Modulation of luminescence intensity of lanthanide complexes by photoinduced electron transfer and its application to a long-lived protease probe

Article abstract of DOI:10.1021/ja060729t

Luminescent lanthanide complexes (Tb³⁺, Eu³⁺, etc.) have excellent properties for biological applications, including extraordinarily long lifetimes and large Stokes shifts. However, there have been few reports of lanthanide-based functional probes, because of the difficulty in designing suitable complexes with a luminescent on/off switch. Here, we have synthesized a series of complexes which consist of three moieties: a lanthanide chelate, an antenna, and a luminescence off/on switch. The antenna is an aromatic ring which absorbs light and transmits its energy to the metal, and the switch is a benzene derivative with a different HOMO level. If the HOMO level is higher than a certain threshold, the complex emits no luminescence at all, which indicates that the lanthanide luminescence can be modulated by photoinduced electron transfer (PeT) from the switch to the sensitizer. This approach to control lanthanide luminescence makes possible the rational design of functional lanthanide complexes, in which the luminescence property is altered by a biological reaction. To exemplify the utility of our approach to the design of lanthanide complexes with a switch, we have developed a novel protease probe, which undergoes a significant change in luminescence intensity upon enzymatic cleavage of the substrate peptide. This probe, combined with time-resolved measurements, was confirmed in model experiments to be useful for the screening of inhibitors, as well as for clinical diagnosis.

Full text of DOI:10.1021/ja060729t

Published on Web 05/06/2006
Modulation of Luminescence Intensity of Lanthanide
Complexes by Photoinduced Electron Transfer and Its
Application to a Long-Lived Protease Probe
Takuya Terai,†,‡ Kazuya Kikuchi,§ Shin-ya Iwasawa,† Takao Kawabe,|
Yasunobu Hirata,| Yasuteru Urano,†, and Tetsuo Nagano*,†,‡
Contribution from the Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, CREST, JST, 4-1-8 Honcho,
Kawaguchi, Saitama, 332-0012, Japan, Department of Material and Life Science, DiVision of
AdVanced Science and Biotechnology, Graduate School of Engineering, Osaka UniVersity,
2-1 Yamada-oka, Suita, Osaka, 565-0871, Japan, Department of Internal Medicine, Graduate
School of Medicine, The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan,
and PRESTO, JST, 4-1-8 Honcho, Kawaguchi-shi, Saitama, 332-0012, Japan
Received January 31, 2006; E-mail: tlong@mol.f.u-tokyo.ac.jp
Abstract: Luminescent lanthanide complexes (Tb3+, Eu3+, etc.) have excellent properties for biological
applications, including extraordinarily long lifetimes and large Stokes shifts. However, there have been few
reports of lanthanide-based functional probes, because of the difficulty in designing suitable complexes
with a luminescent on/off switch. Here, we have synthesized a series of complexes which consist of three
moieties: a lanthanide chelate, an antenna, and a luminescence off/on switch. The antenna is an aromatic
ring which absorbs light and transmits its energy to the metal, and the switch is a benzene derivative with
a different HOMO level. If the HOMO level is higher than a certain threshold, the complex emits no
luminescence at all, which indicates that the lanthanide luminescence can be modulated by photoinduced
electron transfer (PeT) from the switch to the sensitizer. This approach to control lanthanide luminescence
makes possible the rational design of functional lanthanide complexes, in which the luminescence property
is altered by a biological reaction. To exemplify the utility of our approach to the design of lanthanide
complexes with a switch, we have developed a novel protease probe, which undergoes a significant change
in luminescence intensity upon enzymatic cleavage of the substrate peptide. This probe, combined with
time-resolved measurements, was confirmed in model experiments to be useful for the screening of inhibitors,
as well as for clinical diagnosis.
such probes, the targets of which include Ca2+,1 Zn2+,2 NO,3
and enzymes.4 These probes, however, have common disad-
Introduction
Sensitive detection of compounds of interest is one of the
greatest challenges not only in analytical chemistry but also in
biology and medicine. For decades, scientists have been
developing a wide range of sophisticated spectroscopic ap-
proaches to address this issue. Fluorescence is one of the most
widely used techniques in this field, because it offers many
advantages, including sensitivity, simplicity, and robustness.
From the early 1980s,1 functional fluorescence probes have been
developed, which change their fluorescence properties in
response to their reaction with the chemical species of interest.
Since they make possible a sensitive, safe, rapid, quantitative,
and facile detection of the species both in vitro and in vivo,
considerable attention has been devoted to the development of
vantages involving background fluorescence and scattered light.
In the physiological environments such as cytoplasm and serum,
sources of background fluorescence are present everywhere.
Light scattering can also be a difficult problem when a
conventional fluorescence probe, whose Stokes shift is only 20-
30 nm, is used. These factors considerably impair the quality
of measurements, especially when the scale of the assay is small
or the probe concentration is low.
Luminescent lanthanide complexes5 provide an attractive
solution to these problems. If an appropriate chelator is used, a
lanthanide (especially Tb3+ or Eu3+) complex has an extraor-
dinarily long luminescence lifetime of the order of milliseconds,
in contrast to a typical organic compound, which has a short
(2) Minta, A.; Kao, J. P. Y.; Tsien, R. Y. J. Biol. Chem. 1989, 264, 8171-
8178.
† Graduate School of Pharmaceutical Sciences, The University of Tokyo.
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Chem. Soc. 2005, 127, 10197-10204. (b) Gee, K. R.; Zhou, Z. L.; Qian,
W. J.; Kennedy, R. J. Am. Chem. Soc. 2002, 124, 776-778.
(4) Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi,
H.; Hirata, Y.; Nagano, T. Anal. Chem. 1998, 70, 2446-2453.
(5) Mizukami, S.; Kikuchi, K.; Higuchi, T.; Urano, Y.; Mashima, T.; Tsuruo,
T.; Nagano, T. FEBS Lett. 1999, 453, 356-360.
‡ CREST, JST.
§ Osaka University.
| Graduate School of Medicine, The University of Tokyo.
PRESTO, JST.
