New class of bioluminogenic probe based on bioluminescent enzyme-induced electron transfer: BioLeT

Article abstract of DOI:10.1021/ja511014w

Bioluminescence imaging (BLI) has advantages for investigating biological phenomena in deep tissues of living animals, but few design strategies are available for functional bioluminescent substrates. We propose a new design strategy (designated as bioluminescent enzyme-induced electron transfer: BioLeT) for luciferin-based bioluminescence probes. Luminescence measurements of a series of aminoluciferin derivatives confirmed that bioluminescence can be controlled by means of BioLeT. Based on this concept, we developed bioluminescence probes for nitric oxide that enabled quantitative and sensitive detection even in vivo. Our design strategy should be applicable to develop a wide range of practically useful bioluminogenic probes.

Full text of DOI:10.1021/ja511014w

Communication
pubs.acs.org/JACS
New Class of Bioluminogenic Probe Based on Bioluminescent
Enzyme-Induced Electron Transfer: BioLeT
Hideo Takakura,†,‡,○,¶ Ryosuke Kojima,†,◆,¶ Mako Kamiya,‡,∇ Eiji Kobayashi,§ Toru Komatsu,†,∇
Tasuku Ueno,† Takuya Terai,† Kenjiro Hanaoka,† Tetsuo Nagano,∥ and Yasuteru Urano*,†,‡,⊥,#
‡
∥
†Graduate School of Pharmaceutical Sciences, Graduate School of Medicine, Open Innovation Center for Drug Discovery, The
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
#
⊥Basic Research Program, CREST, ∇PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama
332-0012, Japan
§Department of Organ Fabrication, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
S
* Supporting Information
However, rational design strategies for bioluminescence
ABSTRACT: Bioluminescence imaging (BLI) has advan-
tages for investigating biological phenomena in deep
tissues of living animals, but few design strategies are
available for functional bioluminescent substrates. We
propose a new design strategy (designated as bio-
luminescent enzyme-induced electron transfer: BioLeT)
for luciferin-based bioluminescence probes. Luminescence
measurements of a series of aminoluciferin derivatives
confirmed that bioluminescence can be controlled by
means of BioLeT. Based on this concept, we developed
bioluminescence probes for nitric oxide that enabled
quantitative and sensitive detection even in vivo. Our
design strategy should be applicable to develop a wide
range of practically useful bioluminogenic probes.
probes are currently quite limited compared to those for
fluorescence probes. Various principles have been applied to
rational development of fluorescence probes, including photo-
induced electron transfer (PeT),16,17 Forster resonance energy
̈
transfer (FRET),17,18 spirocyclization,19,20 and intramolecular
charge transfer,21,22 whereas currently available rational design
strategies for bioluminescence probes rely mostly on the “caged
luciferin” concept, i.e., release or generation of D-luciferin
(native firefly luciferin) or aminoluciferin (AL) after reaction of
the probe with the target molecule. Probes based on this idea
include 6′-O-ether analogues of D-luciferin10,11,14 and
others12,13,23−25 (Figure S1 and also see SI for another
bioluminescence probe). Thus, the limitations of current design
strategies continue to hamper the development of bio-
luminescence probes, despite the potential advantages of such
probes, and it is generally agreed that new rational design
strategies are urgently needed.26,27 Here, we report a novel
principle for controlling the luminescence properties of AL
derivatives by utilizing electron transfer from a benzene moiety
to the excited luminophore (Figure 1A). Based on this idea, we
designed, synthesized, and evaluated bioluminogenic probes for
nitric oxide (NO). Our design concept should be applicable to
develop a wide range of practically useful bioluminogenic
probes.
n vivo optical imaging is a powerful tool to study complex
Iphysiological and pathological processes in living organ-
isms,1,2 and bioluminescence imaging (BLI), which utilizes
genetically encodable luciferase as an internal light source, has a
number of advantages for this purpose. Unlike fluorescence
imaging, which requires external illumination, a biolumines-
cence signal generated deep inside the body can be detected
with high signal-to-noise ratio and is unaffected by tissue
absorption/scattering of excitation light or background
autofluorescence of tissues. So, the luciferin-luciferase system,
especially that of firefly, has been widely used in BLI to monitor
gene expression, to track cells, and to access tumor progression
in in vivo systems.3−5 Functional BLI, which can be achieved by
off/on switching of bioluminescence in response to a specific
biological phenomenon, can provide even more information.
