A fluorescent probe for estimation of adenosine diphosphate and monitoring of glucose metabolism

Article abstract of DOI:10.1039/c3ob42505c

An ADP selective fluorescent probe working in aqueous medium was identified and the change in fluorescence as a function of ADP concentration was standardized. Using this probe, all the steps of glycolysis coupled with ATP/ADP inter-conversion and oxidative breakdown of pyruvate in the mitochondria were monitored and the consumption/production of ATP/ADP at each step was quantified. The quantity of ADP present in the mitochondria, taken from different body parts of a pig, was also determined. It is hypothesized that an appropriate modification of the technique may provide a diagnostic tool for monitoring biochemical pathways as well as for quick estimation of ADP in the mitochondria and other cell organelles.

Full text of DOI:10.1039/c3ob42505c

Organic &
Biomolecular Chemistry
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PAPER
A fluorescent probe for estimation of adenosine
diphosphate and monitoring of glucose
metabolism†
Cite this: Org. Biomol. Chem., 2014,
12, 3071
Arun Kumar, Parteek Prasher and Palwinder Singh*
An ADP selective fluorescent probe working in aqueous medium was identified and the change in fluo-
rescence as a function of ADP concentration was standardized. Using this probe, all the steps of glycolysis
coupled with ATP/ADP inter-conversion and oxidative breakdown of pyruvate in the mitochondria were
monitored and the consumption/production of ATP/ADP at each step was quantified. The quantity of ADP
present in the mitochondria, taken from different body parts of a pig, was also determined. It is hypo-
thesized that an appropriate modification of the technique may provide a diagnostic tool for monitoring
biochemical pathways as well as for quick estimation of ADP in the mitochondria and other cell
organelles.
Received 14th December 2013,
Accepted 4th March 2014
DOI: 10.1039/c3ob42505c
www.rsc.org/obc
Introduction
Results and discussion
Monitoring of a metabolic process carries immense biological
and medicinal significance in exploring the working of natural
systems and providing useful diagnostic information about the
functioning of a biochemical pathway. Since most of the bio-
chemical reactions are coupled to ATP–ADP inter-conversion,1
approximately 0.005 ng of ATP is used in a typical cell every
1–2 min making a total of 50–75 kg of ATP to hydrolyze in the
body everyday along with an equivalent amount of production
of ATP as well.2 Therefore, besides the use of tracer tech-
niques3 for monitoring biochemical processes, the quantifi-
The idea to increase the fluorescence intensity of the com-
pound by having two acridone moieties in the molecule in
comparison with one in our previous ATP selective probe4m led
us to synthesize dimeric type compounds 2 and 3 by linking
the two acridones through simple spacer groups. Synthesis of
acridone from anthranilic acid and chlorobenzene was fol-
lowed by treatment with epichlorohydrin to procure compound
1. Further reaction of compound 1 with piperazine under
microwave irradiation provided compound 2 (Scheme 1). In
order to see the effect of the spacer group between two acri-
dones on the nucleotide selectivity of the compound, another
linker was chosen and compound 3 was prepared by the reac-
tion of compound 1 with 2-[2-(2-aminoethoxy)ethoxy]ethyl-
amine under microwave irradiation.
cation of formation/consumption of ATP/ADP during
a
biochemical reaction provides an alternative method to
examine the working of a metabolic pathway. Although a
number of ATP/ADP selective probes are reported only a few of
them are employed for the monitoring of metabolic events.4
Here, we report a highly fluorescent, ADP selective probe used
for quantification of consumption/production of ATP/ADP
during various steps of glycolysis and further breakdown of
pyruvate to CO2 in the mitochondrial system. The probe was
also employed to quantify and compare the ADP present in the
mitochondria taken from different body parts of a pig and a
good correlation between the amount of ADP and the function
of those particular tissues was observed.
