71), are some 100 times more
classification, most of these elements are actually abundant in the Earth’s crust than gold.2

542 ACCOUNTS OF CHEMICAL RESEARCH 542-552 April 2009 Vol. 42, No. 4 Published on the Web 03/26/2009 www.pubs.acs.org/acr
10.1021/ar800211j CCC: $71.50 © 2009 American Chemical Society
 From Antenna to Assay Moore et al.

FIGURE 1. Observed increase in hydration enthalpy (left axis, red FIGURE 2. The antenna effect for Ln(III) sensitization, illustrated
squares) and decrease in ionic radii (right axis, black circles) for the using the chromophoric chelate (right) and pendant chromophore
trivalent Ln(III) series. (left) ligand designs (see text).
petes with hydrolysis, and the stability of Ln(III) complexes are
The lanthanides (together with the actinides) are unique in
typically measured using potentiometric or spectrophotometric
that they have very similar chemical properties, such as hydra-
methods, with results evaluated in terms of the equilibrium con-
tion enthalpies, common oxidation state, and ionic radius.
stant for the complexation reaction:
Nonetheless, variations are observed. For example, from Z )
57 to 71, the hydration enthalpies of the trivalent cations [MmLlHh]
mM + lL + hH+ T MmLlHh
mlh )
increase almost linearly3 while the atomic radii decrease reg- [M]m[L]l[H+]h
ularly across the series in the well-known lanthanide contrac-
For example, the log
110 value (i.e., ML complex forma-
tion (Figure 1).4
tion) for La(III) with ethylenediamine tetraacetic acid (EDTA) is
In aqueous solution, the lanthanides exist almost exclu-
15.46 (25 °C, 0.1 M supporting electrolyte),7 which increases
sively in their trivalent state, Ln(III), due to the balance
steadily across the Ln(III) series (log
110 ) 19.80 with Lu(III)),
between hydration enthalpy of the trivalent cations and the
due to the increased Lewis acidity. Alternatively, the stability
sum of the first three ionization energies. The majority of
of metal complexes with differing ligand and complex acidi-
the electronic wave function for the 4f orbitals lies beneath the
ties can be evaluated via conditional stability constants, or
occupied 5s and 5p orbitals. Hence, the 4f valence electrons
pMs, defined as the negative log of the free metal concentra-
interact minimally with their environment, and bonding in
tion, pM7.4 ) -log10[M]free, under conditions of 10 µM total
Ln(III) complexes is mainly electrostatic and nondirectional.
ligand, 1 µM total metal, and pH 7.4.
This same property imparts great flexibility on Ln(III) com-
plexes, in terms of coordination number and geometry, since
Spectroscopic Properties
the requirement of maximizing orbital overlap is relaxed.
One significant aspect of the lanthanides is their unusual spec-
The Ln(III) ions are “hard” Lewis acids and display a prefer-
troscopic properties. Trivalent cations display absorption and
ence for hard, negatively charged donor atoms such as oxygen
emission bands that correspond to Laporte-forbidden f-f tran-
(e.g., OH-, H2O) or fluoride (F-) compared with softer bases such
sitions. Because the 4f orbitals are relatively insensitive to the
as sulfur (e.g., S-) or nitrogen (e.g., NH3), when they act as mono-
ligand field, these optical bands are line-like and characteris-
dentate ligands. Coordination numbers range from CN ) 3 to 12
tic of each metal. As a consequence of the parity rule (and
in the solid state and are largely determined by the ionic radius
sometimes due to the change in spin multiplicity), these
of the cation and the charge and steric requirements of the
absorptions have very low molar absorptivities (ε < 1 M-1
ligand(s), with 8 and 9 being the most common in solution.3,5 In
cm-1), making the direct excitation of the metals very ineffi-
aqueous solutions, Ln(III) cations readily hydrolyze above ca. pH
cient unless high-power laser excitation is utilized. This prob-
6 to form hydroxo species:6
lem is overcome by using a strongly absorbing chromophore
Ln3+(aq) + H2O(l) T Ln(OH)2+(aq) + H+(aq) to sensitize Ln(III) emission in a process known as the antenna
effect (Figure 2); initial excitation occurs via the chromophore,
[Ln(OH)2+][H+]

1 ) which transfers energy to the Ln(III), populating the metal-cen-
[Ln3+]
tered excited state, which then emits. Given the short lifetimes
with reported5 log
1 values ranging from -8.5 for La(OH)2+ to of the excited singlet states for organic chromophores, energy
-7.6 for Lu(OH)2+. Complex formation with organic ligands com- transfer is usually discussed as proceeding from the excited

Vol. 42, No. 4 April 2009 542-552 ACCOUNTS OF CHEMICAL RESEARCH 543
From Antenna to Assay Moore et al.

