A selective luminescent probe for the direct time-gated detection of adenosine triphosphate

Article abstract of DOI:10.1021/ja304373u

A molecular probe for the luminescent detection of adenosine nucleotides is presented. The probe, Tb-DOTAm-Phen, readily distinguishes among the three adenosine nucleotides in buffered aqueous conditions at neutral pH, a requirement for the direct monitoring of enzymatic reactions converting adenosine triphosphate (ATP) to adenosine diphosphate or adenosine monophosphate. The probe is most efficient under millimolar concentrations of ATP which are relevant to intracellular conditions. Moreover, the long luminescence lifetime of the probe readily enables time-gating experiments.

Full text of DOI:10.1021/ja304373u

Communication
pubs.acs.org/JACS
A Selective Luminescent Probe for the Direct Time-Gated Detection
of Adenosine Triphosphate
Evan A. Weitz, Jennifer Y. Chang, Adam H. Rosenfield, and Valerie C. Pierre*
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States
S
* Supporting Information
complete removal of background fluorescence, including those
arising from fluorescent drug candidates.
ABSTRACT: A molecular probe for the luminescent
detection of adenosine nucleotides is presented. The
probe, Tb-DOTAm-Phen, readily distinguishes among the
three adenosine nucleotides in buffered aqueous con-
ditions at neutral pH, a requirement for the direct
monitoring of enzymatic reactions converting adenosine
triphosphate (ATP) to adenosine diphosphate or
adenosine monophosphate. The probe is most efficient
under millimolar concentrations of ATP which are relevant
to intracellular conditions. Moreover, the long lumines-
cence lifetime of the probe readily enables time-gating
experiments.
The generic aspect and the need to screen large protein
substrates can both be met by designing a probe that binds
reversibly and with differential affinity to ATP, ADP, and
adenosine monophosphate (AMP). If the probe is designed in
such a way that binding of the nucleotide affects its
luminescence, then the different binding affinities can
advantageously be used to monitor conversion from one
nucleotide to another. A turn-on response for ATP to ADP
conversion can be achieved if both nucleotides quench the
luminescence of the probe and if the probe has higher affinity
for ATP than ADP. Lastly, for the probe to be efficient at the
high millimolar concentration of ATP typically found intra-
cellularly, the probe must have weak affinity for the nucleotides
in the millimolar range.
Herein, we present a small terbium molecular probe that, by
making use of this design strategy, can detect with different
binding affinity ATP, ADP, and AMP in buffered aqueous
media. It fulfills all nine of the user-defined requirements. More
specifically, our probe takes advantage of three parameters: (1)
the necessity, for practical application, to sensitize terbium
luminescence with a nearby antenna whose triplet state is
slightly higher in energy than the 5D state of the lanthanide;15,16
(2) the ability of adenosine to quench the luminescence of
certain chromophores;15,17−24 and (3) differential weak electro-
static interactions between a positively charged metal complex
and negatively charged nucleotides.
inases currently represent one of the two largest classes of
1,2
Ktargets for drug discovery.
In the field of oncology, the
FDA’s approval of the first kinase inhibitor, Herceptin, in 1998
started an extensive search for cancer drugs that target cellular
signaling pathways.3 The search for new kinase inhibitors,
critical tools in cancer therapy, often begins with the high-
throughput screening (HTS) of libraries followed by the
evaluation of the potency of potential leads and their selectivity
for a desired kinase. These studies require the widespread
availability of efficient and affordable screens for kinase activity.
A recent survey highlighted the most important features
coveted by researchers in such a platform.4 It must concurrently
(1) be affordable, (2) be non-radioactive, (3) be label-free, (4)
be antibody-free, (5) be generic (able to screen all classes of
kinases), and (6) enable the study of large protein substrates. In
addition, (7) time-resolved or time-gated luminescence
detection is highly preferred, as are (8) assays that measure
the accumulation of adenosine diphosphate (ADP) as opposed
to the phosphorylation of a peptide or the displacement of a
biomarker. None of the current commercial assays fulfill all
eight of these requirements.5−13 Moreover, none can be
performed at higher concentrations of adenosine triphosphate
(ATP), closer to the intracellular concentrations of 1−10 mM
needed to study inhibitors of low-affinity kinases.14
Our probe, Tb-DOTAm-Phen (1), consists of a macrocyclic
polyaminocarboxylate terbium complex conjugated to a
phenanthridine antenna which is designed as follows (Figure
1): The macrocyclic DOTA-type chelate gives the complex its
high thermodynamic and kinetic stability, thereby minimizing
transmetalation with enzymes or peptides.25 The three
remaining ligand arms not conjugated to the phenanthridine
antenna are converted to amides, thereby giving the complex an
overall +3 charge. In the “off” state, that is in the absence of any
nucleotide, excitation of the phenanthridine antenna at 346 nm,
followed by intersystem crossing from its singlet state to its
5
triplet state and subsequent energy transfer to the terbium D
In terms of design, in order to fulfill each of the above
parameters, the probe must consist of a small molecule which is
luminescent and non-radioactive. Lanthanide complexes such
as terbium or europium are preferred as their long
luminescence lifetimes, typically in the ms range, enable facile
time-gated experiments. Time-gating is a favored technique in
drug discovery and chemical biology because the delay between
the excitation pulse and the emission measurement enables the
