Detection of NAD(P)H-dependent enzyme activity with dynamic luminescence quenching of terbium complexes

Article abstract of DOI:10.1039/c5cc01613d

We discovered that positively charged terbium complexes bearing 1,4,7,10-tetraazacyclododecane functionalized with amide ligands are highly sensitive to dynamic luminescence quenching by NAD(P)H. We exploited this phenomenon to establish a general time-re

Full text of DOI:10.1039/c5cc01613d

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Detection of NAD(P)H-dependent enzyme activity
with dynamic luminescence quenching of terbium
complexes†
Cite this:DOI: 10.1039/c5cc01613d
Received 23rd February 2015,
Accepted 1st April 2015
ab
ab
a
ab
ac
Hiroki Ito, Takuya Terai,* Kenjiro Hanaoka, Tasuku Ueno, Toru Komatsu,
d
abe
Tetsuo Nagano and Yasuteru Urano*
DOI: 10.1039/c5cc01613d
www.rsc.org/chemcomm
We discovered that positively charged terbium complexes bearing of NAD(P)H at 460 nm is another option, but is rarely used in
,4,7,10-tetraazacyclododecane functionalized with amide ligands practice, partly due to poor sensitivity at low NAD(P)H concen-
1
are highly sensitive to dynamic luminescence quenching by NAD(P)H. trations and an inner filter effect at high NAD(P)H concentra-
4
We exploited this phenomenon to establish a general time-resolved tions. Although a few chemodosimeters for NAD(P)H have been
5
luminescence-based assay platform for sensitive detection of reported, they are not suitable for monitoring enzyme activity in
NAD(P)H-dependent enzyme activities.
real time. Hence, we thought that a novel strategy to detect
NAD(P)H that overcomes these disadvantages would have many
Reduced nicotinamide adenine dinucleotide (phosphate), or potential applications, especially for high-throughput screening
NAD(P)H, is an important cofactor of oxidoreductases, which (HTS) in drug discovery and for clinical diagnostic tests.
transfer electrons from NAD(P)H to their substrates, affording
NAD(P) and the products. These enzymes function in many plexes. Lanthanide (Tb or Eu ) complexes have long luminescence
For this purpose, we focused on luminescent lanthanide com-
+
3+
3+
6
metabolic pathways, and are considered potential therapeutic lifetimes of millisecond order, which enables sensitive detection
1
,2
targets. Assay of their activities is also helpful for clinical by measuring only the long-lived component of luminescence
3
diagnosis. However, current assays of NAD(P)H-dependent (i.e. time-resolved measurement). By exploiting this capability,
enzyme activities are mostly based on absorption measurement, functional luminescent probes for various analytes including
7
8
9
and this strategy has many drawbacks. For example, monitoring ions and enzymes have been developed, but no luminescent
the absorption of NAD(P)H itself at 340 nm, which is a common probes for NAD(P)H have yet been reported. Interestingly, Parker
approach, gives poor sensitivity in small volumes, such as 384- or et al. recently showed that some lanthanide complexes are
4
1
536-well microplates, and absorbance in the UV region is susceptible to dynamic quenching by a few biological reductants
1
0,11
influenced by many extraneous factors. Colouring of tetrazolium such as ascorbate and urate, probably via exciplex formation.