After more than 15 years of development, homogeneous

time-resolved fluorescence (HTRF) assays have become wide-
spread. Of these, a recent survey ranked CisBio’s HTRF tech-
nology, which relies on fluorescence resonance energy
transfer (FRET), as the most frequently used generic assay,10
and the general format of this assay is shown in Figure 4.
When only the Ln(III)-labeled antigen is present, it binds the
antibody and efficient FRET between the lanthanide and
acceptor takes place, resulting in enhanced emission of the
organic dye. When the target antigen is present, the labeled
antigen is displaced and the ratio of organic dye to Ln(III)
emission is altered. With the long-lived luminescence of a lan-
thanide, the sensitivity of the assay can be vastly improved by
using time-gated excitation and detection techniques. This
FIGURE 3. A summary of electronic excited-state energy levels for
the Ln(III) series.
minimizes false signals due to direct excitation of the organic
triplet state, although singlet and charge transfer (CT) states dye molecule and increases sensitivity by removing back-
may also play a role.8 ground autofluorescence from other components present in
The ground and excited states of the Ln(III) ions are shown biological media.
in Figure 3. While the metal ions on the periphery (Ce-Pm, When a lanthanide-based label is developed for a homo-
Ho-Yb) have relatively small energy gaps between adjacent geneous assay, either of two general strategies can be
levels, the central metals (Sm-Dy) display larger gaps, partic- employed in ligand design (Figure 2). The first approach,
ularly for Gd(III). Hence, for the former, nonradiative deactiva- which we term the chromophoric chelate, utilizes one or more
tion processes such as coupling to solvent vibrational modes aromatic groups, which absorb incident light and also con-
are more efficient, and these ions are only weakly emissive. tain coordinating atoms. As such, these chromophores are
For the central group, the luminescent electronic transitions directly bound to the Ln(III) cation. Early examples are the tris-
also involve a change in spin multiplicity. As a result, their bipyridyl cryptands developed by Lehn and co-workers11 (Fig-
luminescence is particularly long-lived (microseconds to mil- ure 5), which have been commercially developed by CisBio.
liseconds). For the purposes of this Account, we will focus on However, these complexes often require an augmenting agent
only Eu(III) and Tb(III), since only these two metals typically (e.g., F-) to optimize their luminescence.12
have the sizable quantum yields (>10%) and long-lived lumi- In the design that we term the pendant chromophore, one or
nescence lifetimes (0.1-1 ms) desirable for commercial appli- more chromophores are linked to a multidentate ligand such as
cations in biotechnology. diethylenetriaminepentaacetic acid (DTPA)13 or 1,4,7,10-tetraaza-
cyclotetradecane-1,4,7-triacetic acid (DO3A),14 which bind the
Lanthanide Complexes in Ln(III) ion and saturate the metal’s coordination sphere (Figure 5).
Fluoroimmunoassays The chromophore is covalently attached to the binding unit but
Both Eu(III) and Tb(III) are used in the field of biotechnology, not necessarily bound to the lanthanide. By this approach, a
where the ability to assay for analytes at small concentrations number of chromophores have been attached to various alkyl
and in a complicated matrix presents a challenging problem. amine or macrocyclic platforms, which form stable and kineti-
Lanthanide luminescence is amenable to these applications cally inert complexes, facilitating their use in biological applica-
since it can be detected at nano- to picomolar concentrations tions. Both strategies allow for incorporation of multiple
by utilizing spectral and temporal discrimination. One of the chromophores, which increases the overall absorbance of the
first assay techniques developed was the dissociation complex; however, in the latter, when the chromophore is not
enhanced lanthanide fluoro immuno assay (DELFIA). This directly attached to the metal cation, ligand-to-Ln(III) energy trans-
assay, however, is not ideal because it is heterogeneous and fer necessarily proceeds via a through-space Förster-type mech-
the signal cannot be traced until the end of the assay.9 Hence, anism, while for the former approach, both Förster15 and
the preparation of efficient lanthanide sensitizers that form sta- Dexter16 mechanisms are possible.
ble complexes in dilute aqueous solution is an active area, To yield highly luminescent Ln(III) complexes, an effective
since they can be used in homogeneous formats. antenna ligand must be optimized in several areas. The ligand

544 ACCOUNTS OF CHEMICAL RESEARCH 542-552 April 2009 Vol. 42, No. 4
 From Antenna to Assay Moore et al.

FIGURE 4. A cartoon illustration of the general principle for homogenous time-resolved fluoro-immunoassay (HTRF) format in the presence
(left) and absence (right) of the target antigen.