state yields characteristic terbium phosphorescence with four
sharp bands, the most intense centered at 545 nm (Figure 2).
In the presence of ATP, stacking of the purine nucleobase on
the antenna favors photoelectron transfer (PET) from the
adenosine to the phenanthridine, thereby preventing energy
Received: May 5, 2012
Published: September 20, 2012
© 2012 American Chemical Society
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Journal of the American Chemical Society
Communication
a
Scheme 1. Synthesis of Tb-PhenDOTAm (1)
Figure 1. Chemical structure and mode of action of the ATP sensor
Tb-DOTAm-Phen (1). In the absence of the nucleotide, excitation of
the phenanthridine followed by energy transfer to the lanthanide
enables bright luminescence centered at 545 nm. Stacking of the
adenosine on the phenanthridine favors PET quenching of the
antenna and, consequentially, also that of the Tb luminescence.
a
Experimental conditions: (a) (PPh3)4Pd, Na2CO3, H2O, dimethoxy-
ethane, reflux, 3 h; (b) Pd/C, H2, CH3OH, 12 h, rt; (c) CH3C(O)Br,
LiOH, CH2Cl2, 0 °C→rt, 2 h; (d) H3PO4, 150 °C, 2.5 h; (e) N-
bromosuccinimide, hν, rt, 45 min; (f) NaN3, acetone, rt, 1 h; (g) Pd/
C, H2, CH3OH, 3.5 h, rt; (h) ClCH2C(O)Cl, CH2Cl2, NEt3, rt, 2 h;
(i) cyclen, Cs2CO3, CH3CN, DMA, 90 °C, 4 h; (j) 2-chloro-N-
methylacetamide, Cs2CO3, CH3CN, 80 °C, 34 h; (k) TbCl3, LiOH,
H2O, 70 °C, 17 h.
methylphenanthridine (4) in the presence of polyphosphoric
acid. Bromination under standard radical conditions, followed
by reaction with sodium azide and reduction yielded
phenanthridin-6-ylmethanamine (6) which was then conju-
gated to the macrocycle following standard conditions in two
steps. First, reaction with chloroacetyl chloride yielded the
chloride (7) which was then conjugated to the cyclen backbone
under basic anhydrous conditions. Addition of the remaining
three acetamide arms yielded the hygroscopic ligand DOTAm-
Phen (8). The lanthanide complex, Tb-DOTAm-Phen (1), was
formed quantitatively by refluxing the ligand and 1 equiv of
TbCl3 in water at neutral pH.
A typical time-gated titration of the probe with ATP is shown
in Figure 2. The probe works most efficiently with ATP
concentrations between 1 and 10 mM, concentrations which
are within the intracellular range and which are therefore more
relevant to drug screening. Moreover, the long luminescence
lifetime of the terbium complex readily enables time-delayed
experiments, whereby the background luminescence of the
sample (enzyme, peptide and/or fluorescent drugs) can be
readily gated out, thereby yielding more accurate measure-
ments, not influenced by the autofluorescence of the system.
The Stern−Volmer plot of the time-gated luminescence of
the terbium probe in the presence of the three adenosine
nucleotides is shown in Figure 3. Each nucleotide quenches
both terbium phosphorescence (Figure 2) and the antenna’s
luminescence (Supporting Information, Figure S2) efficiently.
The fact that the antenna’s luminescence is also quenched
strongly suggests that terbium phosphorescence is quenched via
a mechanism that involves PET from the purine to the
phenanthridine (Figure 1). Importantly, the three nucleotides,
ATP, ADP, and AMP, can be readily distinguished and present
noticeably different Stern−Volmer constants, KSV (Table 1). At
the intracellularly relevant concentration of 1 mM, ATP
Figure 2. Time-delayed luminescence spectra of Tb-DOTAm-
Phen·ATP. Experimental conditions: excitation at 346 nm, time-
delay = 0.1 ms, [Tb-DOTAm-Phen] = 10 μM, water, [Tris] = 10 mM,
pH 7.0, T = 20 °C.
transfer from the antenna to the lanthanide and consequently
quenching terbium luminescence. All three adenosine nucleo-
tides were expected to quench the probe’s luminescence.
Differentiation among ATP, ADP, and AMP is achieved via
weak electrostatic interactions between the +3 charged terbium
complex and the negatively charged nucleotides. The higher the
number of phosphates, the more negative the charge of the
nucleotide, and the higher its affinity for the positively charged
probe. Although these interactions are weak in water, we
postulated that they would be sufficient to distinguish the three
nucleotides at their millimolar intracellular concentra-
tions.17−20,26
The molecular probe Tb-DOTAm-Phen (1) was synthesized
according to Scheme 1. Suzuki coupling of 2-bromonitroben-
zene with phenylboronic acid yielded the nitrobiphenyl (2)
which was subsequently reduced to an amine and acetylated to
the acetamide (3). The amide was subsequently cyclized to 6-
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Journal of the American Chemical Society
Communication
from ADP and AMP under physiologically relevant conditions
renders it an attractive candidate for direct monitoring of
enzymatic reactions involving ATP hydrolysis such as those of
kinases and ATPases. We are currently investigating its
application in high-throughput screening and kinetic studies
of inhibition of kinases.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed experimental procedures, characterization of Tb-
DOTAm-Phen (1), and fluorescence spectra of Tb-DOTAm-
Phen·ATP titration. This material is available free of charge via
the Internet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
pierre@umn.edu
■
Notes
Figure 3. Stern−Volmer plot of the time-delayed luminescence of Tb-
DOTAm-Phen (1) by the adenosine nucleotides ATP (filled circles),
ADP (open triangles), and AMP (gray diamonds). Experimental
conditions: excitation at 346 nm, emission integrated from 450 to 650
nm, time-delay = 0.1 ms, [Tb-DOTAm-Phen] = 10 μM, water, [Tris]
= 10 mM, pH 7.0, T = 20 °C, error bars represent SD, n = 3.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Institutional Research Grant No.
118198-IRG-58-001-52-IRG80 from the American Cancer
Society and the University of Minnesota.
Table 1. Stern−Volmer Constants of Tb-DOTAm-Phen (1)
a
with Adenosine Nucleotides
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