salts upon NAD(P)H-dependent reduction is also used in cell Because they were interested in the use of luminescent com-
proliferation and toxicity assays, but chemical instability of these plexes in cells, they regarded this quenching as undesirable,
4
12
reagents limits their applicability. Fluorescence measurement with one exception, and they developed complexes that were
1
0
tolerant of reductants. However, we thought that we could
make use of this kind of intermolecular quenching to develop a
novel and general strategy to detect NAD(P)H-dependent enzyme
activity. In principle, this should be feasible, because NAD(P)H is
one of the most potent biological reductants (Table S1, ESI†),
a
Graduate School of Pharmaceutical Sciences, The University of Tokyo,
-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
7
b
c
CREST, JST, 7 Gobancho, Chiyoda-ku, Tokyo 102-0076, Japan
PRESTO, JST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
Open Innovation Center for Drug Discovery, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan
d
+
which would efficiently quench luminescence, whereas NAD(P) ,
an oxidized form of NAD(P)H, would not. Herein, we show that
e
Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: t-terai@mol.f.u-tokyo.ac.jp,
uranokun@m.u-tokyo.ac.jp
3
+
Tb complexes are indeed dynamically quenched by NAD(P)H
and the magnitude of the quenching can be modulated by
†
Electronic supplementary information (ESI) available: Experimental proce- adjusting the overall charge of the complex (Fig. 1a). Based on
dures, synthetic protocols and characterization of compounds, photophysical
properties of complexes, absorption and luminescence spectra of complexes
upon addition of NADH, the effect of analogous compounds, an energy diagram
this finding, we have developed a novel general luminescence
intensity- and lifetime-based assay for NAD(P)H-dependent
enzyme activities.
We first hypothesized that the efficiency of the expected
quenching by NAD(P)H would depend on the structure of the
3+
of Tb complexes, fluorescence spectra of the antenna moiety, additional data of
LDH assay, detection of other enzymes, and the reversible nature of the method.
See DOI: 10.1039/c5cc01613d
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3+
Table 1 Photophysical properties of Tb complexes
a
À1
a
À1 À1
Probe
Charge
t (ms)
K
sv (M
)
k
q
(M
s
)
5
8
A1Tb
A2Tb
A3Tb
+3
+1
0
1.52
1.96
1.87
1.9 Â 10
4.0 Â 10
1.7 Â 10
1.2 Â 10
2.1 Â 10
9.1 Â 10
4
4
7
6
a
K
q
sv and k were calculated from Stern–Volmer plots of luminescence
lifetimes upon titration of NADH. Excitation wavelength was 340 nm.
only moderate decreases in luminescence intensity and lifetime
upon titration of NADH up to 100 mM (Fig. S2 and S3, ESI†).
We further investigated the photophysical properties
3
+
of these Tb complexes and the bimolecular quenching con-
stants (k ) upon titration of NADH. No significant differences
3+
q
Fig. 1 (a) Schematic representation of the interaction of the Tb complex
were observed in the shapes of the absorption spectra and
emission spectra, which may imply that the coordination
structures of the three complexes are very similar (Fig. S4 and
and NAD(P)H. (b) Chemical structure of complexes.
chelating moiety, in particular its charge. To test this idea, we Table S2, ESI†). As summarized in Table 1, the more positively
3
+
designed and synthesized three Tb complexes with different charged complex was more sensitive to NADH. The trend
net charges (Fig. 1b). Negatively charged NAD(P)H is expected to was more clearly seen in quenching of A1Tb by NADPH,
show greater electrostatic attraction towards a more positively which possesses greater negative charge than NADH, and is
1
3
charged complex, as previously reported for ATP. As a scaffold therefore expected to have stronger electrostatic interaction
complex, we selected A3Tb, previously reported by Parker than NADH with positively charged compounds (compare
1
1b,12
and prepared a series of side-chain derivatives with Tables S3 and S4, ESI†). On the other hand, 100 mM NAD+
et al.,
altered net charge, because 1,4,7,10-tetraazacyclododecane and had little effect on luminescence intensity and no effect on
its derivatives form kinetically and thermodynamically stable lifetime (Table S5, ESI†). We also measured the effect of several
1
4
complexes with lanthanide ions. Three of the ring nitrogen adenine-containing biological compounds, and showed that
atoms were substituted with amide and/or carboxylate groups they do not cause dynamic quenching of the complex (Table
3
+
that coordinate to Tb . Azaxanthone was used as an antenna S5, ESI†). All these data suggest that electrostatic attraction
3
+
15
3+
moiety because it sensitizes Tb with high efficiency. The between Tb and phosphate groups, as well as reductive
changes in luminescence intensity and lifetime of A1Tb, the electron transfer or charge-transfer exciplex formation between
3
+
complex with the highest positive charge, upon titration of Tb and the reduced nicotinamide moiety at the excited
NADH are illustrated in Fig. 2. Notably, the values of both state, is important for the dynamic quenching by NAD(P)H.