FIGURE 5. Selected examples of compounds based on the cryptate (top left), macrocyclic (e.g., DO3A, top and bottom right), or linear
aminocarboxylate (e.g. DTPA, bottom left) platforms utilized for Ln(III) sensitization.

must form stable complexes over a wide pH range and resist ogy is preferred, which will also confer stability at nano- to
aqueous hydrolysis at subnanomolar concentrations. Further- picomolar concentrations.
more, the ligand should saturate the Ln(III) coordination sphere In terms of the desired photophysical properties, the chro-
to prevent water from coordinating to the metal ion, since mophore should possess a high molar absorptivity, ε, and the
bound solvent effectively quenches luminescence. For Eu(III) excitation wavelength, λex, should be above ca. 350 nm to
and Tb(III), the inner sphere hydration number, q, can be quan- facilitate the use of inexpensive excitation sources and avoid
tified using the observed luminescence lifetimes, τ, in H2O and excitation of other chromophores in biological media (tryp-
D2O respectively, using the general relationship, q ) A(1/τH2O tophan, NADH, etc). Since sensitization typically occurs via the
- 1/τD2O - B). Empirical values of A proposed by Horrocks17 excited triplet state, the antenna should possess a high inter-
are 1.05 ms-1 for Eu(III) and 2.1 ms-1 for Tb(III), with B ) 0. system crossing efficiency (ηisc) and the ligand triplet state
The B term was introduced by Beeby and Parker et al. to should be at least 1850 cm-1 higher in energy than the Ln(III)
account for second sphere water molecules, and constants of emitting state to minimize thermal repopulation of the trip-
A ) 1.2 ms-1, B ) 0.25 for Eu(III) and A ) 5.0 ms-1, B ) 0.06 let.20 For Eu(III), the energetic requirements of the antenna
for Tb(III) were proposed,18 which were later further refined for chromophore are less straightforward, since the metal can
Eu(III) by Horrocks et al.19 to be A ) 1.11 ms-1, B ) 0.31. Due accept energy via the 5D2, 5D1, and 5D0 electronic states (Fig-
to solvent-induced quenching, a multidentate ligand topol- ure 3). Also, while the quenching effects of thermal back trans-

Vol. 42, No. 4 April 2009 542-552 ACCOUNTS OF CHEMICAL RESEARCH 545
From Antenna to Assay Moore et al.

FIGURE 6. Generalized structures of the 2-hydroxyisophthalamide

(IAM, left) and 1-hydroxy-pyridin-2-one (1,2-HOPO, right) chelates
featuring exclusively oxygen donor sets (red), where R is typically
an alkyl amine backbone. FIGURE 7. X-ray structures of [Fe(Bis-Tren-IAM)] (left) and [Eu(Bis-
5
fer can be observed when the ligand triplet and D0 levels of Tren-IAM)2] (right) (selected H atoms omitted).

Eu(III) are too close in energy, low-lying ligand-to-metal

charge-transfer (LMCT) states can be readily accessed when
higher excitation energies (e.g., g30 000 cm-1) are used, due
to the increased ease of photoreduction to form Eu(II) at
shorter wavelengths.21 In addition to governing the sensitiza-
tion efficiency (ηsens) of the complex, the ligand can also exert
control over the coordination environment and hence sym-
metry of the Ln(III) ion, which may affect the overall quan-
tum yield (Φtot) by influencing the intrinsic quantum yield of
the metal center (ΦLn).

