Ubiquinone-rhodol (UQ-Rh) for fluorescence imaging of NAD(P)H through intracellular activation

Article abstract of DOI:10.1002/anie.201311192

The nicotinamide adenine dinucleotide (NAD) derivatives NADH and NADPH are critical components of cellular energy metabolism and operate as electron carriers. A novel fluorescent ubiquinone-rhodol derivative (UQ-Rh) was developed as a probe for NAD(P)H. By using the artificial promoter [(η⁵- C₅Me₅)Ir(phen)(H₂O)]²⁺, intracellular activation and imaging of NAD(P)H were successfully demonstrated. In contrast to bioorthogonal chemistry, this bioparallel chemistry approach involves interactions with native biological processes and could potentially be used to control or investigate cellular systems. Active detection: NADH and NADPH are critical components of cellular energy metabolism and operate as electron carriers. A novel fluorescent ubiquinone-rhodol derivative (UQ-Rh) was developed as a probe for NAD(P)H. By using the artificial promoter [(η⁵-C₅Me₅)Ir(phen)(H ₂O)]²⁺, intracellular activation and imaging of NAD(P)H were successfully demonstrated.

Full text of DOI:10.1002/anie.201311192

Angewandte
Chemie
DOI: 10.1002/anie.201311192
Imaging Agents
Ubiquinone-Rhodol (UQ-Rh) for Fluorescence Imaging of NAD(P)H
through Intracellular Activation**
Hirokazu Komatsu,* Yutaka Shindo, Kotaro Oka, Jonathan P. Hill, and Katsuhiko Ariga*
Abstract: The nicotinamide adenine dinucleotide (NAD)
derivatives NADH and NADPH are critical components of
cellular energy metabolism and operate as electron carriers. A
novel fluorescent ubiquinone-rhodol derivative (UQ-Rh) was
developed as a probe for NAD(P)H. By using the artificial
promoter [(h5-C5Me5)Ir(phen)(H2O)]2+, intracellular activa-
tion and imaging of NAD(P)H were successfully demon-
strated. In contrast to bioorthogonal chemistry, this “biopar-
allel chemistry” approach involves interactions with native
biological processes and could potentially be used to control or
investigate cellular systems.
The activation of biological molecules (e.g., acetyl CoA)
in cells by using an artificial promoter and promoter-assisted
molecular imaging has been proposed.[10] This approach has
been adopted here as an imaging strategy that can be applied
to NAD(P)H.
It has been reported that NAD(P)H can be activated
in vitro by using an Ir complex,[11] although this method has
not been applied in an intracellular context. On the other
hand, a quinone-based molecular probe (indolequinone
derivative) has been reported.[12] We hypothesized that
a ubiquinone derivative might react with NAD(P)H based
on its biological role and reduction potential. Furthermore,
enhanced sensitivity can be obtained by using an Ir-complex-
based artificial promoter, which can activate NAD(P)H.
Therefore, we designed the ubiquinone-rhodol conjugate
UQ-Rh, which contains ubiquinone as an NAD(P)H-reactive
site and the rhodol fluorophore as a novel biocompatible
fluorescent probe, the fluorescence emission of which occurs
in the visible region (Figure 1).[13]
T
he nicotinamide adenine dinucleotide (NAD) derivatives
NADH and NADPH (general term: NAD(P)H) play an
important role in cellular metabolism and energy systems as
electron carriers.[1] NADH is generated by the tricarboxylic
acid (TCA) cycle, with three NADH molecules produced per
cycle. The phosphorylated form of NADH, NADPH, is
involved in photosynthesis and in the Entner–Doudoroff
pathway[2] of glycolysis.[3] NADH has also been investigated
as a therapeutic agent for the treatment of Alzheimerꢀs[4] and
Parkinsonꢀs diseases.[5]
Several imaging methods for intracellular NAD(P)H,
including an autofluorescence-based method,[6] have been
investigated. However, those methods are implemented using
ultraviolet (UV) light, thus resulting in low sensitivity and
interference by intracellular materials. A green fluorescent
protein (GFP)-based technique for imaging the NADH/
NAD+ ratio has also been developed.[7] In that case, the
mechanism of sensing depends on conformational variation of
the protein and requires intracellular gene expression.
Although several other methods for NAD(P)H determina-
tion exist, including enzymatic reaction kits,[8] electrochemical
methods, and chemosensors, these have been developed for
use in vitro and are not suitable for cellular use.[9] To date,
molecular-probe-based imaging of intracellular NAD(P)H
has not been reported.
