ATP selective acridone based fluorescent probes for monitoring of metabolic events

Article abstract of DOI:10.1039/c1cc10253b

Acridones carrying an appropriate substituent at N-10 showed significant fluorescence changes on interacting with ATP in HEPES buffer at pH 7.2. The selectivity and sufficient binding of these probes with ATP could be useful for monitoring of metabolic processes.

Full text of DOI:10.1039/c1cc10253b

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COMMUNICATION
ATP selective acridone based fluorescent probes for monitoring of
metabolic eventsw
Jatinder Kaur and Palwinder Singh*
Received 14th January 2011, Accepted 18th February 2011
DOI: 10.1039/c1cc10253b
Acridones carrying an appropriate substituent at N-10 showed
significant fluorescence changes on interacting with ATP in
HEPES buffer at pH 7.2. The selectivity and sufficient binding
of these probes with ATP could be useful for monitoring of
metabolic processes.
P-gp, ATP and Mg2+). It was envisaged that increasing the
length of the N-10 substituent of acridone and introducing more
H-donor/acceptor sites thereon might result in their better
interactions with ATP. Compounds 1–3 (Chart 1) were
prepared by the reaction of acridone with epichlorohydrin
followed by ring opening with benzyl-/o-aminobenzyl-/
p-aminobenzylamine (Scheme S1, Fig. S1–S3, ESIw).
Fluorescence signalling is an important phenomenon for the
monitoring of biological processes through selective recognition
of a crucial component of the process. In fact this is the
primary step towards the development of drugs, probes,
molecular machines, etc. Amongst various biological anions,
adenosine triphosphate (ATP)1 is the energy currency of the
cell and various cellular processes are linked to the interchange
between ATP and adenosine diphosphate (ADP). To name a
few, the glycolytic pathway of glucose metabolism,2 Kreb’s
cycle,3 conversion of ribose sugar to deoxyribose sugar (raw
material for DNA synthesis),4 transporting activities of
proteins,5 working of the Na+/K+ pump3 are the fundamental
activities of life involving generation/consumption of ATP.
The monitoring of these activities genuinely helps in exploring
the working of natural systems and secrets of life.6 The
fluorogenic sensing and quantitative measurement of ATP
could be a versatile method for studying the metabolic pathways.
Irrespective of a number of chromogenic as well as fluorogenic
sensors of ATP7 there are few reports where a molecule could
selectively interact with ATP and only one commercialized
‘luciferin–luciferase bioluminescence assay’ for ATP determi-
nation is available.8 Here, working under aqueous conditions,
the new molecules selectively interact with ATP and have
successfully been demonstrated for the monitoring of two
metabolic events and hence an issue of biological and medicinal
importance was addressed.
The UV-visible spectrum of compound 1 at 50 mM concen-
tration in HEPES buffer (pH 7.2) exhibited absorption bands
at 253, 387 and 405 nm. Addition of 0–3 equivalents of ATP to
the above solution of compound 1 resulted in significant
decrease in absorbance at 253 nm indicating interactions of
this compound with ATP (Fig. S4, ESIw). Similar changes in
the absorption spectra of compounds 2 and 3 (Fig. S5 and S6,
ESIw) were observed on incremental addition of ATP. The
fluorescence spectrum of a solution of compound 1 (0.1 mM,
F = 0.67) in HEPES buffer (pH 7.2) showed emission bands
at 417 nm and 440 nm when excited at 253 nm. Upon
incremental addition of ATP (0–17 equiv.) (0–1.7 mM) to the
solution of compound 1, the fluorescence gets quenched
(97.5%) at 417 nm as well as at 440 nm (Fig. 1). The
Benesi–Hildebrand12 plot indicated 1 : 1 stoichiometry of 1Á
ATP complex (Fig. S7, ESIw). Similar emission bands and
quenching of fluorescence were observed in the fluorescence
spectra of compounds 2 (F = 0.60) and 3 (F = 0.66) upon
incremental addition of ATP (Fig. S8 and S9; Table S1, ESIw).