For example, advances in luciferase engineering technology,
such as split luciferase complementation6,7 and biolumines-
cence resonance energy transfer,8,9 have made it possible to
quantify protein−protein interactions and to detect biomole-
cules by bioluminescence. Chemical probes utilizing bio-
luminescence have also been used to visualize various molecular
activities such as those of β-galactosidase,10 β-lactamase,11 and
proteases,12,13 as well as hydrogen peroxide (H2O2),14,15 in
vivo.
In order to develop a flexible design strategy for bio-
luminescence probes, we focused on electron transfer as an off/
on switching mechanism, since PeT-based switching has been a
versatile design strategy for fluorescence probes targeting a
wide range of biomolecules, including NO,17 singlet oxygen,17
and metal ions.16 The principle of PeT is that an electron-
transfer process to or from the excited state of a fluorophore
diminishes the fluorescence from the singlet excited state
(Figure 1A, upper). As a working hypothesis, we considered
that the luminescence of luciferase substrates would be similarly
modulated, since both fluorophore and bioluminophore take
singlet excited states, although they differ in the driving force,
i.e., light illumination or enzymatic reaction (Figure 1A, lower).
Therefore, we investigated the feasibility of employing electron
Received: October 26, 2014
© XXXX American Chemical Society
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DOI: 10.1021/ja511014w
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Figure 1. Concept of quenching of the excited state of a luminophore
by BioLeT. (A) Schematic diagram of a fluorescent molecule and a
bioluminescence substrate bearing a benzene moiety with a high
HOMO energy level. The excited luminophore can be quenched by
BioLeT, just as the excited fluorophore is quenched by PeT. (B)
Chemical structures of substrate 1 and 2 and comparison of
luminescence upon reaction with luciferase.
transfer to the excited state of a luminophore as a principle for
controlling luminescence.
Figure 2. Verification of BioLeT with AL derivatives. (A) Chemical
structures of AL derivatives used for verification of the occurrence of
BioLeT. (B) Relationship of HOMO energy level of the benzene
moiety and luminescence intensity of the substrates. Error bars
represent SD (n = 3).
In our studies of PeT,28,29 we showed that the rate of
electron transfer is strongly dependent on the HOMO energy
level of the electron donor. To examine whether the
luminescence of luciferin analogues could be similarly
quenched through an electron-transfer process, we first
prepared substrate 1 and its urethane derivative 2 by
introducing an electron-donating aniline moiety or a less
electron-donating anilide moiety into the AL scaffold (Figure
1B, Scheme S1). AL was selected because a range of
functionalities can be introduced at the 6′-N position without
loss of the luminescence (Figure S1).30−32 Upon reaction with
luciferase from Photinus pyralis, substrate 2 showed 70-fold
higher luminescence than 1 (Figure 1B), even though 2 has a
bulky additional moiety compared with 1, and 1 was completely
consumed by the enzymatic reaction (Figure S2). These results
indicated that substrate 1 reacted with luciferase to yield an
excited-state intermediate but did not emit strong bio-
luminescence, presumably because the electron-transfer process
occurs much faster than the light-emitting process. This
supports the idea that luciferase-dependent luminescence can
be modulated by means of an electron-transfer process. As
there is no previous report of electron transfer to the excited
state of a luminophore, we designated this process as
bioluminescent enzyme-induced electron transfer (BioLeT).