The fluorescence spectrum of compounds 2 and 3 and their
non-selectivity/selectivity for ADP
The fluorescence spectrum of a solution of compound 2
(10 µM) in DMSO–HEPES buffer (1 : 4) exhibited strong emis-
sion at 427 nm (Φ = 0.63) when excited at 250 nm. Incremental
(12 µL) addition of adenosine diphosphate (ADP) (0.5 ng in
12 µL HEPES buffer, 0.1 µM) to the solution of compound 2
(1.2 ml in DMSO–HEPES buffer, 1 : 4) decreased the fluo-
rescence intensity of the solution (trace A to trace B, Fig. 1)
after the addition of 360 μL ADP. No further change in the
fluorescence intensity was observed when more ADP was
added. However, addition of 360 μL ATP (the same concen-
tration as for ADP) to the solution of compound 2 almost
quenched the fluorescence (trace A to trace C, Fig. 1). It means
UGC Sponsored Centre for Advanced Studies, Department of Chemistry, Guru Nanak
Dev University, Amritsar-143005, India. E-mail: palwinder_singh_2000@yahoo.com;
Fax: +91-183-2258820; Tel: +91-183-2258802-09 x 3495
†Electronic supplementary information (ESI) available: NMR, Mass spectral data
of compound
2 and 3 alone and in combination with ADP. See DOI:
10.1039/c3ob42505c
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Scheme 1 Synthesis of compounds 2 and 3.
the selectivity of compound 3 for ADP (Fig. 2B). Addition of
ADP to a solution of compound 3 in the presence of ATP/AMP
decreased the fluorescence intensity to the same extent as with
ADP alone pointing towards non-interference of ATP/AMP
during the interaction of compound 3 with ADP (Fig. 2B). The
1H NMR spectrum of compound 3 in the presence of ADP
showed small changes in the chemical shifts of aromatic
protons as well as the protons of the spacer group (Fig. S1†),
while no such changes in chemical shifts were observed in the
presence of ATP. On comparing the previously reported ATP
selective monomeric type compounds4m with compounds 2
and 3, it is apparent that the interaction of these compounds
with ATP/ADP may be influenced by the orientation and nature
of the substituent on acridone. Furthermore, to rationalize the
selectivity of the compounds for ATP/ADP, some physico-
chemical parameters5 of compounds 2 and 3 and the com-
pounds reported previously4m were calculated (Table S1†). It
was observed that compound 3 has Log P and total polar
surface area (TPSA) quite different from the other compounds
but relatively close to the Log P and TPSA of ADP. It may be
due to this similarity in the topology of the two molecules that
compound 3 is compatible to interact with ADP. The Benesi–
Hildebrand plot indicated a 1 : 1 stoichiometry of the 3·ADP
complex (Fig. S2, ESI†) and their binding constant (K) was cal-
culated as 2.14 × 105 M−1 (ESI†). Moreover, the solution of
compound 3 did not exhibit any change in fluorescence on
addition of Mg2+, Co2+, Zn2+, Mn2+, Fe2+, Fe3+, Cu2+, Hg2+,
Pd2+, Ag+, Ni2+, Na+, and K+, the common biological cations.
The high resolution electrospray mass spectrum (ESI-HRMS)
of the solution of compound 3 with ADP also showed for-
mation of the 3·ADP complex (Fig. 2C). Hence, a fluorescent
probe, workable in aqueous medium and showing consider-
able selectivity for ADP, is identified which proved helpful in
Fig. 1 Change in the fluorescence intensity of the solution of com-
pound 2 (10−5 M) in DMSO–HEPES buffer (1 : 4), pH 7.2 (trace A) on
addition of ADP (trace B), and addition of ATP (trace C). Excitation wave-
length 250 nm.
compound 2 is responding to both ATP and ADP and exhibited
non-selectivity between the two nucleotides.
The fluorescence spectrum of the solution of compound 3
(10 µM) in DMSO–HEPES buffer (1 : 4) also showed a strong
emission band at 430 nm (Φ = 0.68) when excited at 250 nm.
Stepwise (12 µL) addition of adenosine diphosphate (ADP)
(0.5 ng in 12 µL HEPES buffer, 0.1 µM) to the solution of com-
pound 3 (1.2 ml in DMSO–HEPES buffer, 1 : 4) (final concen-
tration of ADP in 1.2 ml solution was 0.001 µM) resulted in a
decrease in fluorescence intensity of the solution until com-
plete quenching of fluorescence had taken place when 153 ng
ADP (addition of 360 µL of ADP, 1 µM) was added to the solu-
tion (Fig. 2A). However, no change in the fluorescence of solu-
tion of compound 3 was observed in the presence of even
300 ng (in 12 µL HEPES buffer) ATP (45 µM), AMP (72 µM) and
other nucleotides (GTP, GDP, CTP, CDP, UTP, UDP), indicating
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Fig. 2 (A) Decrease in the fluorescence intensity of the solution of compound 3 (10−5 M) in DMSO–HEPES buffer (1 : 4), pH 7.2 on stepwise addition
of ADP. Excitation wavelength 250 nm. (B) Fluorescence change of the solution of compound 3 in DMSO–HEPES buffer (1 : 4, pH 7.2) in the presence
of other nucleotides (45 µM) showing selective and competitive binding of compound 3 with ADP. (C) HRMS of the solution of compound 3 and
ADP showing formation of the 3 + ADP complex with m/z 1078.3378 (calcd m/z 1078.3471). (D) Normalized curve for the change in fluorescence
intensity of solution of compound 3 as a function of ADP concentration (black line). The red line indicates the actual position of data points.