1
7
3
+
parameters were greatly decreased in the presence of as little However, considering the energy diagram of Tb complexes
as 1 mM NADH, and the addition of 100 mM NADH almost totally (Fig. S5, ESI†), which starts with antenna excitation, followed
quenched the luminescence. The quenching was concentration- by intersystem crossing and intramolecular energy transfer,
dependent, and the Stern–Volmer constants (Ksv) of lumines- and results in the excited state of Tb , it is possible that
cence intensity and lifetime were almost identical at low NADH NAD(P)H also acts on the antenna moiety. Therefore, we further
concentrations (Fig. S1, ESI†), indicating that the quenching examined whether the excited state of the antenna is involved
3
+
3
+ 16
3+
process primarily occurs in the excited state of the Tb . The in the quenching process by direct excitation of the Tb ion at
3
+
data demonstrate that Tb complexes with appropriate struc- 488 nm (Tables S3 and S4, ESI†). The obtained quenching
tures are indeed dynamically quenched by NADH with high constants were the same for direct excitation (488 nm) and
efficiency. On the other hand, complexes with less positive antenna excitation (340 nm), supporting the idea that NAD(P)H
charge and neutral charge, A2Tb and A3Tb, respectively, showed only acts in the excited state of Tb . In accordance with this
view, the fluorescence of the antenna moiety, 2-Me azaxanthone,
3
+
was hardly quenched in the presence of 10 mM NADH (Fig. S6,
ESI†). Importantly, the k
q
value of A1Tb for NAD(P)H (over 1 Â
8
À1 À1
10 M
s ) was much larger than those for GSH and ascorbate,
so A1Tb is tolerant of these reductive reagents, which are often
used in enzyme assay (Table S1, ESI†).
Next, we tested whether this quenching can be used to
monitor enzyme reactions, because A1Tb responds to 1–100 mM
NAD(P)H, which is the optimal range for applications such as
HTS. The selected model enzyme, lactate dehydrogenase (LDH,
EC 1.1.1.27), is a NADH-dependent enzyme that catalyzes the
reduction of pyruvate to L-lactate while converting NADH into
Fig. 2 Luminescence spectra (left) and luminescence lifetime (right) of
A1Tb upon titration of NADH from 0 mM (dark blue) to 100 mM (aqua). Error
bars represent S.D. (n = 3).
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measurements for alcohol dehydrogenase (ADH, EC 1.1.1.1)
and malate dehydrogenase (MDH, EC 1.1.1.37), and confirmed
that A1Tb could also probe their activity on microplates with
small S.D. values (Fig. S9 and S10, ESI†). In principle, our probe
can monitor the activities of hundreds of NAD(P)H-dependent
enzymes, and the number could be further increased by coupling
these enzymes to other enzymes. A further advantage of our probe
is that it is reversible (i.e. the luminescence can be turned on and
off repeatedly). For example, when the NADH concentration in the
sample was initially increased and then decreased, the probe
could readily report these changes in real time (Fig. S11, ESI†).
This is in sharp contrast to reaction-based fluorescence and
5
luminescence sensors.