Sensitization of Tb(III) with the IAM

Chromophore FIGURE 8. Observed emission from Ln(III) cations sensitized by an
IAM ligand irradiated with a standard laboratory UV lamp (λex )
Our interest in ligand-sensitized Ln(III) luminescence originated 365 nm). From left to right, ligand only, Tb(III), Eu(III), Dy(III), and
with the 2-hydroxyisophthalamide (IAM) binding unit (Figure Sm(III) complexes.
6), developed to investigate the salicylate binding mode in properties (ε ) 26 800 M-1 cm-1, Φtot ) 59%, τ ) 2.6 ms)
Fe(III) complexes.22 The X-ray structure of the Fe(III) complex seen with Bis-Tren-IAM.
of the Bis-Tren-IAM ligand shows each 2-hydroxyisophthala- To quantify the stability of this new class of luminescent
mide arm is bidentate, binding through the deprotonated phe- species, it was necessary to improve their aqueous solubility,
nolate oxygen and an adjacent carbonyl oxygen. The which was achieved by replacing the methyl amide with a
remaining amide group is oriented so the amide proton forms 2-methoxyethyl (MOE) amide group (Figure 9). For the
an intermolecular H-bond with the phenolate oxygen (Figure [Ln(H(2,2)-IAM-MOE)] complexes, log
110 values of 14.7(1)
6), which stabilizes the structure by predisposing the ligand and 14.4(1) were measured for Tb(III) and Eu(III), with corre-
toward metal binding. In addition, we found that Bis-Tren-IAM sponding pM values of 14.7 and 14.3.24 Spectrophotometric
forms highly luminescent complexes when mixed with solu- titration data was used to assign the protonation sequence of
tions of Ln(III) salts,23 which sparked our ongoing interest in the ligand, revealing a central amine in the backbone to be
this area. the most basic site, with a pKa value of ca. 9.5. The high basic-
Remarkably, the IAM chromophore can sensitize all four of ity of the amine is retained in the Ln(III) complexes, which
the visible-emitting Ln(III) cations, Tb(III), Eu(III), Sm(III), and have pKa values of 9.4 and 9.2 with Tb(III) and Eu(III), respec-
Dy(III) (Figure 8). The Tb(III) complex in particular displays a tively. The Ln(III) affinities of the fully deprotonated versus
quantum yield in aqueous solution of Φtot ) 61%, which monoprotonated ligand suggest that the latter benefits from
remains one of the highest values reported. The X-ray struc- a stabilizing hydrogen bond involving the tertiary backbone
ture of the Eu(III) complex reveals that Bis-Tren-IAM acts as a amines, predisposing the ligand for metal binding.24 Another
tetradentate ligand, binding in a 2:1 stoichiometry, with only important result was that the photophysical properties of the
two of the three 2-hydroxyisophthalamide units chelated (Fig- more soluble [Tb(H(2,2)-IAM-MOE)] complex remain essen-
ure 7).23 To better suit the Ln(III) coordination requirements, tially unchanged (ε ≈ 27 000 M-1 cm-1, Φtot ) 56%, τ ) 2.6
an octadentate ligand, H(2,2)-IAM (Figure 9), was prepared, ms). The Gd(III) complexes of both H(2,2)-IAM and H(2,2)-IAM-
and its Tb(III) complex retains the favorable photophysical MOE were prepared to measure the zero phonon energy of

546 ACCOUNTS OF CHEMICAL RESEARCH 542-552 April 2009 Vol. 42, No. 4
 From Antenna to Assay Moore et al.

FIGURE 9. Chemical structures of ligands based on the 2-hydroxyisophthalamide (IAM) and 1-hydroxy-pyridin-2-one (1,2-HOPO) chelates.

the lowest energy ligand triplet state (T0-0), resulting in val- longer propyl and butyl linkages between the two central
ues of ca. 23 350 and 23 170 cm-1 respectively. The lowest amines have a single water molecule bound to the metal,
excited singlet and triplet states for the IAM ligands are quite leading to decreased quantum yields.
close in energy (∆Eavg(S1fT0-0)) 850 cm-1), suggesting a rela- Modifying the IAM amide linkage can also introduce chiral-
tively high rate of intersystem crossing due to the similarity of ity into the ligand, yielding Ln(III) complexes with circularly
the excited-state geometries, which may contribute to the high polarized luminescence (CPL) activity. Employing both enan-
quantum yields observed. Furthermore, the mean T0-0 energy tiomers of R-methylbenzylamine, we produced the chiral
is only ca. 2750 cm-1 higher in energy than the Tb(III) 5D4 ligands H(2,2)-IAM-R(+)-R-BnMe and H(2,2)-IAM-S(-)-R-BnMe
emitting state, which is within the optimal region for efficient (Figure 9).25 Their Ln(III) complexes retain the brightness of the
ligand to Tb(III) energy transfer.20 parent achiral forms and also display CPL activity, reported as
Altering the IAM chromophore via modification of the sec- the luminescence dissymmetry factor, glum, which measures
ondary amide group on the “open” face of the ligand does not the difference in left and right circularly polarized emission (IL
alter the photophysical properties of the Tb(III) complexes. or IR) compared with the total emission;
However, in the same report,24 we showed that altering the
∆I IL - IR
length of the central amine backbone can have a dramatic glum ) )
1 1
impact on the photophysical behavior of the metal complexes I (I + IR)
2 2 L
by altering the solvent accessibility. Estimations of q for the
Tb(III) complexes using the equations derived by Beeby and These [Ln(H(2,2)-IAM-BnMe)] complexes display very high
Parker et al.18 showed that for the [Tb(H(2,2)-IAM-MOE)] com- |glum| values of ca. 0.3 with Eu(III), and we also reported the
plex, there are no bound water molecules; in contrast, com- first CPL-active Sm(III) complexes formed from chiral ligands.
plexes formed with analogous ligands that have slightly One drawback of these complexes is their low solubility, pre-

Vol. 42, No. 4 April 2009 542-552 ACCOUNTS OF CHEMICAL RESEARCH 547
From Antenna to Assay Moore et al.