(1) (a) Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem. 1985, 260,
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9
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Modulation of Lanthanide Complex Luminescence Intensity
A R T I C L E S
Figure 1. Schematic representations of our strategy to control the luminescence of lanthanide complexes. (a) Energy scheme for the luminescence process
in a typical Ln3+ complex. (b) Schematic representation of the relationship between the HOMO energy level and photoinduced electron transfer. (c and d)
Ln3+ complexes with a luminescence off/on switch. (c) Switch “off” model where PeT occurs, resulting in no Ln3+ luminescence. (d) Switch “on” model
where PeT does not occur, resulting in strong Ln3+ luminescence.
fluorescence lifetime in the nanosecond region. Taking advan-
tage of this feature, the influence of short-lived background
fluorescence and scattered light can be reduced to a negligible
level by the method termed time-resolved fluorescence (TRF)
measurement.6 In TRF measurement, the fluorescence signal is
collected for a certain gate time after an appropriate delay time,
following a pulsed excitation. By employing this method, it is
readily possible to distinguish long-lived lanthanide lumines-
cence from other contaminating signals. Furthermore, lanthanide
complexes have large Stokes shifts (>200 nm), which also helps
to reduce the background. Luminescent lanthanide complexes
have been exploited as luminescent tags for TRF measurements
in various fields,7 especially immunoassays8 and high throughput
screenings,9 where both high sensitivity and small assay scale
are essential.
Since the lanthanide f-f transitions have a low absorption
coefficient because of the Laporte selection rule, sensitized
emission is often used to achieve high luminescence.5 Here, a
chromophore incorporated in the ligand (called a sensitizer or
an antenna) absorbs excitation light with a large absorption
coefficient and transfers its energy to the metal by intersystem
crossing, whereby the metal enters the emission state (Figure
1a).10 By choosing an appropriate antenna, it is possible to
obtain a highly emissive lanthanide complex.10a,11
organic fluorophores, however, only a limited number of these
probes have so far been reported, though their targets include
pH,12 cations,13 anions,14 and others.15 The lack of a coherent
strategy for the design of luminescent sensors slows the
development of highly sensitive probes suitable for practical
use.
Photoinduced electron transfer (PeT) is a comprehensively
reported mechanism for excited-state quenching, in which an
electron is transferred from an electron donor moiety to an
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Currently, there is growing interest in lanthanide-based
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Terai et al.
Figure 2. Structures of [Ln-1] to [Ln-12]. Calculated HOMO energy levels of the off/on switch moiety are also shown.
acceptor moiety.16 If the electron donor has a high HOMO level,
the fluorescence of the electron acceptor is quenched (Figure
1b). The PeT mechanism has been examined in detail as a
fluorescence off/on switch for fluorescein derivatives,17 and it
has become possible to rationally develop fluorescein-based
fluorescence probes with finely controlled photophysical proper-
ties.18
In the case of lanthanide complexes, PeT can take place in
several ways: ligand to metal,19 ligand to ligand,12a,15a,20
extramolecular species to metal or ligand,21 and others. Among
them, intramolecular PeT within the ligand moiety should be
most useful in rationally developing a luminescence probe,
because using this kind of PeT as a switch eliminates the need
to incorporate the reactive moiety in the antenna, which is a
great limitation in designing a probe. In other words, it is
possible to optimize the reactive moiety for the target species
independently of the antenna structure. Surprisingly, this sort
of PeT has not been investigated in detail before.
With the aim of establishing a rational and convenient
methodology for the development of long-lived luminescence
probes, we set out to scrutinize the potent role of PeT within
the ligand moiety as a luminescence off/on switch. We have
synthesized a series of complexes, [Ln-X] (Ln ) Eu, Tb) (X
) 1-12), the ligands of which consist of three moieties: a
chelator, an antenna, and a luminescence on/off switch, of which
the latter corresponds to the reactive moiety of the probe. Clear
dependence of lanthanide luminescence on the switch HOMO
level was observed, which shows that the luminescence intensity
of the lanthanide complexes can indeed be controlled by
intramolecular PeT within the ligand.
To demonstrate the feasibility of the PeT-based design of
luminescence probes, a novel luminescence probe, [Ln-13] (Ln
) Tb, Eu), for a protease, microsomal leucine aminopeptidase
(LAP), was developed. This complex drastically changes its
luminescence property in response to the enzymatic activity of
LAP. Applications of the probe for inhibition activity assay,
microplate assay, and clinical diagnosis are described. To our
knowledge, this is the first functional long-lived luminescence
probe for enzymatic activity, controlled by the PeT mechanism.
Results and Discussion
Design and Synthesis of [Ln-X]. To examine the effect of
PeT within the ligand moiety, we designed and synthesized a
series of complexes [Ln-X] (Ln ) Eu, Tb) (X ) 1-12), which
consist of three parts: a Ln3+ chelate, an antenna, and a
luminescence on/off switch (Figure 2). As a chelator, diethyl-
enetriaminepentaacetic acid (DTPA) was chosen. DTPA forms
a highly stable (KML ) 1022.39 and 1022.71 for Eu3+ and Tb3+
,
respectively)22 complex with a Ln3+ ion, with a coordination
number of 8.23
It also prevents solvent water molecules from coupling away
energy from the excited state of the lanthanide ion. As an
antenna, 7-amino-4-methyl-2(1H)-quinolinone (cs124) was se-
lected, because the DTPA-amide of this compound works as
an efficient sensitizer for both Tb3+ and Eu3+ in aerated
water.11a,24 The luminescence on/off switch is a substituted
benzene of appropriate electron density, which was linked to
the methyl group of the antenna to avoid conjugative coupling
of the two aromatic rings. We have synthesized 12 compounds
as described in detail in the Supporting Information, followed
by complexation with Ln3+
.
(16) Callan, J. F.; de Silva, A. P.; Magri, D. C. Tetrahedron 2005, 61, 8551-
8588.
Calculation of HOMO Energy Level. The likelihood of PeT
can be judged from the change in free energy (∆GeT), which is
calculated from the Rehm-Weller eq 1
(17) (a) Ueno, T.; Urano, Y.; Setukinai, K.; Takakusa, H.; Kojima, H.; Kikuchi,
K.; Ohkubo, K.; Fukuzumi, S.; Nagano, T. J. Am. Chem. Soc. 2004, 126,
14079-14085. (b) Miura, T.; Urano, Y.; Tanaka, K.; Nagano, T.; Ohkubo,
K.; Fukuzumi, S. J. Am. Chem. Soc. 2003, 125, 8666-8671.