quantification of ADP during a biochemical reaction as well as
in biological samples. Besides a recent report by Feng et al.4b
about a mononuclear zinc complex appended with two anthra-
cene groups as a highly selective ADP sensor, compound 3 is
amongst the very few ADP selective probes reported so far. In
order to utilize the results of these experiments for monitoring
ATP–ADP coupled biochemical reactions, a curve showing
changes in the fluorescence intensity of a solution of com-
pound 3 as a function of ADP concentration was standardized
(Fig. 2D).
Monitoring of glucose metabolism (Phase I)
A
quick review of the process of glucose metabolism
(Scheme 2) indicates formation of ADP in steps 1 and 3 and
consumption of ADP in steps 6, 8 and 9. Starting with calcu-
lated amounts of glucose and other reagents required for reac-
tions 1, 3, 6, 8 and 9, all these steps of glucose metabolism
were monitored using probe 3.
Scheme 2 Various steps of glucose metabolism showing formation/
consumption of ADP/ATP.
Phosphorylation of glucose
A solution of D-glucose (50 µM), hexokinase (80 units), Mg2+
(50 µM) and compound 3 (10 µM in DMSO) in HEPES buffer
(2 ml) showed the usual fluorescence intensity of compound 3
(trace A, Fig. 3A), indicating no effect of glucose and hexo-
kinase on fluorescence behaviour of the compound. The
moment 100 µL ATP (4 ng ATP in 100 µL HEPES buffer; 0.0036
µM in 2 ml final solution) was added to this solution, the fluo-
rescence intensity started decreasing and became constant at
8000 A.U. (trace B, Fig. 3A). As calculated from the standard
curve (Fig. 2C), the amount of ADP in the solution is ∼3.1 ng
(0.0036 µM) which indicates that all the ATP in the solution
have been changed to ADP. With the same amount of reactants
(as used above), kinetic analysis of this enzymatic reaction
was studied by varying the concentration of hexokinase and
it was found to be first order (Fig. 3B). The Lineweaver–Burk
plot (Fig. 3C) gave the Michaelis–Menten constant (Km) of
125 µM corresponding to the reported one 160 µM.6 HRMS of
the reaction mixture in this step of glucose metabolism
(Fig. 3D) did not show any peak due to the mass of ATP while
the peak at m/z 261.0355 corresponding to the mass of phos-
phorylated glucose (calcd m/z 261.0370 [M + H]+) is quite
intense. As a control experiment, the same reaction was per-
formed in the absence of hexokinase. No change in the fluo-
rescence intensity was observed in this experiment. Therefore,
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Fig. 3 (A) Change in fluorescence from A to B during the reaction in the first step of glucose metabolism. (B) Initial rate as a function of the amount
of hexokinase. (C) Lineweaver–Burk plot. (D) A part of HRMS of the reaction mixture in the first step of glucose metabolism.
a simple protocol for quantification of consumption of ATP from the standard curve, the quenching of fluorescence corre-
and production of ADP helped in monitoring the enzymatic sponds to the presence of 3.1 ng ADP (0.0036 µM) in the solu-
phosphorylation of glucose.
tion. Kinetic analysis of this reaction indicates it to be a first
order reaction (Fig. 4B). The Michaelis–Menten constant Km
was calculated as 7 µM (lit.7 3 µM) (Fig. 4C). HRMS of the reac-
tion mixture showed a peak at m/z 339.9961 which corresponds
to the mass of fructose diphosphate (calcd m/z 339.9955 [M]+)
(Fig. 4D). Hence, showing the quantitative conversion of ATP
to ADP, the second phosphorylation step of glucose meta-
bolism was also successfully mimicked. In this experiment
too, no change in fluorescence was observed in the absence of
phosphofructokinase.