FIGURE 10. Chemical structure of CyH(2,2)-IAM (left) and EtH(2,2)-1,2-HOPO (right) ligands for aqueous CPL measurements.

venting studies in water. An alternative approach where the

chiral center was incorporated into the ligand backbone
(CyH(2,2)-IAM, Figure 10) displays both improved aqueous sol-
ubility and an increased |glum| value of 0.078 compared with
[Tb(H(2,2)-IAM-BnMe)] (|glum| ) 0.048).26
We were also interested in determining how the photo-
physical properties of the antenna can be further optimized.
For example, it remains challenging to easily and reliably pre-
dict the effects of antenna modification on the ligand and
hence ensuing photophysical properties. A series of IAM
FIGURE 11. Representative emission spectrum of a Tb(III)-IAM
ligands substituted para to the phenolate group (IAM-X) were complex in aqueous solution, pH 7.4.
developed to determine the relationship between chro-
mophore modification and the resulting photophysical prop-
erties of the complexes.27 This was achieved using a
simplified tetradentate 5LI-IAM (Figure 9) ligand scaffold,
which forms ML2 complexes with Tb(III) that retain most of the
favorable photophysical properties (ε ) 25 100 M-1 cm-1, Φtot
) 36%, τ ) 2.5 ms) compared with the octadentate
derivatives.
A wide selection of X groups covering a broad range of
electron-withdrawing and electron-donating abilities was cho-
sen. With the exception of the nitro-substituted ligand (5LI-
IAM-NO2) (vide infra), the ligand absorption, fluorescence, and
phosphorescence energies were all found to increase linearly
with the resonance component of the substituent Hammett
parameter (R).27 The overall quantum yield of the Tb(III) emis-
Tb FIGURE 12. Results from TD-DFT (B3LYP/6-311G++(d,p)) for
sion, Φtot , was also found to increase with the triplet energy
simplified model “[Na(IAM)]” (top) and “[Na(1,2-HOPO)]” (bottom)
of the ligand, an effect that we attribute in part to a decrease chromophores, revealing the π f π* character of the low-lying
in nonradiative deactivation caused by thermal repopulation excited singlet and triplet states.
of the ligand excited triplet state. The luminescence behavior tions agree (within ca. 5%) with the experimentally deter-
observed for the Tb(III) complexes (Figure 11) agrees well with mined absorption maxima and triplet energies. An
results reported by Latva et al.,20 which show that quantum examination of the orbitals involved in the transitions (Figure
yield values display marked decreases when the ligand trip- 12) also confirmed that they can be described as essentially
let energy falls below ca. 22 000 cm-1. π-π*, except in the case of the IAM-NO2. The excited-state
To complement these findings and to develop a more gen- transitions for this chromophore showed significant intrali-
eral way to screen antenna ligands, time-dependent density gand charge transfer (ILCT) character, which explains why this
functional theory (TD-DFT) calculations were performed27 on ligand fails to adhere to the trends seen for the remainder of
simplified bidentate IAM chelates, using Na+ in place of Tb(III). the IAM-X ligand series. Together the experimental and com-
The singlet and triplet values obtained from these calcula- putational results serve as complementary techniques useful

548 ACCOUNTS OF CHEMICAL RESEARCH 542-552 April 2009 Vol. 42, No. 4
 From Antenna to Assay Moore et al.

FIGURE 14. X-ray structures of [Eu(5LIO-1,2-HOPO)2]- (left) and

[Eu(Tren-1,2-HOPO)(H2O)2] (right) (selected H atoms omitted).
FIGURE 13. Representative emission spectrum of a Eu(III)-1,2-HOPO
complex in aqueous solution, pH 7.4. appropriately positioned to sensitize the 5D1 state of Eu(III),
to quickly and accurately assess the photophysical proper- located at 19 030 cm-1 (∆E(T0-0f5D1)) 2230 cm-1). Using the
ties of potential antenna chromophores prior to ligand same TD-DFT technique developed for the IAM ligands, we
synthesis. have performed calculations on a simplified Na+ adduct of a
bidentate 1,2-HOPO chromophore (Figure 12).31 The close
Sensitization of Eu(III) with the “1,2-HOPO” agreement (within ca. 5%) observed between the experimen-
Chromophore tal T0-0 energy and the calculated value of 21 365 cm-1 is
Given the success with the IAM chromophore as a sensitizer further evidence to support the generality of this approach in
for Tb(III), an improved chromophore for Eu(III) was sought. A making a rapid assessment of the potential of a given antenna
systematic screening of ligands developed in our laboratory system prior to synthetic efforts.
was undertaken, and a ligand utilizing a 6-amide derivative of Utilizing the same ligand backbone employed for the IAM
1-hydroxy-pyridin-2-one (1,2-HOPO) (Figure 6) was found to chromophore, we prepared an octadentate ligand, H(2,2)-1,2-
sensitize Eu(III) with high efficiency.28 Previous examples of HOPO (Figure 9), which was expected to form a more thermo-
sensitized Tb(III) and Eu(III) emission using 1,2-HOPO exist, but dynamically stable complex than the tetradentate analogs.33
the chromophore lacked the 6-amide group.29,30 Since our ini- The low aqueous solubility (>0.1 mM) of [Eu(H(2,2)-1,2-
tial communication, we have progressed in the development HOPO)]- below pH 7 prevented stability measurements by
and characterization of complexes that feature the 1,2-HOPO potentiometry, so stability was assessed by competition batch
chelate group. titration versus DTPA, at pH 6.1, 7.4, and 8.5, from which log
The tetradentate ligand, 5LI-1,2-HOPO (Figure 9), is remark-
110 ) 21.8(5), log
111 ) 31.2(3), and log
112 ) 38.1(2) val-
able since its EuL2 complex combines both exceptional pho- ues were extracted. These can be compared directly with
tophysical properties (ε ) 18 800 M-1 cm-1, Φtot ) 20.7%, those for the corresponding [Tb(H(2,2)-IAM-MOE)]- complex of
τH2O) 737 µs, q ) 0.1) with excellent aqueous stability (log log
110 ) 14.7(1), log
111 ) 24.1(1), and log
112 ) 30.3(1),