The design of new molecules was based on our previous
reports9 concerned with the development of multi-drug resis-
tance modulators.10 Those molecules (A, Chart 1) inhibit the
efflux of R6G from the cell and seemed to work through the
inhibition of the P-glycoprotein efflux pump11 (constituted by
Chart 1
Department of Chemistry, Guru Nanak Dev University, Amritsar-
143005, India. E-mail: palwinder_singh_2000@yahoo.com;
Fax: +91 183 2258819; Tel: +91 183 2258802x3495
w Electronic supplementary information (ESI) available: Synthetic
procedure, experimental data and NMR spectra of compound 1–3,
fluorescence spectra for selectivity and competitive binding of
compounds 1–3 with ATP. See DOI: 10.1039/c1cc10253b
Fig. 1 Fluorescence spectra of 1 (0.1 mM) on incremental addition of
ATP.
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The change in the UV spectra as well as in the fluorescence
quenching of these compounds on addition of ATP seems to
be due to their p–p and electrostatic interactions with ATP.
To look into the probable mode of binding of compounds
1
1–3 with ATP, H NMR titrations of these compounds upon
addition of ATP were carried out. 1H NMR spectra of
compounds 1–3 (DMSO + D2O) in the presence of 3 equiv.
of ATP showed a downfield shift by 0.13–0.25 ppm of
two CH2 groups attached to NH, 0.05–0.13 ppm of H attached
to C-12 and 0.094–0.116 ppm of the aromatic protons
(Fig. S10–S12; Table S2, ESIw). It seems as ATP is overlapping
(Fig. S13, ESIw) compound 1 with polar interactions between
the two tails (a substituent at N-9 of adenine and N-10 of
acridone) and p–p interactions between the head groups
(adenine and acridone), photoinduced electron transfer occurs
between adenine and acridone fragments resulting in quench-
ing of fluorescence. No such overlapping in the energy mini-
mized structures of 1 with ADP and AMP was observed. The
Fig. 3 Fluorescence response of compound 1 to various homologues/
analogues of ATP. Pink bars: (1) 1, (2) 1 + adenosine, (3) 1 + AMP,
(4) 1 + ADP, (5) 1 + GTP, (6) 1 + GDP, (7) 1 + ITP, (8) 1 + IDP,
(9) 1 + CTP, (10) 1 + CDP, (11) 1 + UTP, (12) 1 + UDP. Blue bars
represent subsequent addition of 17 equivalents of ATP to the solution
(1) 1 + ATP, (2) 1 + adenosine + ATP, (3) 1 + AMP + ATP,
(4) 1 + ADP + ATP, (5) 1 + GTP + ATP, (6) 1 + GDP + ATP,
(7) 1 + ITP + ATP, (8) 1 + IDP + ATP, (9) 1 + CTP + ATP, (10) 1 +
CDP + ATP, (11) 1 + UTP + ATP, (12) 1 + UDP + ATP.
and the conversion of phosphoenolpyruvate (PEP) to pyruvate
in the presence of pyruvate kinase (PK) and Mg2+ coupled
with the transformation of ADP into ATP (Scheme 1).
A modeled reaction was carried out in HEPES buffer at pH 7.2
and compound 1 was used as fluorescent probe. ADP, PEP
and PK did not exhibit fluorescence either individually or in
combination with one another (overlapped green, violet,
maroon traces; inset of Fig. 4). Compound 1 (0.1 mM) in
combination with ADP and PEP exhibited usual fluorescence
when excited at 253 nm (uppermost red trace, Fig. 4). Addition
of PK and Mg2+ to the above solution (compound 1 + ADP
+ PEP) resulted in quenching of fluorescence (Fig. 4). Further
increase of concentration of ADP in this solution resulted in
a stepwise quenching of fluorescence in the same pattern as
qthe quenching of fluorescence of compound 1 on incremental
addition of ATP (Fig. 1). Therefore compound 1 was employed
to monitor the transformation of PEP into pyruvate, accom-
panied with the efficient conversion of ADP to ATP, which
resulted in quenching of fluorescence in a time-dependent
manner (Fig. 5).
binding constants of compounds 1–3 for ATP (6–8 Â 107 MÀ1
)
are significantly better than the reported ones7a,b,f (Table S3,
ESIw).