Figure 1. The reduction of UQ-Rh by NAD(P)H.
UQ-Rh was synthesized from a ubiquinone derivative and
rhodol (see the Supporting Information). It possesses an
absorbance maximum at 492 nm (e = 1.66 ꢁ 104 cmÀ1mÀ1) and
a fluorescence maximum at 518 nm with a quantum yield of
0.733 in PBS buffer at pH 7.4 (Figure 2, blue lines).
To observe the fluorescence response as a result of
quinone reduction, UQ-Rh was dissolved in PBS buffer and
the quinone substituents were reduced by using Na2S2O4.[14]
Following reduction, the fluorescence dropped to 1/30 of the
starting value (Figure 2, red lines). The attenuation of the
fluorescence is considered to be due to intramolecular
photoinduced electron transfer (PET)[15] from hydroquinone
to rhodol.
[*] Dr. H. Komatsu, Dr. J. P. Hill, Prof. Dr. K. Ariga
MANA, National Institute for Materials Science
1-1 Namiki, Tsukuba-city, Ibaraki, 305-0044 (Japan)
E-mail: KOMATSU.Hirokazu@nims.go.jp
ARIGA.Katsuhiko@nims.go.jp
Dr. Y. Shindo, Prof. Dr. K. Oka
Graduate School of Science and Technlolgy, Keio University
3-14-1Hiyoshi,Kohoku-ku, Yokohama 223-8522 (Japan)
The NADPH fluorescence response of UQ-Rh was
measured with no promoter, in the presence of a promoter
(1 mm [(h5-C5Me5)Ir(phen)(H2O)]2+), or in the presence of
2 mgmLÀ1 quinone reductase in PBS.
[**] This work was supported by JST-CREST.
Supporting information for this article (including experimental
details) is available on the WWW under http://dx.doi.org/10.1002/
anie.201311192.
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reaction of NADPH was 1.7-fold faster than of NADH
(Figure S2). This difference may be due to the presence of the
negatively-charged phosphate group in NADPH, which likely
strengthens the interaction of NADPH with the positively-
charged Ir complex. Overall, the response difference between
NADPH and NADH is not so large.
The selectivity of UQ-Rh for NAD(P)H over potentially
interfering intracellular apecies was also investigated. Intra-
celluar cations (Na+, K+, Ca2+, Mg2+) and glutathione (at
intracellular concentrations) gave no fluorescence response
with UQ-Rh (Figure S2). Furthermore, we applied the
fluorescent probe UQ-Rh and the promoter [(h5-C5Me5)Ir-
(phen)(H2O)]2+ to the intracellular activation and imaging of
NAD(P)H in HeLa cells. UQ-Rh was introduced by incubat-
ing UQ-Rh with the HeLa cells. Fluorescence-microscopy
imaging revealed that although some UQ-Rh seemed to be
localized in organelles, the majority of the UQ-Rh appeared
to stain the cytosol.
The addition of pyruvate (5 mm), which activates the TCA
cycle in cells and increases the intracellular NADH concen-
tration, resulted in a decrease in cellular fluorescence, thus
indicating that UQ-Rh can indeed be used to measure NADH
increases in cells simply by monitoring fluorescence (Fig-
ure S3).
Figure 2. A) Fluorescence excitation spectra (detected at 520 nm), and
B) fluorescence emission spectra (excited at 488 nm) of UQ-Rh in the
presence and absence of Na2S2O4 in PBS (pH 7.4). The normalized
fluorescence intensity was plotted.
The fluorescence emission intensity of UQ-Rh decreased
in 1 mm NADPH. Moreover, in the presence of the promoter
[(h5-C5Me5)Ir(phen)(H2O)]2+, a 4-fold enhancement of the
reaction rate was observed and UQ-Rh was considered to be
effectively reduced. However, quinone reductase was not so
effective (1.45-fold enhancement), probably because of the
poor fit of UQ-Rh to the pocket of this enzyme (Figure S1 in
the Supporting Information).
The dependence of the UQ-Rh response on NADPH
concentration was determined in the presence of 0.5 mm [(h5-
C5Me5)Ir(phen)(H2O)]2+ (Figure 3). A weak response was
The promoter [(h5-C5Me5)Ir(phen)(H2O)]2+ was added to
the UQ-Rh stained HeLa cells and the time dependence of
the fluorescence intensity was imaged. Prior to the addition of
[(h5-C5Me5)Ir(phen)(H2O)]2+, a slight decrease in fluores-
cence was observed. This result is considered to be due to the
reaction of NAD(P)H occurring at resting levels in the cells.