120 ) 22.6(1), pM ) 18.4).31 As an example, the metal-cen- showing that the 1,2-HOPO-based chelate is ca. 7 orders of
tered emission spectrum observed for [Eu(5LI-1,2-HOPO)2]- in magnitude more stable than the IAM derivative.
aqueous solution is shown in Figure 13. As a rationale for the The increased stability toward dilution achieved using an
impressive aqueous stability of this complex, we note the use octadentate ligand rather than a tetradentate scaffold was also
of an all-oxygen donor atom set and that the bidentate 1,2- highlighted by comparing the properties of the Eu(III) com-
HOPO chelating units form a five-membered ring with Eu(III). plexes of H(2,2)-1,2-HOPO and a structurally similar tetraden-
This binding mode of the 1,2-HOPO chelate has been con- tate ligand, 5LINMe-1,2-HOPO (Figure 9). For a fixed tolerance
firmed in X-ray structures with Eu(III), Sm(III), and Gd(III) (Fig- of 1% free Eu(III) at pH 7.4, the resistance of the octadentate
ure 14)31,32 and is in contrast to the less favorable six- [Eu(H(2,2)-1,2-HOPO)]- complex toward aqueous hydrolysis is
membered ring formed in IAM complexes. From the 77 K improved by ca. 8 orders of magnitude compared with the tet-
phosphorescence spectrum of [Gd(5LIO-1,2-HOPO)2]-, the radentate [Eu(5LINMe-1,2-HOPO)2]- complex.33
ligand T0-0 energy was measured at 21 260 cm-1, explain- The photophysical properties of [Eu(H(2,2)-1,2-HOPO)]-
ing the observations that 6-amide 1,2-HOPO derivatives do were measured in aqueous solution, and while the absorp-
not efficiently sensitize luminescence from Tb(III) at room tem- tion properties (ε ) 18 200 M-1 cm-1) and emission spectra
perature. The ligand triplet state is too close in energy to the are similar to those observed for the Eu(III) complexes with 5LI-
5
D4 accepting state of Tb(III) (∆E(T0-0f5D4)) 760 cm-1) but is and 5LIO-1,2-HOPO, the overall quantum yield is significantly

Vol. 42, No. 4 April 2009 542-552 ACCOUNTS OF CHEMICAL RESEARCH 549
From Antenna to Assay Moore et al.