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T. Tetrahedron 2004, 60, 11067-11073. (b) Urano, Y.; Kamiya, M.; Kanda,
K.; Ueno, T.; Hirose, K.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 4888-
4894. (c) Kamiya, M.; Urano, Y.; Ebata, N.; Yamamoto, M.; Kosuge, J.;
Nagano, T. Angew. Chem., Int. Ed. 2005, 44, 5439-5441.
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769. (b) Aime, S. et al. J. Chem. Soc., Dalton Trans. 1997, 3623-3636.
(c) Dickins, R. S.; Howard, J. A. K.; Maupin, C. L.; Moloney, J. M.; Parker,
D.; Riehl, J. P.; Siligardi, G.; Williams, J. A. G. Chem.sEur. J. 1999, 5,
1095-1105.
∆GeT ) Eox - Ered - ∆E0,0 - wp
(1)
where Eox and Ered are the oxidation and reduction potentials
of the electron donor (on/off switch moiety) and acceptor
(22) Moeller, T.; Thompson, L. C. J. Inorg. Nucl. Chem. 1962, 24, 499-510.
(23) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Chem. ReV. 1999,
99, 2293-2352.
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700-705.
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D. Org. Biomol. Chem. 2005, 3, 1013-1024.
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Modulation of Lanthanide Complex Luminescence Intensity
A R T I C L E S
Figure 3. Spectroscopic properties of [Ln-X]. Plot of luminescence intensity as a function of the calculated HOMO level of the switch moiety in the cases
of (a) Tb3+ and (b) Eu3+ complexes. (c) Absorption and (d) emission spectra of [Tb-8] (green line) and [Tb-1] (black line) 5 µM in aqueous solution (100
mM HEPES buffer, pH 7.3, I ) 0.1 (NaNO3)). Excitation wavelength was 330 nm. The attribution of each band is shown in the figure (see text).
(antenna moiety), ∆E0,0 is the singlet excited energy, and wp is
the work term for the charge separation state.25 If a fixed
acceptor is used, low Eox leads to a negative ∆eT value and
facilitates the electron transfer, as can easily be understood from
eq 1. Hence, the fluorescence intensity of the compound
primarily depends on the oxidation potential of the electron
donor moiety. This parameter can be readily evaluated from
the HOMO energy level of the donor moiety, as shown for
fluorescein derivatives.18a,b
Our working hypothesis was that the strategy used in
fluorescein derivatives would also be applicable to the synthe-
sized lanthanide complexes [Ln-X]. We anticipated that,
provided that the HOMO level of the switch moiety is high
enough to induce PeT to the antenna, the singlet excited state
of the antenna would be quenched, resulting in no luminescence
from Ln3+ (Figure 1c). In contrast, a complex with a switch
having a low HOMO level would not suffer from this quenching
mechanism and would emit strong luminescence (Figure 1d).
Bearing this in mind, the HOMO energy level of the
luminescence on/off switch of 1-12 was calculated by the DFT
method using the B3LYP/6-31G(d) basis set. The results are
shown in Figure 2. The values cover a wide range from -4.98
eV of 1 to -7.77 eV of 12.
Photochemical Properties of [Ln-X]. For each synthesized
complex, the quantum yield of luminescence in neutral aqueous
buffer was determined and plotted against the calculated HOMO
energy level of the switch moiety (Figure 3). As expected, a
clear dependence of quantum yield on the HOMO level was
observed for both Tb3+ and Eu3+ complexes. The threshold level
of luminescence on and off switching was shown to be around
-5.8 eV for Tb and -5.5 eV for Eu. Only the complexes with
a HOMO level of the switch moiety below the threshold have
strong luminescence, and their quantum yields are similar to
those of the complexes without the switch moiety (12.1% and
4.3% for Tb3+ and Eu3+ complexes, respectively). The slight
difference of the threshold level between Tb3+ and Eu3+ is
probably due to the effect of the metal on the π-system of the
antenna moiety. Such a phenomenon has been suggested before
in a complex in which the antenna directly binds to the
lanthanide ion.12a Considering that the 7-amide group of cs124-
DTPA-Ln is known to participate in metal binding,24 our result
is not surprising.
To scrutinize the mechanism of quenching, the luminescence
and chemical properties of [Ln-X] were investigated. For
clarity, the absorption and emission spectra of [Tb-8] (PeT
off state) and [Tb-1] (PeT on state) are shown in Figure 3c
and 3d, respectively. The UV-vis absorption spectra (over
(25) Rehm, D.; Weller, A. Isr. J. Chem. 1970, 8, 259-271.
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Terai et al.
Figure 4. Schematic representation of the probe. Ln ) Tb, Eu.
310 nm) did not change among the complexes, showing that
the ground state of the antenna is not affected by the switch
moiety. For [Tb-8], short-lived emission of the antenna moiety
(labeled as fl. in Figure 3d) was detected at around 370 nm, as
well as four luminescence bands of Tb3+ at 490, 546, 583, and
621 nm. These bands correspond to the transition from the 5D4
excited state to the 7F6, 7F5, 7F4, and 7F3 ground state,
respectively. No emission, however, was observed from [Tb-
1], including the fluorescence of the antenna at around 370 nm.
In the case of Eu3+ complexes, essentially identical results were
obtained, except that the observed transition from the Eu3+
excited state occurred from 5D0 to 7FJ (J ) 0, 1, 2, 3, 4) (Figure
S1). This quenching of antenna fluorescence, in cooperation with
the luminescence from Ln3+, indicates a presence of a quenching
pathway, such as PeT, to the singlet excited state of the antenna
moiety.
In other words, we can screen and optimize the structure of the
probe in silico. Compared with the previously exploited trial
and error approach, this rational method should have many
advantages, including savings of cost and time.
Design and Photochemical Properties of an LAP Probe.
Based on the above findings, we set out to develop a protease
probe based on PeT. A protease is an enzyme which cleaves a
specific peptide bond of a substrate peptide or protein. There
are enormous numbers of enzymes in this category,28 many of
which are related to various diseases.29 Although several
protease substrates for chromogenic and fluorogenic assays have
been reported,30 none of them has a long-lived luminescence
except for a few31 based on RET (resonance energy transfer).