In contrast to the response of compounds 1–3 to ATP,
addition of adenosine, AMP, ADP, GDP, IDP, CDP, UDP,
GTP, ITP, CTP, and UTP separately to the solutions of
compounds 1–3 resulted in no change in their fluorescence
spectra pointing towards the selective recognition of ATP
(amongst its homologues/analogues) by compounds 1–3
(Fig. 2). This indicates that the nucleobase as well as the
phosphate group(s) contribute to interaction between the
compound and nucleotide.
Moreover, to check the practical applicability of compounds
1–3, their competitive binding with ATP, in the presence of
homologues/analogues of ATP, was studied which showed
that these compounds bind to ATP even in the presence of
ADP, AMP, adenosine, GTP, GDP, ITP and IDP (Fig. 3;
Fig. S14 and S15, ESIw).
Therefore, with the acridone derivatives 1–3, working under
aqueous conditions, 94–97% quenching of fluorescence was
observed on addition of ATP, which along with their selectivity
for ATP could be utilized for the monitoring/controlling of
cellular processes involving generation/consumption of ATP.
Here we demonstrated how an acridone derivative is utilized
for in vitro real time monitoring of fluorescence in response to
the generation and consumption of ATP in two enzymatic
reactions.
Scheme 1 Enzymatic reaction involving formation of ATP.
First enzymatic reaction (last step of the payoff phase of
glucose metabolism) is the phosphate transfer reaction, involving
the transfer of phosphate from phosphoenolpyruvate to ADP
Fig. 4 Fluorescence change of compound 1 in a phosphate transfer
reaction catalyzed by enzyme PK in 0–90 min. The inset shows the
expanded part: ADP (green line), ADP + PEP (violet line), ADP +
PEP + PK (maroon line).
Fig. 2 Selectivity of compounds 1–3 for ATP amongst adenosine,
AMP, ADP, GDP, IDP, CDP, UDP, GTP, ITP, CTP and UTP.
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The reaction rate was accelerated by increasing the concen-
tration of ADP. The Lineweaver–Burk plot (Fig. 6) gave a
Michaelis constant (Km) of 249 mM which agrees with the
reported one.13
The first step of the preparatory phase of glucose metabolism,
involving phosphorylation of glucose in the presence of
hexokinase (HK) and Mg2+ coupled with the conversion of
ATP to ADP (Scheme 2), was also monitored using compound
1. Here, reverse of Scheme 1 occurred, the non-fluorescent
solution of ATP, glucose and compound 1 gains about 50%
fluorescence intensity (w.r.t. the pure compound) upon addition
of hexokinase in a time dependent manner indicating the
conversion of ATP to ADP (Fig. 7 and 8).
Fig. 8 Representation of fluorescence intensity change of compound
1 in a phosphorylation reaction.
Kinetic analysis indicated that the initial rate of reaction is
proportional to the amount of HK which is based on first-
order reaction kinetics (Fig. 9).
Fig. 9 Plot of the initial rate (v0, mM minÀ1) as a function of the
amount of HK.
Therefore, we have developed ATP selective acridone based
fluorescent probes workable under physiological conditions
and through simple experiments, their use for the monitoring
of enzymatic reactions was studied. This paves the way for
their more applications in studying the metabolic pathways
involving generation/consumption of ATP.
The authors are thankful to DST, New Delhi, for the
research grant. JK thanks CSIR, New Delhi, for SRF.
Notes and references
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Fig. 5 Representation of fluorescence intensity change of compound
1 in a phosphate transfer enzyme reaction.
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Scheme 2 Enzymatic reaction involving formation of ADP.
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Fig. 7 Fluorescence change of compound 1 (red line) in a phosphor-
ylation reaction catalyzed by enzyme HK in 0–50 min. The inset shows
glucose + ATP (overlapped blue line), glucose + ATP + HK +
Mg2+ (overlapped pink line).
c
4474 Chem. Commun., 2011, 47, 4472–4474
This journal is The Royal Society of Chemistry 2011