In conclusion, we have developed a novel assay platform
3
+
for NAD(P)H-dependent enzymes using luminescent Tb com-
plexes. Though many luminescent probes for biological mole-
7
–9,12
cules have been reported,
most of them are specific to
limited analytes and do not have practical utility for general
1
3
assays. In contrast, probes for coenzymes including NAD(P)H
are expected to have high generality. We believe that this is the
first report to describe the interaction between NAD(P)H and
1
7
luminescent lanthanide complexes. We showed that NAD(P)H
Fig. 3 (a) Schematic illustration of LDH assay using lanthanide lumines-
3
+
cence. (b) Time course of luminescence intensity (left) or lifetime (right) of dynamically quenches the luminescence of Tb complexes,
4
mM A1Tb in the presence (black diamond) and absence (gray circle)
and this phenomenon can be used to monitor the activities
of NAD(P)H-dependent enzymes. Although it is not suitable
for imaging cellular NADH, our probe can monitor enzyme
reactions in real time with good sensitivity. In this communica-
tion, we have focused on optimizing the net charge of the
complexes, and the influence of other factors still needs to be
À1
of LDH (50 mU mL ). Initial concentrations of pyruvate and NADH were
À1
1
25 mg mL and 100 mM, respectively. (c) Comparison of time-resolved
luminescence (left) and NADH absorption (right) measurements on micro-
plates after incubation for 25 minutes. Error bars represent S.D. (n = 3).
Asterisks indicate p o 0.05 vs. 0 mU mLÀ1
.
studied to elucidate the quenching mechanism in more detail.
NAD (Fig. 3a); it plays a key role in the anaerobic pathway of We are currently synthesizing Tb complexes with other antenna
glucose metabolism. Because cancer cells tend to use this or chelator moieties to optimize sensitivity, and we aim to apply
+
3+
1
pathway instead of the aerobic pathway (Warburg effect), a them for inhibitor screening and clinical diagnostic assays of
specific subtype of this enzyme, LDH-A, is considered as an NAD(P)H-dependent enzymes.
1
attractive target for anticancer drugs. The results of LDH assay
This work was supported by KAKENHI (Grant 22000006 to
with A1Tb in a cuvette are shown in Fig. 3b. Addition of LDH T.N., 24689003 and 24659042 to K.H., 24655147 to K.T., 25104506
was accompanied by a monotonic increase in both lumines- to T.T., and 23249004 to Y.U.), by SENTAN, JST (K.H.), Grant-in-
cence intensity and lifetime of A1Tb, while no change was Aid for JSPS Fellows (H.I.) and by Astellas Foundation for Research
observed in the absence of the enzyme. Unaltered LDH activity on Metabolic Disorders (T.T.).
in the presence of A1Tb was confirmed by measuring NADH
absorption (Fig. S7, ESI†), which indicates that the probe does not
Notes and references
interfere with the reaction of LDH. Also, the reaction products,
+
L-lactate and NAD , had negligible effect on the luminescence
1
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and Fig. S8, ESI†). These data demonstrate that the conversion
(
2
+
of NADH into NAD by LDH changed the luminescence properties
3
S. L. Upstone, Encyclopedia of Analytical Chemistry, John Wiley &
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whether the method is suitable for HTS. As little as 1 mU mL
Sons Ltd, Chichester, 2000.
À1
4 M. J. V ´a zquez, S. Ashman, F. Ram ´o n, D. Calvo, A. Bardera,
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À1
incubation for 25 minutes. In contrast, 10 mU mL was the
lowest concentration that could be detected by absorption
measurement of NADH at 340 nm (Fig. 3c). Thus, A1Tb
provides a more sensitive measurement of enzyme activity than
this classical method.
The scope of A1Tb is not limited to LDH. To demonstrate its
generality, we performed preliminary time-resolved luminescence
6
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1
3+
3+
16 Replacing Tb with Eu greatly reduced the extent of quenching, as
10,11a
1
has been reported for other reductants.
3+
5
, 2975; (b) G.-L. Law, D. Parker, S. L. Richardson and K.-L. Wong,
by NADH on the film (T. Basova, I. Jushina, A. G. G u¨ rek, V. Ahsen
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1
Chem. Commun.
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