In contrast to RET-based probes, which require two chro-
mophores at appropriate sites, probes using PeT should provide
simple and robust assays and should also be applicable to
exopeptidases.
Among the proteases, microsomal leucine aminopeptidase
(LAP) was selected as the target enzyme. LAP (EC 3.4.11.2,
also known as aminopeptidase M or aminopeptidase N) is a
widespread exopeptidase, which removes N-terminal amino
acids (preferably leucine and alanine) from almost all unsub-
stituted oligopeptides.28 It is well-known that LAP plays
important roles in tumor-cell invasion,32 tumor metastasis,33
and maturation of MHC class I epitopes.34 Although chromo-
genic and fluorogenic substrates, L-pNA35 and L-MCA,36
Other parameters listed in Table S1 (Tb3+ complexes) and
S2 (Eu3+ complexes) are also consistent with the idea that the
luminescence of [Ln-X] is controlled by PeT from the off/on
switch moiety to the antenna singlet state. The triplet energy
level of the antenna, calculated from the phosphorescence
spectra measured in MeOH/EtOH ) 1:1 at 77 K did not change
at all, which excludes the possibility that the quenching of
luminescence is due to a change of the nature of the antenna
triplet state. In addition, either in H2O or in D2O, the
luminescence lifetimes of the complexes with different switches
did not vary, demonstrating that the excited state of the
lanthanide ion remains intact. It is well-known that the O-H
oscillator of solvent coordinated to the metal center can quench
(28) Barrett, A. J.; Rawlings, N. D.; Woessner, J. F. Handbook of Proteolytic
Enzymes, 2nd ed.; Elsevier Academic Press: Amsterdam, The Netherlands,
2004.
(29) (a) Southan, C. Drug DiscoV. Today 2001, 6, 681-688. (b) Artal-Sanz,
M.; Tavernarakis, N. FEBS Lett. 2005, 579, 3287-3296. (c) Singh, R. B.;
Dandekar, S. P.; Elimban, V.; Gupta, S. K.; Dhalla, N. S. Mol. Cell.
Biochem. 2004, 263, 241-256.
(30) (a) Erlanger, B. F.; Kokowsky, N.; Cohen, W. Arch. Biochem. Biophys.
1961, 95, 271-278. (b) Zimmerman, M.; Yurewicz, E.; Patel, G. Anal.
Biochem. 1976, 70, 258-262. (c) Leytus, S. P.; Patterson, W. L.; Mangel,
W. F. Biochem. J. 1983, 215, 253-260.
the luminescence from Ln3+ 26,27
For every complex, however,
.
the number of coordinated water molecules (q values) at the
metal center was estimated to be approximately 1, using the
equation proposed by Parker et al.27 This shows that the off/on
switch does not change this nonradiative decay of Ln3+ by
bound water molecules.
Figure 3a and 3b provide us with highly significant informa-
tion for designing a luminescence probe. To develop a practical
probe with a large change of luminescence in response to the
target, what we need to do is to find a reactive moiety whose
HOMO level is changed across the threshold by the reaction.
(31) (a) Karvinen, J.; Elomaa, A.; Ma¨kinen, M.-L.; Hakala, H.; Mukkala, V.-
M.; Peuralahti, J.; Hurskainen, P.; Hovinen, J.; Hemmila¨, I. Anal. Biochem.
2004, 325, 317-325.(b) Karvinen, J.; Laitala, V.; Ma¨kinen, M.-L.; Mulari,
O.; Tamminen, J.; Hermonen, J.; Hurskainen, P.; Hemmila¨, I. Anal. Chem.
2004, 76, 1429-1436.
(32) Saiki, I.; Fujii, H.; Yoneda, J.; Abe, F.; Nakajima, M.; Tsuruo, T.; Azuma,
I. Int. J. Cancer 1993, 54, 137-143.
(33) Fujii, H.; Nakajima, M.; Saiki, I.; Yoneda, J.; Azuma, I.; Tsuruo, T. Clin.
Exp. Metastasis 1995, 13, 337-344.
(34) Saric, T.; Chang, S.-C.; Hattori, A.; York, I. A.; Markant, S.; Rock, K. L.;
Tsujimoto, M.; Goldberg, A. L. Nat. Immunol. 2002, 3, 1169-1176.
(35) Tuppy, H.; Wiesbauer, U.; Wintersberger, E. Hoppe-Seyler’s Z. Physiol.
Chem. 1962, 329, 278-288.
(26) Horrocks, W. DeW., Jr.; Sudnick, D. R. Acc. Chem. Res. 1981, 14, 384-
392.
(27) Beeby, A.; Clarkson, I. M.; Dickins, R. S.; Faulkner, S.; Parker, D.; Royle,
L.; de Sousa, A. S.; Williams, J. A. G.; Woods, M. J. Chem. Soc., Perkin
Trans. 2 1999, 493-503.
(36) Kanaoka, Y.; Takahashi, T.; Nakayama, H. Chem. Pharm. Bull. 1977, 25,
362-363.
(37) Melhuish, W. H. J. Phys. Chem. 1961, 65, 229-235.
9
6942 J. AM. CHEM. SOC. VOL. 128, NO. 21, 2006

Modulation of Lanthanide Complex Luminescence Intensity
A R T I C L E S
Table 1. Photochemical Properties of the Probes
One possible application of a spectroscopic probe for a
protease is to evaluate inhibitory activities of candidate com-
pounds. Indeed, protease inhibitors are widely used as thera-
peutic drugs.39 In particular, LAP inhibitors are suggested to
have antitumor angiogenesis activity.39b Thus, we have inves-
tigated the applicability of [Tb-13] for the assessment of
inhibitory activity. Several compounds are known to inhibit the
activity of LAP, including actinonin,40 amastatin,41 and besta-
tin.42 The inhibitory activities of these three inhibitors were
probed with L-pNA, L-MCA, and [Tb-13], respectively, and
the inhibition constants Ki were determined on the assumption
that these inhibitors were noncompetitive (Table S4).43 Similar
results were obtained regardless of the probes used.