Phosphorylation of fructose 6-phosphate
Similar to the monitoring of the above reaction, phosphoryl-
ation of fructose-6-phosphate (step 3, Scheme 2) was per-
formed. The fluorescent solution of compound 3 (10 µM),
fructose-6-phosphate (F-6-P) (50 µM) and ATP (4 ng ATP in
100 µL HEPES buffer; 0.0036 µM in 2 ml final solution) under-
went a decrease in fluorescence (trace A to trace B, Fig. 4A)
when phosphofructokinase (80 units) was added. Estimated
Fig. 4 (A) Change in fluorescence from A to B during the reaction in the third step of glucose metabolism. (B) Initial rate as a function of the
amount of phosphofructokinase. (C) Lineweaver–Burk plot. (D) A part of HRMS of the reaction mixture in the third step of glucose metabolism.
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Formation of 3-phosphoglycerate
rescence as observed for the solution of compound 3 alone.
This change in fluorescence intensity of the solution clearly
indicated the consumption of all the ADP (formation of
∼55 ng ATP, 0.05 µM). Here also the reaction was found
to be first order and the Michaelis–Menten constant Km
250 µM corresponds to the reported value (260 µM)9 (Fig. 6C).
Formation of pyruvate (as sodium pyruvate) was also ascer-
tained from HRMS of the reaction mixture (Fig. 6D) (calcd m/z
132.9872 [M + Na]+).
Therefore, using fluorescent probe 3, all those steps of
glucose metabolism, which involve either consumption of ADP
or generation of ADP (steps 1, 3, 6 and 8; Scheme 2) were
monitored. It is hypothesized that starting from the calculated
amount of glucose and by performing all the steps in a
sequence (transferring the solution from the first vial to the
next one), phase I of glucose metabolism, as shown in the ani-
mated figure, could be demonstrated in one go (animated
Fig. S1, see ESI†). It is worth mentioning that the formation of
ADP during steps 1 and 3 and ATP during the reactions of 6th
and 8th steps was also quantified with the help of LC-MS
using a QuantAnalysis2.0. The results of mass spectral experi-
ments and fluorescence experiments were very close
(Table S2†) with a difference of <4%.
During the dephosphorylation steps of glucose metabolism,
the measurement of the increase in fluorescence intensity of
the non-fluorescent solution of compound 3 (in the presence
of ADP) also gave quantitative information about ADP con-
sumed and the consequent formation of ATP during the reac-
tions 6, 8 and 9 in Scheme 2. To demonstrate step 6 of glucose
metabolism, a solution of 1,3-bisphosphoglycerate (50 µM),
ADP (50 ng ADP in 100 µL HEPES buffer; 0.058 µM in 2 ml
final solution) and compound 3 (10 µM) was taken in 2 ml
HEPES buffer. The moment phosphoglycerokinase (80 units)
was added to this solution; its fluorescence started increasing
and attained the same intensity as that of the pure compound
(trace A to trace B, Fig. 5A). This observation clearly indicated
the consumption of almost all the ADP (formation of ∼55 ng
ATP, 0.05 µM in 2 ml final solution) during this reaction while
formation of 3-phosphoglycerate was also detected in HRMS of
the reaction mixture (Fig. 5D) (calcd m/z 208.9822 [M + Na]+).
Kinetic analysis indicated this reaction to be first order
(Fig. 5B) and the experimental Michaelis–Menten constant
(142.85 µM) corresponds to the reported one (180 µM)8
(Fig. 5C).
