Lanthanide-based protease activity sensors for time-resolved fluorescence measurements

Article abstract of DOI:10.1021/ja800322b

Lanthanide-based luminescent sensors are noteworthy because their long-lifetime luminescence enables time-resolved fluorescence measurements. After exploring suitable antenna groups, we designed and synthesized lanthanide-based luminogenic sensors detecti

Full text of DOI:10.1021/ja800322b

Published on Web 10/08/2008
Lanthanide-Based Protease Activity Sensors for Time-Resolved Fluorescence
Measurements
Shin Mizukami, Kazuhiro Tonai, Masahiro Kaneko, and Kazuya Kikuchi*
DiVision of AdVanced Science and Biotechnology, Graduate School of Engineering, Osaka UniVersity,
2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Received January 15, 2008; E-mail: kkikuchi@mls.eng.osaka-u.ac.jp
Time-resolved fluorescence measurements with long-lifetime
luminescent lanthanide complexes1 have attracted great attention
from scientists. This is because the time-resolved luminescence
signals are highly sensitive and are scarcely affected by other
fluorescent compounds that coexist in the sample, which often
interfere with steady-state fluorescence measurements. This method
is quite useful, especially in high-throughput drug screening.2
Protease inhibitors can be important drug targets for diverse
diseases, including cancer, AIDS, inflammatory disorders, and so
on. Therefore, lanthanide-based luminescent sensors that detect
protease activities assume great significance.
Figure 1. (a) Structures of synthesized compounds; (b) emission spectra
of [1-Tb3+] and [2-Tb3+]; λex ) 250 nm.
Scheme 1. Structures and Schematic Representation of the
Probes for Detecting Protease Activity
Karvinen et al. reported a protease assay based on time-resolved
fluorescence resonance energy transfer (TR-FRET).3 However, the
synthesis of TR-FRET probes is generally laborious because it
involves the attachment of both the luminescent lanthanide complex
and the appropriate quencher on the substrate peptides. Additionally,
when the probe concentration is high, this system is associated with
the risk of diffusion-enhanced intermolecular FRET.4 Although
another simpler assay based on quenching by photoinduced electron
transfer is known, the luminescent substrate becomes nonlumines-
cent after enzyme reaction in this case.5 In principle, such
quenching-type fluorescent probes are inferior to fluorogenic probes,
which fluoresce after enzyme reaction, because fluorescence is
generally quenched by several factors such as collisional quenching,
energy transfer, electron transfer, and so on. Besides TR-FRET
probes, there exist no other fluorogenic protease probes based on
lanthanide luminescence. Therefore, fluorogenic probes such as
substrate peptides attached with the short-lifetime fluorophore MCA
(4-methylcoumarinyl-7-amide) are widely used in protease assays,
despite the above-described limitations.6 We here report simple-
structure luminogenic lanthanide probes that detect protease
activities.
The long-lifetime luminescence of lanthanide ions is due to the
forbidden f-f transitions of metal electrons. Generally, energy
transfer from an adjacent chromophore to a lanthanide ion is utilized
for the efficient excitation of lanthanide ions. The structures of the
antenna chromophores regulate the lanthanide luminescence inten-
sity.7 Thus, the antenna groups can be strategic targets for designing
luminogenic lanthanide probes. By modifying the antenna structures,
several kinds of lanthanide-based probes have been developed so
far, for instance, for detecting pH,8 metal ions,9 and other
molecules.10 To develop luminogenic lanthanide probes for detect-
ing protease activities, the candidate antenna groups should fulfill
two requirements: (1) antenna groups with an amino group should
yield strong lanthanide luminescence and (2) the lanthanide
luminescence should be very weak when the amino group is
protected with an acyl group. We thoroughly investigated the known
antenna groups for the above requirements. We found that very
few antenna groups that have an amino group could emit lanthanide
luminescence in aqueous solution.11 We considered this is because
amino groups generally function as quenching groups for lanthanide
luminescence.5,12,13 Generally, amino substituents cause the red shift
of the absorption spectra, and long-wavelength dyes are not
adequate for the efficient excitation of Tb3+ or Eu3+ 12
Thus, we
.
hypothesized that only simple aromatic compounds with an amino
group can act as efficient antenna groups for luminescent lanthanide
ions such as Tb3+ and Eu3+. Since tyrosine is known to sensitize
Tb3+ and Eu3+ 14
we expected that even aniline derivatives could
,
serve as good antenna groups.
For confirming the hypothesis, we designed and synthesized
antenna-chelator conjugates 1 and 3, which have simple aniline
derivative groups as the antennas, and the corresponding acetylated
compounds 2 and 4 (Figure 1a). We chose DO3A (1,4,7-tricar-
boxymethyl-1,4,7,10-tetraazacyclododecane) as the chelator for its
strong binding ability to trivalent lanthanide ions.15 The complex
[1-Tb3+] showed the characteristic emission spectrum of lanthanide
complexes besides the antenna fluorescence at 350 nm (Figure 1b).
Meanwhile, [2-Tb3+]sthe Tb3+ complex of the acetylated com-
pound 2sdid not show lanthanide luminescence. On the other hand,
although [3-Tb3+] did not show lanthanide luminescence, [4-Tb3+
]
showed strong lanthanide luminescence (see Supporting Informa-
tion). Since the two acetylated compounds 2 and 4 are the model
compounds of peptide conjugates, the above results indicated that
the 4-aminobenzyl group can be a suitable antenna for luminogenic
lanthanide probes detecting protease activities and that 4-aminoben-
zoylmethyl group can be an antenna for quenching-type protease
probes. In addition, none of the Eu3+ complexes of compounds
1-4 showed lanthanide luminescence (data not shown).
Next, we designed Suc-LY-Abd-Tb to detect calpain activity,
as shown in Scheme 1. Calpains are a family of intracellular cysteine
proteases that are involved in many cellular processes.16 Calpains
9
14376 J. AM. CHEM. SOC. 2008, 130, 14376–14377
10.1021/ja800322b CCC: $40.75  2008 American Chemical Society