Upon the addition of 0.5 mm [(h5-C5Me5)Ir(phen)(H2O)]2+,
an increase in fluorescence was observed (probably owing to
injection shock) with a subsequent rapid decrease in fluores-
cence intensity (Figure 4A). Hydride was thus transferred
Figure 3. A) The time-dependence and dose-dependence (in terms of
NADPH) of the fluorescence intensity (normalized) of UQ-Rh in the
presence of 0, 0.25, 0.5, 1.0, 2.5, 5, and 10 mm of NADPH (10 mm UQ-
Rh, 0.5 mm[(h5-C5Me5)Ir(phen)(H2O)]2+, PBS pH 7.4).
first observed after the addition of 0.5 mm NADPH. At 10 mm
NADPH, an 8.6-fold decrease in fluorescence intensity was
observed after 10 min. By preparing a calibration curve for
fluorescence intensity after the addition of NADPH in the
presence of 0.5 mm [(h5-C5Me5)Ir(phen)(H2O)]2+, a linear
correlation was found between 0.5 mm and 5 mm NADPH.
NADPH can thus be quantitatively determined in this
concentration range. Because of its response to NADPH,
UQ-Rh can be used to quantify NADPH with a sensitivity
that can be enhanced by adding the Ir complex as a promoter.
The NADH response was also measured and compared
with the response to NADPH. NADH produced a decrease in
fluorescence intensity similar to that observed for NADPH.
In the presence of 0.5 mm [(h5-C5Me5)Ir(phen)(H2O)]2+, the
Figure 4. Fluorescence imaging of HeLa cells. A) Normalized fluores-
cence intensity before and after addition of the Ir complex [(h5-
C5Me5)Ir(phen)(H2O)]2+ (0.5 mm) at 2 min. ([UQ-Rh]=10 mm, excita-
tion at 488 nm; average of 9 cells). B) Fluorescence images (excitation
at 488 nm).
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from NAD(P)H to the Ir complex by the artificial promoter
and could be imaged by observing the fluorescent probe.
Further observation of the cells following NAD(P)H
activation revealed that 4 min after the addition of [(h5-
C5Me5)Ir(phen)(H2O)]2+, leaching of the cell contents oc-
curred, which was followed after 7 min by cell death.
NAD(P)H activation induced ubiquinone reduction or an
NAD(P)H shortage in the cells might have affected cell
homeostasis, thus resulting in cell necrosis (Figure 4B).
The ubiquinone-rhodol-derived fluorescent probe UQ-
Rh responds to NADPH with an 8.6-fold decrease in
fluorescence intensity over 10 min in the presence of [(h5-
C5Me5)Ir(phen)(H2O)]2+. UQ-Rh was introduced into HeLa
cells and, following the addition of [(h5-C5Me5)Ir(phen)-
(H2O)]2+, the activation of NAD(P)H could be imaged by
observing the decrease in fluorescence. Although the use of
this probe for the quantification of intracellular NAD(P)H is
not easy, we have demonstrated the first use of an artificial
fluorescent probe for the activation and imaging of NAD(P)H
in cells. The activation of NAD(P)H induces necrosis by
inducing errors in cell homeostasis.
The activation of molecules within cells can induce novel
chemical reactions, an approach that contrasts with the
conventional concept of bioorthogonal chemistry[16] in the
field of chemical biology. Bioorthogonal chemistry denotes
chemical reactions that occur without interfering with native
biological process. By contrast, in our system, interactions do
occur (i.e. activation of NAD(P)H). Considering these
interactions, we hope to investigate novel aspects of living
systems by applying activation and functionalization within
cells to influence their intracellular metabolic processes. To
distinguish this from the widely known bioorthogonal reac-
tions, we have applied the label “bioparallel chemistry” and
believe that it may be possible to use this strategy to control
cellular systems or living systems in general. This system
could form the basis of a new approach in medicine, with the
development of novel artificial promoters and reagents.
Keywords: fluorescent probes · imaging agents ·
in vivo imaging · NAD(P)H · redox chemistry
.
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Received: December 25, 2013
Revised: January 10, 2014
Published online: March 5, 2014
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ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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