Conversion of phosphoenolpyruvate to pyruvate
Quantification of ADP in the mitochondria
For monitoring the last step of the payoff phase of glycolysis
viz. conversion of phosphoenolpyruvate to pyruvate (step 8, A solution of 20 µL of compound 3 (DMSO) and 10 mg mito-
Scheme 2), a solution of phosphoenolpyruvate (50 µM), com- chondrial mass in 100 µL resuspended buffer (extracted from
pound 3 (10 µM) and ADP (50 ng ADP in 100 µL HEPES buffer; the pig liver and refined as per the reported procedure10) in
0.058 µM in 2 ml assay solution) in 2 ml HEPES buffer 1 ml final solution in HEPES buffer (compound 3 is of 10 µM)
(pH 7.2) was prepared. After the addition of pyruvate kinase showed fluorescence as per trace B of Fig. 7. This decrease in
(80 units) to the reaction mixture, the solution slowly turned fluorescence intensity of the otherwise fluorescent solution of
fluorescent (Fig. 6A) and within 2 min, it gained the same fluo- compound 3 (trace A, Fig. 7) was assumed to be due to the
Fig. 5 (A) Change in fluorescence from A to B during the reaction in the sixth step of glucose metabolism. (B) Initial rate as a function of the
amount of phosphoglyceratekinase. (C) Lineweaver–Burk plot. (D) A part of HRMS of the reaction mixture showing the formation of phosphoglyce-
rate with m/z 208.9835 (calcd m/z 208.9822 [M + Na]+).
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Fig. 6 (A) Change in fluorescence from A to B during the reaction corresponding to conversion of phosphoenolpyruvate to pyruvate. (B) Initial rate
as a function of the amount of pyruvatekinase. (C) Lineweaver–Burk plot. (D) A part of HRMS of the reaction mixture showing the formation of
sodium pyruvate with m/z 132.9878 (calcd m/z 132.9872 [M + Na]+).
Breakdown of pyruvate in the mitochondria (phases II and III
of glucose metabolism)
Interesting observations were recorded while performing
experiments corresponding to phases II and III of glucose
metabolism viz. breakdown of pyruvate to CO2 (step 9,
Scheme 2). In order to perform the experiment for breakdown
of pyruvate in the mitochondria, 30 mg mitochondria (carrying
∼150 ng ADP), extracted from the pig liver, was taken in
100 ml HEPES buffer (pH 7.2) in a two necked round bottom
flask (rbf) (Fig. S5†). Through a rubber tube, one end of the
Fig. 7 Change in fluorescence of the solution of compound 3 from
trace 2 to trace 1 when mitochondrial solution was added.
rbf was connected to a test tube containing lime water. On
addition of pyruvate solution (1.5 mg in 5 ml HEPES buffer)
through a dropping funnel fixed in the second neck of rbf, the
solution in the rbf started turning fluorescent and bubbling
was observed in lime water. Within 2–3 min, the solution in
presence of ADP in the mitochondrial system because almost
the rbf became fluorescent (Fig. 8) and a solid appeared in
all the other components associated with the mitochondria
lime water. Since CO2 is generated during the breakdown of
were removed.10 As per the standard curve (Fig. 2D), ∼50 ng of
ADP seems to be there in the tested sample of mitochondria. A
high resolution mass spectrum of mitochondrial solution also
showed a mass peak at m/z 428.0377 corresponding to the
mass of ADP (calcd m/z 428.0367, [M + H]+) (Fig. S4†) and
48 ng ADP in 10 mg mitochondrial mass was quantified
by LC-MS using QuantAnalysis2.0. For comparison,
mitochondria from heart muscles and leg muscles of a pig
were also isolated. Performing the same experiment as for
the liver mitochondria, here, respectively 100 ng and 40 ng of
ADP were estimated in 10 mg mitochondrial mass extracted
from heart muscles and leg muscles. The amount of ADP esti-
mated in three types of tissues taken from different body parts
seems to be almost consistent with the functions of these
tissues. Hence the experiments demonstrated here provide a
Fig. 8 Change in fluorescence of the solution of compound 3 and
practical approach for estimation of ADP in the biological
samples.
mitochondrial mass from trace A (non-fluorescent in the presence of
mitochondrial mass) to trace B on addition of pyruvate.
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pyruvate in the mitochondria, 4 mg CaCO3 was isolated from with the help of probe 3. Interestingly, the amount of ATP
the lime water which corresponds to the formation of produced in this experiment was in excellent agreement
0.052 mmol of CO2. Consistent with oxidative transformation with that calculated from the LC-MS experiment using
of pyruvate (eqn (1)), in this experiment, the total available QuantAnalysis.
pyruvate (1.5 mg) had undergone breakdown to generate CO2.
Therefore, the results of this experiment demonstrated the
part of glucose metabolism which takes place in the
mitochondria.