TRF Assay on Microplate. Microplates (96-well, 384-well,
etc.) are an indispensable tool for biological and environmental
assays. They can handle a large number of samples simulta-
neously with a small sample volume (microliter order) and are
suitable for automated assay systems. They are, however, more
likely to suffer from autofluorescence of the device and scattered
light. Therefore, TRF measurements with low background
signals are expected to be highly advantageous compared with
the use of organic fluorescent compounds.9a,44
After confirming that LAP activity on a 96-well plate could
be monitored with 2.5 µM [Tb-13] by TRF measurement
(Figure S5), we conducted experiments with lower concentra-
tions of [Tb-13], such as at nanomolar level. The experiments
were repeated using L-MCA to compare the results. At 50 nM,
[Tb-13] could clearly detect the presence of inhibitor (P <
0.001 by Student’s t-test), while L-MCA failed to show a
statistically significant difference of fluorescence intensity (P
> 0.05) between with and without inhibitor, due to the large
background signal (Figure 6).
We also performed experiments with smaller amounts of
LAP. With [Tb-13], as little as 1 µU of LAP could be detected
(Figure S6a) in a concentration-dependent manner. With L-
MCA, however, no increase of fluorescence intensity was
observed when the amount of LAP was lower than 50 µU,
presumably due to the influence of the background signal
(Figure S6b).
a
absmax
(nm)
emmax
(nm)
Φlum
(%)
τH
(ms)
τD
O
2
(ms)
O
2
ꢀ
qb
[Tb-13]
[Tb-1]
[Eu-13]
[Eu-1]
330
330
330
330
13 000
13 000
13 000
13 000
547
547
614
614
6.8
0.01
4.8
1.12
n.d.c
0.62
n.d.c
1.70
n.d.c
2.10
n.d.c
1.20
n.d.c
0.96
n.d.c
0.01
a Quantum yields were determined using quinine sulfate (Φ ) 0.546 in
1 N H2SO4)37 for Tb and [Ru(bpy)3]Cl2 (Φ ) 0.028 in air-equilibrated
water)38 for Eu complexes as standards. Measurements were performed in
100 mM HEPES buffer (pH 7.4) at 25 °C. bq values were estimated using
-1
-1
-1
- 0.06) (for Tb) and q ) 1.2(τ
H2O
the equation q ) 5(τ
- τ
H2O
D2O
-1
- τD O - 0.25 - 0.075) (for Eu).27 c Not determined.
2
respectively, have been reported, there is so far no substrate
suitable for TRF measurement.
The design of the probe is shown in Figure 4. The substrate
peptide sequence of LAP, L-Leu, was attached through a peptide
bond to the amino group of the luminescence off/on switch
moiety of [Ln-1] (for detailed synthetic procedure, see Sup-
porting Information). The synthesized compound, [Ln-13], was
expected to have strong luminescence, because the HOMO
energy level of its switch moiety (-5.89 eV) was similar to
that of [Ln-6]. After the cleavage of the peptide by LAP, [Ln-
13] would be converted to [Ln-1], whose luminescence would
be quenched by PeT.
The photochemical properties of [Ln-13], compared with
those of [Ln-1], were summarized in Table 1. As expected,
the parameters do not differ from each other except for the
quantum yields of luminescence. The quantum yields of [Ln-
13] were close to those of [Ln-6], indicating the accuracy of
the prediction from the calculated HOMO energy level. For both
Tb3+ and Eu3+, the luminescence intensity of the complexes
before and after the LAP-mediated reaction differed by ap-
proximately 500-fold, indicating that this probe is potentially
useful for the sensitive detection of LAP activity. Because the
quantum yield of [Tb-13] is higher than that of the corre-
sponding Eu3+ complex, most of the following experiments were
performed with [Tb-13].
Enzymatic Reaction and Assessment of Inhibitory Activ-
ity. When the probe was reacted with LAP, the emission from
Ln3+, as well as that from the antenna, decreased in a time-
dependent manner (Figure 5) until complete quenching was
achieved (Figure S2). This decrease was not observed if the
enzyme was absent or if the [Ln-13] complex was replaced
with [Ln-5] (Figure S3). In addition, other proteases, such as
trypsin, chymotrypsin, elastase, caspase-3, and cathepsin B, did
not affect the luminescence of the probe (for trypsin, data are
shown in Figure S3), which shows that [Ln-13] is a specific
probe for LAP activity. The conversion of [Tb-13] to [Tb-1]
was confirmed by analytical HPLC (Figure S4).
Although some influence of the apparatus cannot be excluded,
these results suggest that TRF measurements with [Tb-13] offer
higher reproducibility and sensitivity than conventional fluo-
rescence assays in a system with a high background signal level,
such as microplates.
Application for Clinical Diagnosis. LAP is a clinically
significant enzyme, and elevated levels of LAP activity in serum
are associated with diseases of the liver, bile duct, and
pancreas.45 As LAP activity in serum renders valuable informa-
tion for clinical diagnosis, it is often quantified in hospitals. To
(39) (a) Leung, D.; Abbenante, G.; Fairlie, D. P. J. Med. Chem. 2000, 43, 305-
341.(b) Aozuka, Y.; Koizumi, K.; Saitoh, Y.; Ueda, Y.; Sakurai, H.; Saiki,
I. Cancer Lett. 2004, 216, 35-42.
Kinetic parameters of [Tb-13] were determined and com-
pared with those of commercially available substrates, L-pNA
and L-MCA (Table S3). The Km and kcat of [Tb-13] were found
to be 4.18 × 105 M and 29.3 s-1, respectively. The kcat/Km value
was 7.01 × 105 (M-1 s-1), virtually identical with those of
L-pNA and L-MCA (7.17 × 105 and 6.13 × 105 M-1 s-1). This
result indicates that the reaction rates are almost the same, when
the probe concentration is low.
(40) Umezawa, H.; Aoyagi, T.; Tnanaka, T.; Suda, H.; Okuyama, A.; Naganawa,
H.; Hamada, M.; Takeuchi, T. J. Antibiotics 1985, 38, 1629-1630.
(41) Aoyagi, T.; Tobe, H.; Kojima, F.; Hamada, M.; Takeuchi, T.; Umezawa,
H. J. Antibiotics 1978, 31, 636-638.
(42) Umezawa, H.; Aoyagi, T.; Suda, H.; Hamada, M.; Takeuchi, T. J. Antibiotics
1976, 29, 97-99.