Next, in order to confirm that BioLeT really exists and to
examine whether it is feasible to rationally design bio-
luminescence probes by utilizing this phenomenon, we
prepared a series of AL derivatives bearing benzene moieties
with various HOMO energy levels (Figures 2A and S3,
Schemes S2−5) and examined the correlation between HOMO
energy level and luminescence intensity. Indeed, the
luminescence intensity of the substrates varied greatly depend-
ing on the HOMO energy level of the benzene moiety (Figures
2B and S3c), while the shapes of their bioluminescence spectra
and the Km values of the substrates were almost identical
(Figure S3b,c). As in the case of fluorophores (Figure S4), the
luminescence was quenched as the HOMO energy level of the
benzene moiety increased, which strongly supports the
occurrence of BioLeT-dependent quenching. Further, since
the AL derivatives were almost non-luminescent when the
HOMO energy level of the benzene moiety was more than −5
eV, it should be possible to predict whether or not the
luminescence of substrates would be strongly quenched by
BioLeT by calculation of the HOMO energy level of the
benzene moieties (see SI for further discussion about Figure
2B).
In order to validate BioLeT as a principle for developing a
new class of bioluminescence probes, we aimed to develop a
novel bioluminescent probe by using BioLeT as a luminescence
switching mechanism. For this, we focused on NO as target
molecule, since it is well established that a diaminobenzene
group reacts with NO under air to form benzotriazole, resulting
in a substantial change of the HOMO energy level of the
benzene moiety.33,34 We designed and synthesized diamino-
phenylpropyl-AL (DAL) as shown in Figure 3A and Scheme
S6. We also prepared DAL-T, which is the putative reaction
product of DAL with NO, as a reference substrate. The HOMO
energy levels of the diaminophenyl moiety and benzotriazole
moiety were calculated to be −4.68 and −6.22 eV, respectively,
which indicated that bioluminescence of DAL would be
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of NOC7 (Figure S11). These results demonstrate that DAL
can quantitatively detect NO even in cellulo and support its
availability for BLI in living systems. Then, we examined the
applicability of DAL in transgenic rats ubiquitously expressing
firefly luciferase (luc-Tg rat), whose gene is driven under the
ROSA26 promoter.35 NOC has been reported to release NO
even in vivo,36 so we again used NOC7 as a NO donor. At 10
min after i.p. injection of DAL (1 μmol), a freshly prepared
NOC7 (20 μmol) solution in PBS was i.p. injected. We found
that DAL could detect NO released from a NO donor in the
peritoneal cavity of luc-Tg rat (Figure 4, see Figure S12 for
Figure 3. Development of DAL, a bioluminescence probe for NO. (A)
Molecular design of DAL based on the BioLeT mechanism. (B)
Selectivity of DAL among various ROS. (C) Quantification of NO
released from a NO donor using DAL in vitro (error bars represent
SD n = 4).
Figure 4. Detection of NO released from NO donor with DAL in vivo.
(A) BLI of DAL in luc-Tg rat with NOC7. At 10 min after i.p.
injection of 1 μmol DAL, luc-Tg rats were i.p. injected with or without
20 μmol NOC7. The images were taken at 40 min after the injection
of NOC7. (B) Quantification of luminescence intensity (L.I.) of DAL
from abdomen of luc-Tg rat in (A) (error bars represent SEM n =
3). The intensities were normalized by those just before the injection
of NOC7.
sufficiently quenched by BioLeT, while that of DAL-T would
not be quenched, resulting in recovery of the characteristic
luminescence properties of DAL upon reaction with NO.
As expected, the luminescence intensity of DAL was strongly
quenched, while that of DAL-T was not, and the increase of
DAL-T luminescence compared to that of DAL was about 41-
fold (Figures 3A and S5). We confirmed that the rates of
consumption of DAL and DAL-T by luciferase were almost the
same (Figure S6). These findings strongly support the idea that
the difference of luminescence intensity of DAL and DAL-T
was indeed derived from the difference of luminescence
quantum efficiency from the singlet excited state, which should
be regulated by BioLeT. We also confirmed that effective
quenching of bioluminescence was reduced when a diamino-
benzene moiety was conjugated at a remote site from the
luminophore (Figure S7 and Scheme S7), which again supports
the operation of BioLeT, because the efficacy of quenching of
the singlet excited state by electron transfer is known to
decrease with increasing distance between the electron donor
and the acceptor28 and such a large difference in efficacy of the
quenching in this range of distance (<1 nm) is characteristic of
electron-transfer-based quenching.