Conclusions
A highly versatile approach for monitoring biochemical reac-
tions is demonstrated. Using a fluorescent and ADP selective
probe, the consumption/production of ATP/ADP during the
ð1Þ
four reactions of phase I of glucose metabolism was quanti-
fied. The same probe was also used for estimation of ADP in
the mitochondria of the pig liver, heart and leg muscles and
breakdown of pyruvate to CO2 in the mitochondria was moni-
Combination of the process of glycolysis with oxidative
breakdown of pyruvate
tored. The experimental protocol could be highly useful to
As per the results of above experiments, production/consump-
show breakdown of glucose to CO2, monitor ATP/ADP coupled
tion of ADP can be quantified during various reactions of
phase I (glycolysis) of glucose metabolism. Moreover, the
amount of pyruvate metabolized in the mitochondria can be
calculated, if the step corresponding to conversion of fructose
diphosphate to phosphoenolpyruvate is also achieved; starting
from step 1, sequentially performing all the eight reactions of
biochemical reactions, compare the healthy/unhealthy state of
mitochondria from different body parts and with certain modi-
fications, it may prove as medicinally significant in diagnosis.
Experimental
phase I, the reaction mixture in the last step can be passed
General note
onto mitochondria (phase II/III) and hence breakdown of
glucose to CO2 can be demonstrated (animated Fig. S2, see
ESI†) by looking at the fluorescence change at each step. In
order to check the workability of this connection between
phase I and phase II/III reactions, the reaction mixture in the
last step of phase I of glucose metabolism (containing pyru-
vate, ATP) was subjected to mitochondrial solution. The fluo-
rescent solution of step 8 (Scheme 2) turned non-fluorescent
when it was added to 30 mg mitochondria taken in 50 ml
HEPES buffer (trace A, Fig. 9) indicating the presence of ∼150
ng ADP in the mitochondrial solution. Within 2–3 min, the
solution started turning fluorescent and attained 5000 AU fluo-
rescence (trace B, Fig. 9). This has probably happened due to
the metabolism of pyruvate in mitochondrial solution and
conversion of ADP to ATP. Using the standard curve (Fig. 2D),
it was calculated that 15 ng ADP was present in the solution
and approximately 105 ng was converted to ATP. Hence, it is
possible to link phase I reactions of glucose metabolism to
phase II/III and breakdown of glucose to CO2 could be studied
1H and 13C NMR spectra were recorded on JEOL 300 MHz
NMR spectrometer using DMSO-d6 as a solvent. Chemical
shifts are given in ppm with TMS as an internal reference.
J values are given in hertz. Mass spectra were recorded on a
Bruker micrOTOF Q II Mass spectrometer. The low intensity of
desired peaks was due to more intense HEPES peaks in the
mass spectra because all these reactions were performed in
HEPES buffer. The reactions corresponding to epoxy ring
opening with amines were performed in a microwave synthesi-
zer (BIOTAGE INITIATOR EXP – EU) at 75 watt and 140 °C.
Reactions were monitored by thin layer chromatography (TLC)
on glass plates coated with silica gel GF-254. Column chrom-
atography was performed with 60–120 mesh silica. IR and UV
spectral data were recorded on FTIR (VARIAN 660 IR) and
BIOTEK Synergy H1 Hybrid Reader instruments, respectively.
The fluorescence spectra were recorded on a BIOTEK Synergy
H1 Hybrid Reader spectrofluorophotometer (excitation at
250 nm). Glucose, hexokinase, fructose-6-P, phosphofructo-
kinase, phosphoglycerokinase, pyruvate kinase, phosphoenol-
pyruvate, pyruvate, ATP, ADP, AMP, GTP, CTP, UTP and other
nucleotides were purchased from Sigma-Aldrich. 1,3-Bisphos-
phate glycerate was prepared in step 5 (Scheme 2). Pig tissues
from different body parts were taken from a local butcher’s
shop.
Experimental procedure
N-Phenyl anthranilic acid (5 g) was treated with 10 ml conc.
H2SO4 at 90–100 °C for 1.5 h and 10H-acridin-9-one (5 g) was
procured. NaH (200 mg) was washed with dry hexane and
added to 20 ml DMSO followed by addition of 5 g acridone
and 2 ml epichlorohydrin. The reaction mixture was heated at
Fig. 9 Fluorescence change (from trace A to trace B) when the solution
in step 8 of phase I was subjected to mitochondria.