(43) Barclay, R. K.; Phillips, M. A. Biochem. Biophys. Res. Commun. 1980,
96, 1732-1738.
(44) Gudgin Dickson, E. F.; Pollak, A.; Diamandis, E. P. J. Photochem.
Photobiol., B 1995, 27, 3-19.
(45) (a) Szasz, G. Am. J. Clin. Path. 1967, 47, 607-613. (b) Shay, H.; Sun, D.
C. H.; Siplet, H. Am. J. Digest. Dis. 1960, 5, 217-232. (c) Pineda, E. P.;
Goldbarg, J. A.; Banks, B. M.; Rutenburg, A. M. Gastroenterology 1960,
38, 698-712.
(38) Nakamaru, K. Bull. Chem. Soc. Jpn. 1982, 55, 2697-2705.
9
J. AM. CHEM. SOC. VOL. 128, NO. 21, 2006 6943

A R T I C L E S
Terai et al.
Figure 5. Reaction of [Ln-13] with LAP. Emission spectra of 2 µM (a) [Tb-13] and (b) [Eu-13] at 0, 5, 10, 15, 20, 30, and 60 min after the addition
of LAP (0.01 U). The reaction was performed in 100 mM Tris-HCl buffer (pH 7.4) at 37 °C. The excitation wavelength was 330 nm.
Figure 6. Microplate assay of LAP (0.002 U) with 50 nM (a) [Tb-13] (TRF measurement) and (b) L-MCA. Amastatin (10 µM) was added as an inhibitor.
Fluorescence intensity (ex./em. ) 325/545 nm for (a) and 380/440 nm for (b)) was recorded 60 min after the initiation of the reaction. Data are shown as
mean ( SEM (n ) 5). *** indicates P < 0.001.
examine the feasibility of applying [Tb-13] for clinical diag-
nosis, we conducted experiments using human sera. This is a
challenging experimental condition, since serum contains hun-
dreds of biological compounds and ions. It absorbs more than
90% of excitation light (at 330 nm), emitting a strong
background fluorescence. Using TRF measurements, LAP
activities in sera were quantified in both normal volunteers and
patients with cancer (5 persons each). As can be seen in Figure
7, LAP activity was 1.9-fold higher in sera from patients than
in sera from normal persons (P < 0.01 by Student’s t-test). The
results obtained with this method were shown to have a high
linear correlation (r ) 0.94) with commercially performed
measurements of the same samples, conducted with a chro-
mogenic substrate (Figure S7). Moreover, the coefficient of
variation (CV) for normal patients was 17.7, about half that of
the chromogenic method (30.0). The luminescence intensity of
[Tb-13] did not change at all when amastatin, a known inhibitor
of LAP, was added to the serum, which confirmed that [Tb-
13] specifically measured the LAP activity of human serum
(Figure S8).
Figure 7. Difference of LAP activity in human sera between normal
subjects and patients with cancer. Assay was performed in 10% serum with
1 µM [Tb-13]. Luminescence intensity (ex./em. ) 330/545 nm) was
measured for the initial 10 min using TRF measurement and converted to
enzyme activity. In this case, 1 U was defined as the amount of protein
which is required to hydrolyze 1 µmol of [Tb-13] per min at 37 °C, pH
7.4. Data are shown as mean ( SEM (n ) 5). ** indicates a significant
difference, P < 0.01.
Compared with colorimetric assays, a fluorescence assay has
many advantages. For instance, whereas fluorescence assays can
be performed at submicromolar concentrations of the probes,
colorimetric assays require millimolar levels. Another advantage
is that simultaneous detection of different targets is possible
using multiple fluorescent probes with different excitation or
emission wavelengths. As one of the major disadvantages, i.e.,
background fluorescence, can be eliminated by means of TRF
9
6944 J. AM. CHEM. SOC. VOL. 128, NO. 21, 2006

Modulation of Lanthanide Complex Luminescence Intensity
A R T I C L E S
purchased from SIGMA (St. Louis, MO). Leu-pNA was purchased from
Peptide Institute, Inc. (Osaka, Japan). Leu-MCA was purchased from
Bachem AG (Bubendorf, Switzerland). Silica gel column chromatog-
raphy was performed using silica gel 60N (Kanto Kagaku Co., Ltd.,
(Tokyo, Japan)). ODS column chromatography was performed using
Chromatorex-ODS (Fuji Silysia Chemical Ltd., Japan).
measurements (see previous section), it is expected that TRF
assays using lanthanide complexes such as [Tb-13] will be of
great practical value.
Conclusions
We have designed and synthesized a series of lanthanide
complexes, each consisting of three moieties: a lanthanide
chelate, a luminescence off/on switch, and an antenna. As we
had anticipated, quenching of lanthanide luminescence was
observed as the HOMO energy level of the switch moiety
became higher. By careful examination of the photochemical
properties of these complexes, we demonstrated that the
quenching of the complexes observed here is due to intramo-
lecular PeT from the switch moiety to the antenna. This is the
first report in which the effectiveness of intramolecular PeT as
a mechanism for luminescence off/on switch has been studied.
The luminescence off/on threshold for both Tb3+ and Eu3+
complexes was determined for the first time; this is critical
information for the design of luminescence probes. These
findings are an important step in establishing a rational method
for the development of luminescence-based chemosensors.
By applying this novel approach of calculating the HOMO
level of the switch moiety, we developed a luminescence probe
for LAP, [Ln-13]. It was confirmed to be a sensitive probe
for monitoring LAP activity on microplates, where the high
background fluorescence could be eliminated by TRF measure-
ment. [Ln-13] was also shown to be potentially useful in
clinical diagnosis. It is noteworthy that the peptide sequence of
[Tb-13] can be changed as desired, so that one can easily
develop a luminescence probe for any desired protease.
The greatest advantage of our PeT-based approach is that
the luminescence intensity of the complex can be predicted to
a large extent by calculating the HOMO energy level of the
switch moiety. The protease probe described here is not the
only application of this approach. By selecting appropriate
switch moieties, it can in principle be applied to every target
species, including metal ions, enzymes, ROS, and so on.