C O M M U N I C A T I O N S
In conclusion, we developed novel luminogenic lanthanide probes
for detecting protease activities. The probe design principle could
be widely applicable to time-resolved assays for any proteases.
These lanthanide-based probes could accelerate drug-screening
processes and also contribute to the clarification of biological
systems. Furthermore, achievement of longer wavelength excitation
could enable a microscopic time-resolved fluorescence imaging of
protease activities in living cells.13
Acknowledgment. We thank MEXT of Japan and JST for the
financial supports. We thank Dr. Tomoyoshi Suenobu for the
technical support for time-resolved fluorescence measurement.
Figure 2. (a) Steady-state and (b) time-resolved (delay time, 10 µs; gate
time, 3.0 ms) emission spectral change of Suc-LY-Abd-Tb with calpain I.
λex ) 250 nm.
Table 1. Photophysical Properties of the Synthesized Compounds
Supporting Information Available: Detailed synthetic procedures;
supplementary spectra; physical properties; photostability experiment;
enzyme reaction experiment. This material is available free of charge
via the Internet at http://pubs.acs.org.
λ
abs/nm
ꢀ/M-1 cm-1
Φ
τ/ms
[1-Tb3+
[2-Tb3+
]
]
240
284
254
263
12 000
3 500
11 000
13 000
0.051 1.47
<0.001 1.49
<0.001 1.46
0.012 1.45
Suc-LY-Abd-Tb
Suc-LY-Abd-Tb + calpain Ia
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Finally, to demonstrate the generality of our sensing system, we
synthesized another lanthanide-based probe, Leu-Abd-Tb, for
leucine aminopeptidase (LAP); this enzyme hydrolyzes the peptide
bond of N-terminal hydrophobic amino acids such as leucine
(Scheme 1). Leu-Abd-Tb showed an increase in the time-resolved
luminescence when incubated with LAP (see Supporting Informa-
tion), similar to the case of Suc-LY-Abd.
JA800322B
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