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70 °C for 18 h (TLC monitoring). After the addition of water Phosphorylation of glucose
(20 ml) to the reaction mixture, it was extracted with ethyl
Taking the calculated volume of all the components, an assay
solution of ATP (0.0036 μM), glucose (50 μM) and compound 3
(10 μM) was prepared in 2 ml HEPES buffer (pH 7.2). After
addition of hexokinase (80 units) and Mg2+ (50 μM), the fluo-
rescence intensity (excitation at 250 nm) of the solution was
recorded. A 42% decrease in fluorescence intensity was
observed. The concentration of ADP was calculated from the
change in fluorescence intensity which on dividing by the time
taken for the change gave the initial velocity (v0, μM min−1).
The Michaelis–Menten constant (Km, μM) was obtained from
the Lineweaver–Burk plot.
acetate (5 × 50 ml). The organic phase was dried over anhy-
drous Na2SO4. The solvent was distilled off and the residue
was column chromatographed using ethyl acetate and hexane
as eluents to isolate the pure compound 1. A solution of com-
pound 1 (500 mg) and piperazine/(2-[2-(2-aminoethoxy)ethoxy]-
ethylamine (150 µL) was taken in a reaction vial containing
2 ml dichloromethane and was irradiated in a microwave
synthesizer for 20 min. After the completion of reaction (TLC
monitoring), the reaction mixture was column chromato-
graphed using chloroform and methanol as eluents to isolate
compound 2/3.
Compound 2. Recrystallization from ethanol yielded 58%
light green solid: 159 °C; H NMR (300 MHz, CDCl3 + DMSO-
Phosphorylation of fructose 6-phosphate
1
An assay solution having a final concentration of 0.0036 µM
for ATP, 50 μM for fructose-6-phosphate and 10 μM for com-
pound 3 was prepared in 2 ml HEPES buffer (pH 7.2). After
addition of phosphofructokinase (80 units) and Mg2+ (50 μM),
the fluorescence intensity (excitation at 250 nm) was recorded.
About 43% decrease in the fluorescence intensity was
observed. The concentration of ADP was calculated from the
change in fluorescence intensity which on dividing by the time
taken for the change gave the initial velocity (v0, μM min−1).
The Michaelis–Menten constant (Km, μM) was obtained from
the Lineweaver–Burk plot.
d6): δ = 3.30 (m, 8H, piperazine 4 × CH2), 3.81 (d, 4H, J =
5.4 Hz, 2 × NCH2), 4.39–4.42 (m, 2H, 2 × CHOH), 4.64–4.71 (m,
4H, 2 × NCH2), 5.68 (d, 2H, J = 5.1 Hz, 2 × OH), 7.29 (m, 4H,
ArH), 7.75 (m, 4H, ArH), 7.89 (d, 4H, J = 12 Hz, ArH), 8.44 (d,
4H, J = 7.8 Hz, ArH) ppm; 13C NMR (75 MHz, DMSO-d6): δ =
45.92, 48.03, 67.54, 115.15, 119.89, 121.08, 126.22, 132.68,
141.39, 194.66 ppm; IR (KBr): 1593, 3261 cm−1; HR-ESI-MS for
C36H36N4O4 required 589.2809 [M + H]+. Found 589.2810.
Bis-10-[2-hydroxy-3-(2-methoxy-ethylamino)-propyl]-10H-
acridin-9-one (3). Using the above mentioned procedure, after
recrystallization from ethanol yielded 61% green solid:
1
164–170 °C; H NMR (300 MHz, DMSO-d6): δ = 2.58–2.76 (m,
Conversion of glyceraldehyde-3-phosphate to 1,3-
bisphosphoglycerate
8H, NCH2), 3.46–3.52 (m, 8H, OCH2), 3.90–4.05 (m, 2H,
CHOH), 4.42–4.57 (m, 4H, 2 × NCH2), 7.24–7.28 (m, 4H, ArH),
7.71–7.75 (m, 4H, ArH), 7.88–7.99 (m, 4H, ArH), 8.28–8.45 (m,
4H, ArH) ppm; 13C NMR (75 MHz, DMSO-d6): δ = 46.2, 50.2,
65.3, 66.2, 114.5, 118.7, 119.1, 124.0, 131.3, 140.0, 174.1,
191.3 ppm; IR (KBr): 1559, 3400, 3447 cm−1; HR-ESI-MS for
C38H42N4O6 required 651.3183 [M + H]+. Found 651.3123.