Recently, a few groups have reported time-resolved lumi-
nescence imaging of lanthanide complexes.46 For instance, we
have developed a luminescence microscopy system which can
obtain time-resolved images of cells, and we successfully
visualized intracellular Zn2+ with virtually no background.47 The
development of rationally designed probes for molecules of
interest, in combination with these imaging systems, will open
a new frontier in fluorescence imaging.
1
Instruments. H NMR and 13C NMR spectra were recorded on a
JEOL JNM-LA300. Mass spectra were recorded on a JEOL JMS-
SX102A mass spectrometer (EI+), a JEOL JMS-700 mass spectrometer
(FAB+), or a JEOL JMS-T100LC (ESI+, ESI-). Preparative HPLC
purification was performed on a reversed-phase ODS column (GL
Sciences (Tokyo, Japan), Inertsil Prep-ODS 30 mm × 250 mm) fitted
on a JASCO PU-1587 HPLC system. UV-visible spectra were obtained
on a Shimadzu UV-1600 (Tokyo, Japan). Fluorescence and phospho-
rescence spectroscopic studies were performed with a Hitachi F4500
(Tokyo, Japan). Time-resolved luminescence spectra and lanthanide
luminescence lifetimes were obtained using a Perkin-Elmer LS-55
(Beaconsfield, Buckinghamshire, England). Luminescence assays on
96-well plates were performed using a plate reader instrument coupled
with a Perkin-Elmer LS-55.
UV-visible Absorption Spectral Measurements. Absorption
spectra of the complexes were measured at 5 µM in 100 mM HEPES
buffer (pH 7.3, I ) 0.1 (NaNO3)). Each sample contained less than
0.1% DMSO as a cosolvent. Measurements were performed in 1 cm
quartz cells at 25 °C.
Luminescence Spectral Measurements. Luminescence spectra of
the complexes without a delay time were measured at 5 µM in 100
mM HEPES buffer (pH 7.3, I ) 0.1 (NaNO3)), following excitation at
330 nm. Each sample contained less than 0.1% DMSO as a cosolvent.
Measurements were performed in 1 cm quartz cells at 25 °C. The
excitation slit width was 2.5 nm (for Tb3+ complexes) or 5 nm (for
Eu3+ complexes). The emission slit width was 2.5 nm. The photomul-
tiplier voltage was 950 V.
Time-Delayed Luminescence Spectral Measurements. Time-
delayed luminescence spectra of [Ln-13] (Ln ) Tb, Eu) (5 µM) were
measured in 100 mM HEPES buffer (pH 7.3, I ) 0.1 (NaNO3)), with
a delay time of 50 µs and a gate time of 2.0 ms, after a 10 µs pulsed
excitation at 330 nm. Each sample contained less than 0.1% DMSO as
a cosolvent. Measurements were performed in 1 cm quartz cells at 25
°C. The excitation slit width was 2.5 nm (for Tb3+ complexes) or 5
nm (for Eu3+ complexes). The emission slit width was 2.5 nm.
Quantum Yield Measurements. Quantum yields of the complexes
were estimated by a relative method with reference to a luminescence
standard. For Tb3+ complexes, quinine sulfate (Φ ) 0.546 in 1 N H2-
SO4)37 was used as a standard. For Eu3+, [Ru(bpy)3]Cl2 (Φ ) 0.028 in
air-equilibrated water)38 was used. The quantum yields were calculated
by eq 2,
2
Φx/Φst ) [Ast/Ax][nx /nst2][Dx/Dst]
(2)
Experimental Section
where Φ is the quantum yield (subscript “st” stands for the reference,
and “x”, for the sample), A is the absorbance at the excitation
wavelength, n is the refractive index (if both spectra are obtained in
the aqueous solution, this term is canceled), and D is the area under
the luminescence spectra on an energy scale. The sample and the
reference were excited at the same wavelength (330 nm), where the
absorbance was kept lower than 0.07. The luminescence spectra were
measured at 25 °C in 100 mM HEPES buffer (pH 7.3, I ) 0.1 (NaNO3))
containing less than 0.1% DMSO as a cosolvent. All spectra were
obtained with a Hitachi F4500 spectrofluorometer.
Materials. Boc-L-Leu-OH, HBTU, and HOBT‚H2O were obtained
from Watanabe Chemical Industries, Ltd. (Japan). All other reagents
for organic synthesis were purchased from Tokyo Kasei Co., Ltd.
(Tokyo, Japan) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Microsomal LAP () aminopeptidase M) was purchased from Pierce
Biochemistry, Inc. (Rockford, IL). Unless otherwise stated, 1 U is
defined as the amount of protein which is required to hydrolyze 1 µmol
of L-pNA per min at 25 °C, pH 7.2. Trypsin (from bovine pancreas),
actiononin, amastatin hydrochloride, and bestatin hydrochloride were
(46) (a) Faulkner, S.; Pope, S. J. A.; Burton-Pye, B. P. Appl. Spectrosc. ReV.
2005, 40, 1-31. (b) Weibel, N.; Charbonnie`re, L. J.; Guardigli, M.; Roda,
A.; Ziessel, R. J. Am. Chem. Soc. 2004, 126, 4888-4896. (c) Charbonnie`re,
L.; Ziessel, R.; Guardigli, M.; Roda, A.; Sabbatini, N.; Cesario, M. J. Am.
Chem. Soc. 2001, 123, 2436-2437.
(47) Hanaoka, K.; Kikuchi, K.; Kobayashi, S.; Nagano, T. Manuscript in
preparation.
Estimation of the Lowest Triplet Energy Level. Lowest triplet
energy levels (T1) were calculated from the wavelength of the first
peak of phosphorescence spectra. Phosphorescence spectra of [Gd-
X] (X ) 1-12) (20 µM) were measured at 77 K in MeOH/EtOH )
1:1, following excitation at 330 nm. Experiments were performed with
9
J. AM. CHEM. SOC. VOL. 128, NO. 21, 2006 6945

A R T I C L E S
Terai et al.
a Hitachi F4500 equipped with a low-temperature phosphorescence
measurement unit.