reduced, to Φtot ) 3.6%. Solvent-induced quenching was sus- HOPO)2]-, which could alter either kisc or kEET and decrease the
pected due to the reduced luminescence lifetime in aqueous efficiency of sensitization.
solution (τH2O ) 480 µs), and the presence of an inner sphere
water molecule was confirmed by measurements in D2O Conclusions
(τD2O) 1220 µs, q ) 1.1). Hence, while bis-tetradentate com- Over the past several years, we have made substantial efforts
plexes receive adequate protection from nonradiative deacti- toward further optimizing the IAM chromophore and under-
vation, the H(2,2)-1,2-HOPO complex has a single bound standing the stability and photophysical properties of the
water molecule, strongly enhancing the nonradiative decay Tb-IAM complexes. To a similar extent, considerable progress
rate and explaining the poor luminescence performance. The in understanding the Eu-1,2-HOPO system has also been
addition of ethyl substituents on the arms of the H(2,2) back- achieved. While the IAM group forms slightly less stable com-
bone displacing second sphere water molecules has the ben- plexes than the 1,2-HOPO chelate, they nonetheless retain
efit of imbuing chirality in the ligand and hence CPL activity sufficient aqueous stability and resistance toward hydrolysis
(Figure 10). However, the Eu(III) complex still retains one inner for use in vitro in homogeneous assays. Another key feature
sphere water molecule, and the improved quantum yield was of Tb-IAM complexes is the relative insensitivity of the lumi-
limited to Φtot ) 7.7%.26 nescence lifetime to the ligand topology. By contrast,
To further investigate the photophysical properties of these Eu(III)-1,2-HOPO-based complexes show significant variation,
Eu(III) complexes, we examined the metal-centered radiative in terms of the luminescence lifetimes and also overall quan-
(kr) and nonradiative (knr) decay rate constants using the tum yields. A current limitation of the octadentate [Eu(H(2,2)-
approach reported by Verhoeven34 and Beeby.35 As expected, 1,2-HOPO)(H2O)]- complex is the residual inner sphere water
the rate of nonradiative decay, knr, is significantly higher for molecule, which quenches luminescence. For the IAM group,
the [Eu(H(2,2)-1,2-HOPO)(H2O)]- complex compared with this is less problematic, likely due to the steric protection pro-
[Eu(5LI-1,2-HOPO)2]-, for example, due to the bound inner vided by the methyl amide group on the “open” face of the
sphere water molecule. However, analysis of the kinetic [Tb(H(2,2)-IAM)]- complex. We are currently exploring meth-
parameters also revealed the radiative decay rate constant, kr, ods to increase the steric bulk of 1,2-HOPO-based ligands to
is lower for [Eu(H(2,2)-1,2-HOPO)(H2O)]-, presumably due to improve the performance of octadentate Eu(III) complexes. The
the different symmetries of the coordination environments lower thermodynamic stability of the Tb(III)-IAM complexes
experienced by Eu(III) (e.g., CN ) 8 vs CN ) 9). For complexes can be offset by the kinetic stability of macrotricyclic cryptate
of Eu(III), it has been shown that the steady-state emission complexes.
spectra can be used to probe the solution structure, and our The exceptional performance of Tb-IAM complexes has
analysis of these 1,2-HOPO complexes has shown that, while culminated in the recent commercialization of an IAM-based
the eight coordinate Eu(III) cation in bis-tetradentate ML2 com- cryptate (Lumi4-Tb) by Lumiphore, Inc., and subsequent
plexes such as [Eu(5LI-1,2-HOPO)2]- likely have average D2d marketing of this compound by CisBio for use in their HTRF
site symmetry in solution,31 the nine-coordinate [Eu(H(2,2)- platform. This product is twice as sensitive as the previ-
1,2-HOPO)(H2O)]- complex has local C2 symmetry,33 highlight- ously utilized Eu(III) complexes, as measured by a compar-
ing the importance of symmetry on the photophysical ative IP-One GPCR secondary messenger assay,36 and more
properties of these luminescent Eu(III) complexes. importantly, this comparison was made at a shared com-
From the kinetic analysis, the efficiency of the sensitiza- mon emission wavelength of 620 nm, close to the maxi-
tion process, ηsens, can be quantified by comparing the mum intensity of the Eu(III) 5D 0 f 7F 2 transition, but
observed total quantum yield, Φtotal, with the calculated intrin- accounting for only ca. 8% of the overall Tb(III)-centered
sic quantum yield value using the relationship, ηsens ) ΦtotalEu
/ luminescence (Figure 11). Detection and TR-FRET using the
ΦEu. This is related to both the rate of intersystem crossing, kisc, Lumi4-Tb cryptate can also be performed at 545 nm and
and the kinetics of the electronic energy transfer step, kEET. A 488 nm corresponding to the metal-centered 5D4 f 7F5 and
5
comparison of the data for the Eu(III) complexes with 5LI-1,2- D4 f 7F6 emission bands (which account for ca. 49% and
HOPO and H(2,2)-1,2-HOPO has shown that while the effi- 21%, respectively, of the overall Tb(III) luminescence). Since
ciency of these processes approaches 50% for the former, it FRET is dependent upon the overlap of donor emission and
is as low as ca. 20% for the octadentate complex with Eu(III). acceptor absortion,37 greater efficiency can be expected by
Recent indications are that the T0-0 energy of [Gd(H(2,2)-1,2- using blue-shifted organic dyes as HTRF acceptors (e.g.,
HOPO)(H2O)]- is ca. 875 cm-1 lower than that of [Gd(5LI-1,2- rhodamine B, λabs max ) 543 nm; fluorescein, λmax ) 500 nm),
abs