100 µL of glyceraldehyde-3-phosphate (10−3 M), glyceraldehyde
phosphate dehydrogenase (80 units), 100 µL NAD+ (10−3 M),
100 µL H2PO4 (10−3 M) and 100 µL MgCl2 (10−3 M) were taken
in 2 ml HEPES buffer. Reaction completion was confirmed
from the NADH absorbance at 340 nm in the UV-vis spectrum
of the reaction mixture.
UV-vis and fluorescence studies with ADP
Formation of 3-phosphoglycerate
10−3 M stock solutions of compounds 2 and 3 were prepared
in DMSO. A 10−3 M stock solution of ADP was prepared by dis-
solving NaADP in HEPES buffer. All the solutions were pre-
pared in Eppendorf/glass vials and UV-vis and fluorescence
spectra were recorded by taking 1 ml of the solution in a Bio-
cell of 1 ml capacity. Taking the concentration of compounds
2 and 3 as constant (10 μM), incremental addition of ADP
(0–50 μM) resulted in an increase in absorbance in the UV
spectrum. For recording the fluorescence spectra, in 12 µL
increments, ADP (0.001–1 μM) was added to 10 μM solutions
of compound 3 which resulted in the decrease in fluorescence
up to complete quenching.
Taking the calculated volume of ADP, 1,3-bisphosphoglycerate
and compound 3, their final concentration in 2 ml HEPES
buffer (pH 7.2) was adjusted to 0.058 μM, 50 μM and 10 μM,
respectively. After addition of PGK (80 units) and Mg2+
(50 μM), the fluorescence intensity (excitation at 250 nm) was
recorded. An 87% increase in fluorescence intensity was
observed. The concentration of ADP was calculated from the
change in fluorescence intensity which on dividing by the time
taken for the change gave the initial velocity (v0, μM min−1).
The Michaelis–Menten constant (Km, μM) was obtained from
the Lineweaver–Burk plot.
Selectivity for ADP
Conversion of phosphoenolpyruvate to pyruvate
To check the selectivity of compound 3 for ADP amongst its An assay solution of ADP (0.058 μM), phosphoenol pyruvate
homologues ATP, AMP, etc., the stock solutions (10−3 M) of the (50 μM) and compound 3 (10 μM) was prepared in 2 ml HEPES
nucleotides were prepared in HEPES buffer. Upon addition of buffer (pH 7.2). After addition of PK (80 units) and Mg2+
even 45–60 μM of ATP/AMP/GTP/CTP/UTP to 10 μM solution of (50 μM), the fluorescence intensity (excitation at 250 nm) was
compound 3 to a final volume of 1 ml, no change in the fluo- recorded. An 85% increase in fluorescence intensity was
rescence intensity was observed.
observed. The concentration of ADP was calculated from the
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change in fluorescence intensity which on dividing by the time
taken for the change gave the initial velocity (v0, μM min−1).
The Michaelis–Menten constant (Km, μM) was obtained from
the Lineweaver–Burk plot.
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Breakdown of pyruvate in the mitochondria
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increase in fluorescence intensity was observed.
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LC-MS and QuantiAnalysis
For quantification of ADP/ATP, the standard curve was
obtained by recording the LC-MS of commercial samples of
ADP and ATP. For LC-MS, the Dionex Ultimate 3000 system
was linked to a mass spectrometer. A C-18 column (Acclaim®
120 C18) 5 µm 120 Å (4.6 × 250 mm) was used for HPLC. Aceto-
nitrile–water (1 : 1) was used as the eluent. 2 µL of sample
(injection volume) was loaded onto the column, the flow rate
was kept at 0.2 ml and the absorbance was set at 200, 220 and
254 nm. Sodium formate was used as an internal calibrant. As
per the protocol of QuantAnalysis2.0, the ADP and ATP in the
reaction mixtures were quantified from the standard curves.
Acknowledgements
Financial assistance in the form of a sponsoring research
project by DST, New Delhi and CSIR, New Delhi is gratefully
acknowledged. AK and PP acknowledge CSIR, New Delhi for
fellowships. The authors acknowledge the contributions of
Prof. A.S. Brar, Vice-Chancellor GNDU, for purchasing state of
the art research instruments from the UGC grant under the
University with Potential for Excellence programme.
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