Inertsil 3 ODS column (4.6 mm × 250 mm, GL Sciences, Japan), fitted
on a JASCO PU-980 HPLC system. Conditions: linear gradient from
15% to 45% solvent B (solvent A, 0.1 M triethylammonium acetate
(pH 7.4); solvent B, 80% acetonitrile/20% 0.1 M triethylammonium
acetate (pH 7.4)).
Luminescence Lifetime Measurements. Luminescence lifetimes
were measured in both H2O (100 mM HEPES buffer, pH 7.4) and D2O
(100 mM HEPES buffer, pD 7.4). The luminescence intensity (ex. 330
nm, em. 545 nm (Tb3+ complexes) or 615 nm (Eu3+ complexes)) at
every 10 µs after pulsed excitation was measured using a 10 µs gate
time. Data were fitted to a single-exponential decay curve (eq 3), where
I and I0 are the luminescence intensity at time t and time 0, respectively,
and τ is the luminescence lifetime.
LAP Assay on 96-Well Plates. Experiments were performed on
PLL-coated black plates (clear bottom) (Iwaki, Japan) at room
temperature. First, LAP was added to 100 mM Tris-HCl buffer (pH
7.4). For wells with inhibitor, amastatin (10 µM) was added at the same
time. After 10 min, [Tb-13] was added to the wells, the total volume
of which was 0.2 mL. The plate was incubated for 60 to 90 min, and
then the luminescence intensity (ex. 325 nm, em. 545 nm) was measured
with delay and gate times of 50 µs and 2.0 ms, respectively. The
excitation and emission slits were 10 nm. An assay using Leu-MCA
was performed with the same procedure except for the measured
wavelength (ex. 380 nm, em. 440 nm) and without a delay time.
I ) I0 exp(-t/τ)
(3)
Enzymatic Reaction with LAP. All reactions were performed in a
quartz cell at 37 °C. LAP (0.01 U) was dissolved in 100 mM Tris-HCl
buffer (pH 7.4) and incubated for 5 min, before 2 µM [Ln-13] (Ln )
Tb, Eu) was added. The reaction volume was 3.0 mL, and the solution
contained less than 0.1% DMSO as a cosolvent. Luminescence spectra
were measured at every 5 min after the addition of the probe without
a delay time. Control experiments were performed by replacing LAP
or [Ln-13] with other reagents.
Estimation of Inhibitory Activity. Various amounts of inhibitors
were added to 100 mM Tris-HCl buffer (pH 7.4) along with LAP (0.01
U), and the mixtures were incubated for 5 min at 37 °C, followed by
the addition of [Tb-13] (2 µM). The total reaction volume was 3.0
mL, and the solution contained less than 0.1% DMSO as a cosolvent.
Luminescence intensity (ex. 330 nm, em. 545 nm) was measured
continuously up to 5 min at 37 °C. The initial velocity of the reaction
was calculated from the change of intensity during the first 3 min, where
the luminescence decreased in a linear fashion. Inhibitory activity (%)
was determined by comparing the velocity in the presence and absence
of the inhibitor. Studies using Leu-MCA were performed with the same
procedure except for the measured wavelength (ex. 380 nm, em. 440
nm). For Leu-pNA, probe concentration was 20 µM, and the absorbance
at 405 nm was recorded. The inhibition constant (Ki) was calculated
from the following equation (eq 4), where V ) reaction velocity in the
presence of inhibitor, V0 ) reaction velocity in the absence of inhibitor,
and [I] ) inhibitor concentration.
Assay of Human Serum (Using Tb Complex). Human sera from
5 healthy volunteers (normal) and 5 patients with cancer of the hepato-
biliary-pancreatic systems (patients) were diluted 10 times with Tris-
HCl buffer (0.1 M, pH 7.4). Then 1 µM [Tb-13] was added to the
solution, and a TRF measurement was performed as described above
at 37 °C. Enzymatic activity was calculated from the change of
luminescence over the first 10 min. 1 U was defined as the enzymatic
activity that cleaved 1 µmol of [Tb-13] per min under the assay
conditions.
Assay of Human Serum (Using a Commercially Performed
Method). The following experiments were performed by SRL, Inc.
(Tokyo, Japan). Briefly, the substrate l-leucyl-3,5-dibromo-4-hydroxya-
nilide (L-Leu-DBHA) was converted by LAP in the sample to 3,5-
dibromo-4-hydroxyanilide, which was coupled to N-ethyl-N-(2-hydroxy-
3-sulfopropyl)-m-toluidine (TOOS) by another enzyme, bilirubin
oxidase (BOD), to yield a green pigment. The absorbance of the pigment
at 700 nm was recorded by a Hitachi 7170 autoanalyzer as a measure
of the LAP activity of the sample. Experiments were performed at 37
°C.
Supporting Information Available: Synthetic procedures of
[Ln-X] (Ln ) Tb, Eu) (X ) 1-13); photophysical properties
of the complexes; comparison of kinetic parameters; inhibition
constants of LAP inhibitors; emission spectra of [Eu-8] and
[Eu-1]; comparison of luminescence intensity before and after
the enzymatic reaction; spectral change of [Ln-13] upon
reaction with LAP; emission intensity of [Ln-X] after the
addition of LAP or trypsin; HPLC chart of [Ln-X]; LAP
reaction of [Tb-13] on 96-well plate; limit of detection of LAP
using two probes; correlation between LAP activity measured
by the two methods; serum LAP assay in the presence of
amastatin; list of authors of ref 19b. This material is available
free of charge via the Internet at http://pubs.acs.org.
V
V0
1
)
(4)
1 + [I]/Ki
Determination of Kinetic Parameters. Various concentrations of
the probes () Leu-pNA, Leu-MCA, [Tb-13]) were dissolved in 100
mM Tris-HCl buffer (pH 7.4). LAP (0.001 U/ml, final concentration)
was added to the solution, and the luminescence intensity (or absor-
bance) was recorded continuously as described above. The initial
reaction velocity was calculated, plotted against probe concentration,
and fitted to a Michaelis-Menten curve. Km and kcat were determined
by the least-squares method.
Analytical HPLC. To confirm that enzymatic cleavage of the leucine
moiety had taken place, analytical RP-HPLC was performed using an
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6946 J. AM. CHEM. SOC. VOL. 128, NO. 21, 2006