550 ACCOUNTS OF CHEMICAL RESEARCH 542-552 April 2009 Vol. 42, No. 4
 From Antenna to Assay Moore et al.

which will further enhance the sensitivity achievable in 10 Comley, J. TR-FRET based assays-getting better with age. Drug Discovery World,
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This work was partially supported by the NIH (Grant HL69832) 12 Cross, J. P.; Dadabhoy, A.; Sammes, P. G. The sensitivity of the Lehn cryptand-
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and by the Director, Office of Science, Office of Basic Energy Sci- saturated systems. J. Lumin. 2004, 110, 113–124.
ences, and the Division of Chemical Sciences, Geosciences, and 13 Ge, P.; Selvin, P. R. Carbostyril derivatives as antenna molecules for luminescent
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Biosciences of the U.S. Department of Energy at LBNL under 14 Clarkson, I. M.; Beeby, A.; Bruce, J. I.; Govenlock, L. J.; Lowe, M. P.; Mathieu,
Contract No. DE-AC02-05CH11231. This technology is licensed C. E.; Parker, D.; Senanayake, K. Experimental assessment of the efficacy of
sensitized emission in water from a europium ion, following intramolecular excitation
by the University of California to Lumiphore, Inc., in which some by a phenanthridinyl group. New J. Chem. 2000, 24, 377–386.
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BIOGRAPHICAL INFORMATION 17 Horrocks, W. D.; Sudnick, D. R. Lanthanide ion probes of structure in biology. Laser-
Evan G. Moore completed his B.S. and Ph.D. at the University of induced luminescence decay constants provide a direct measure of the number of
metal-coordinated water molecules. J. Am. Chem. Soc. 1979, 101, 334–340.
Queensland under the supervision of Prof. Paul Bernhardt before 18 Beeby, A.; Clarkson, I. M.; Dickins, R. S.; Faulkner, S.; Parker, D.; Royle, L.; Sousa,
commencing postdoctoral work at U.C. Berkeley. He is currently A. S. d.; Williams, J. A. G.; Woods, M. Non-radiative deactivation of the excited
an independent APD research fellow at the University of Mel- states of europium, terbium and ytterbium complexes by proximate energy-matched
OH, NH and CH oscillators: An improved luminescence method for establishing
bourne, Australia. solution hydration states. J. Chem. Soc., Perkin Trans. 2 1999, 493–504.
Amanda P. S. Samuel received her B.S. in chemistry from Yale 19 Supkowski, R. M.; Horrocks, W. DeW., Jr. On the determination of the number of
water molecules, q, coordinated to europium(III) ions in solution from luminescence
University in 2003 and her Ph.D. from U.C. Berkeley in 2008 decay lifetimes. Inorg. Chim. Acta 2002, 340, 44–48.
under the direction of Prof. Kenneth N. Raymond. She is currently 20 Latva, M.; Takalo, H.; Mukkala, V.-M.; Matachescu, C.; Rodriguez-Ubis, J. C.;
undertaking postdoctoral studies in Prof. Michael R. Wasielews- Kankare, J. Correlation between the lowest triplet state energy level of the ligand
ki’s laboratory at Northwestern University. and lanthanide(III) luminescence quantum yield. J. Lumin. 1997, 75, 149–169.
21 Kusaba, M.; Nakashima, N.; Kawamura, W.; Izawa, Y.; Yamanaka, C. Higher yield of
Kenneth N. Raymond received a B.A. from Reed College in photoreduction from europium (3+) to europium (2+) with shorter wavelength
1964 and his Ph.D. from Northwestern University in 1968 under irradiation. Chem. Phys. Lett. 1992, 197, 136–140.
the direction of Profs. Fred Basolo and James Ibers. He joined the 22 Cohen, S. M.; Petoud, S.; Raymond, K. N. A novel salicylate-based macrobicycle
with a “split personality”. Inorg. Chem. 1999, 28, 4522–4529.
faculty of U.C. Berkeley in 1967 and is currently U.C. Berkeley 23 Petoud, S.; Cohen, S. M.; Bunzli, J.-C.; Raymond, K. Stable lanthanide
Chancellor’s Professor and Director of the Glenn T. Seaborg Cen- luminescence agents highly emissive in aqueous solution: Multidentate 2-
ter, Lawrence Berkeley National Laboratory. hydroxyisophthalamide complexes of Sm3+, Eu3+, Tb3+, Dy3+. J. Am. Chem.
Soc. 2003, 125, 13324–13325.
24 Samuel, A. P. S.; Moore, E. G.; Melchior, M.; Xu, J.; Raymond, K. N. Water-soluble
FOOTNOTES 2-hydroxyisophthalamides for sensitization of lanthanide luminescence. Inorg.
Chem. 2008, 47, 7535–7544.
* Corresponding author. Mailing address: Department of Chemistry, University of Califor-
nia, Berkeley, CA 94720-1460. Phone: +1 510 642 7219. Fax: +1 510 486 5283. 25 Petoud, S.; Muller, G.; Moore, E. G.; Xu, J.; Sokolnicki, J.; Riehl, J. P.; Le, U. N.;
E-mail: [email protected]. 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,
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34 Werts, M. H. V.; Jukes, R. T. F.; Verhoeven, J. W. The emission spectrum and the 36 Trinquet, E.; Gregor, N.; Degorce, F.; Tardieu, J.-L.; Seguin, P. Introduction of a New
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