Next, we examined detection of NO with DAL in vitro. DAL
showed a large luminescence increase upon addition of NO-
bubbled solution (Figure S8) or NOC7 (Figure 3B), a NO
donor that is known to release NO when dissolved in neutral
aqueous solvents. LC-MS analysis of the reaction solution
demonstrated that DAL was converted to DAL-T as the sole
product (Figure S9), providing direct evidence that DAL reacts
with NO and the reaction product is luminescent. Further, the
luminescence increase was linearly correlated with the amount
of NOC7 added to the solution (Figure 3C, detection limit:
1.51 μM) and was selective for NO among various ROS
(Figure 3B), showing that DAL can quantitatively and
selectively detect NO. These ROS did not affect the
luminescence intensity of AL under our conditions (Figure
S10). We further examined the availability of DAL in cellulo.
When HEK293 cells stably expressing luc2 were incubated with
a solution of DAL and various concentrations of NOC7, we
observed a luminescence signal that depended on the amount
kinetics). This result directly demonstrates that DAL is
applicable for BLI in vivo. Further, when injected i.v., DAL
was distributed to the whole body, suggesting that it would be
available to monitor NO production throughout the body
(Figure S13). It should be noted that a red fluorescence probe
for NO, DAR-4M AM, was subject to serious interference by
autofluorescence from fur and by absorbance/scattering of skin
and tissues (Figure S14). On the other hand, the signal of BLI
can be detected even without shaving the fur, and a strong
luminescence increase was observed, indicating that functional
bioluminescence probes should provide much more reliable
data than fluorescence probes in in vivo imaging of rats. Thus,
the BioLeT-based strategy enabled us to design and develop a
new class of bioluminescence probe for NO, DAL, which could
not have been developed based on the caged luciferin strategy.
In conclusion, we present here a new concept for rational
design of functional bioluminescence probes, BioLeT, which
should enable flexible design of a wide range of functionalized
bioluminescence probes for detecting various biomolecules of
interest, just as PeT has been used for development of versatile
fluorescent probes. The validity of this approach was
demonstrated by development of a novel bioluminogenic
probe for NO, DAL. For in vivo imaging, bioluminescence-
based techniques are well-known to have significant advantages
over fluorescence-based ones. We believe that our findings will
extend the applicability of luciferin-luciferase systems by
providing sensitive tools for investigating a broad range of
biological phenomena, especially in vivo.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures, characterization of synthesized
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free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*uranokun@m.u-tokyo.ac.jp
Present Addresses
○Department of Cell Biology, Yale University School of
Medicine, New Haven, CT 06520, United States
◆Department of Biosystems Science and Engineering (D-
BSSE), ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland
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Author Contributions
¶These authors contributed equally.
Notes
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The authors declare the following competing financial
interest(s): Luciferase transgenic rats used in this study are in
the subject of a Japanese patent (patent number: 5198433).
ACKNOWLEDGMENTS
■
This research was supported in part by a Grant-in-Aid for JSPS
Fellows (to H.T. and R.K.), the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (Grant-in-Aid for
Scientific Research (KAKENHI), grants 20117003, 23249004,
and 26111012 to Y.U.), by The Daiichi-Sankyo Foundation of
Life Science (grant to Y.U.), by The Mochida Memorial
Foundation for Medical and Pharmaceutical Research (grant to
M.K.), and by The Tokyo Society of Medical Sciences (grant to
M.K.), by JSPS Core-to-Core Program, Advanced Research
Networks. E.K. is the Chief Scientific Advisor for Otsuka
Pharmaceutical Co., Ltd., Tokyo, Japan.
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DOI: 10.1021/ja511014w
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