Implementation of anion-receptor macrocycles in supramolecular tandem assays for enzymes involving nucleotides as substrates, products, and cofactors

Article abstract of DOI:10.1039/b925192h

A supramolecular tandem assay for direct continuous monitoring of nucleotide triphosphate-dependent enzymes such as potato apyrase is described. The underlying principle of the assay relies on the use of anion-receptor macrocycles in combination with fluorescent dyes as reporter pairs. A combinatorial approach was used to identify two complementary reporter pairs, i.e. an amino-γ-cyclodextrin with 2-anilinonaphtalene-6-sulfonate (ANS) as dye (fluorescence enhancement factor of 17 upon complexation) and a polycationic cyclophane with 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS) as dye (fluorescence decrease by a factor of more than 2000), which allow the kinetic monitoring of potato apyrase activity at different ATP concentration ranges (μM and mM) with different types of photophysical responses (switch-ON and switch-OFF). Competitive fluorescence titrations revealed a differential binding of ATP (strongest competitor) versus ADP and AMP, which constitutes the prerequisite for monitoring enzymatic conversions (dephosphorylation or phosphorylation) involving nucleotides. The assay was tested for different enzyme and substrate concentrations and exploited for the screening of activating additives, namely divalent transition metal ions (Ni²⁺, Mg²⁺, Mn²⁺, and Ca²⁺). The transferability of the assay could be demonstrated by monitoring the dephosphorylation of other nucleotide triphosphates (GTP, TTP, and CTP). The Royal Society of Chemistry.

Full text of DOI:10.1039/b925192h

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Implementation of anion-receptor macrocycles in supramolecular tandem
assays for enzymes involving nucleotides as substrates, products, and
cofactors†
Mara Florea and Werner M. Nau*
Received 28th September 2009, Accepted 5th December 2009
First published as an Advance Article on the web 7th January 2010
DOI: 10.1039/b925192h
A supramolecular tandem assay for direct continuous monitoring of nucleotide
triphosphate-dependent enzymes such as potato apyrase is described. The underlying principle of the
assay relies on the use of anion-receptor macrocycles in combination with fluorescent dyes as reporter
pairs. A combinatorial approach was used to identify two complementary reporter pairs, i.e. an
amino-g-cyclodextrin with 2-anilinonaphtalene-6-sulfonate (ANS) as dye (fluorescence enhancement
factor of 17 upon complexation) and a polycationic cyclophane with 8-hydroxy-1,3,6-pyrene
trisulfonate (HPTS) as dye (fluorescence decrease by a factor of more than 2000), which allow the
kinetic monitoring of potato apyrase activity at different ATP concentration ranges (mM and mM) with
different types of photophysical responses (switch-ON and switch-OFF). Competitive fluorescence
titrations revealed a differential binding of ATP (strongest competitor) versus ADP and AMP, which
constitutes the prerequisite for monitoring enzymatic conversions (dephosphorylation or
phosphorylation) involving nucleotides. The assay was tested for different enzyme and substrate
concentrations and exploited for the screening of activating additives, namely divalent transition metal
ions (Ni2+, Mg2+, Mn2+, and Ca2+). The transferability of the assay could be demonstrated by
monitoring the dephosphorylation of other nucleotide triphosphates (GTP, TTP, and CTP).
receptor with a covalently attached fluorophore, because the
Introduction
specificity of the reaction is determined through the enzyme
Numerous supramolecular receptors for nucleotides have already
itself. This principal insight is similar to that reached by Matile
been introduced with the general aim to achieve a high selectivity
and coworkers, who have followed enzymatic reactions by using
multifunctional synthetic pores embedded in vesicles,26,27 with
the difference that supramolecular tandem assays allow direct
continuous monitoring in homogeneous solution.
and the ultimate goal to construct highly specific biological
chemosensors.1–19 But applications to actual biological systems
have remained scarce. With the tandem assay strategy, we have
recently developed a supramolecular application, which reduces
the key requirement for a receptor from the specificity for a single
analyte to the mere necessity to show a differential binding between
two analytes: the substrate and the product of an enzymatic
reaction.20–23 This affords convenient label-free enzyme assays, in
which the enzymatic conversion is monitored through a reporter
pair composed of a fluorescent dye and a macrocycle, which
responds to the concentration changes affected by the enzymatic
reaction. This involves, drawing inspiration from the indicator-
displacement principle,24,25 either a continuous displacement of the
dye from the macrocycle by the higher affinity product (product-
selective assay), or its uptake into the host, facilitated by the
depletion of the competing substrate (substrate-selective assay).
At least two designed chemosensors with covalently incorpo-
rated chromophores have been previously demonstrated to be
principally suitable for following enzymatic reactions in a non-
continuous manner.1,2 But in order to monitor an enzymatic
reaction, it is in fact not required to employ a highly specific
While our previous examples of tandem assays were limited
to cationic analytes in combination with cation-receptors,20–23
the investigation of anionic metabolites such as nucleotides, and
particularly ATP, requires the use of macrocycles with anion-
receptor properties,28 which we have now investigated.
Results and discussion
We have first screened a combinatorial library consisting of a set
of 10 water-soluble anion receptors, which in part were already
known to bind to nucleotides,11–15 in combination with a set of 9
anionic and neutral water-soluble fluorescent dyes (Charts 1 and
2). To develop practical tandem assays, our aim was to obtain
i) a large fluorescence response upon dye complexation, ii) a
strong macrocycle-dye binding, and iii) a large differentiation in
binding between nucleotide tri- and monophosphates. Our best
(and new) reporter pair consists of the amino-g-cyclodextrin 1
(the amino groups of which are positively charged near neutral
pH) in combination with 2-anilinonaphtalene-6-sulfonate (ANS)
as dye. A complementary reporter pair, originally suggested for
the selective recognition of GTP,10,11 consists of the cyclophane 2
with 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS) as dye.
School of Engineering and Science, Jacobs University Bremen, Campus
Ring 1, D-28759, Bremen, Germany. E-mail: w.nau@jacobs-university.de;
Fax: +494212003229; Tel: +494212003233
† Electronic supplementary information (ESI) available: Additional exper-
imental results. See DOI: 10.1039/b925192h
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Chart 1 Investigated library of macrocycles with anion-receptor properties.
Chart 2 Investigated library of anionic and neutral fluorescent dyes studied in combination with the macrocycles in Chart 1.
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Table 1 Binding constants of 1 and 2 with the substrate as well as
intermediary and final enzymatic products
Guest
K/106 M-1 (1)a
K/103 M-1 (2)b
ATP
ADP
100 60
2.8 0.8
4.5 1.0
< 1
AMP
Orthophosphate
0.011 0.003
0.05 0.01
< 1
< 0.1
a Determined by competitive fluorescence titration using 1/ANS in 50 mM
NaOAc buffer, pH 5.5, cf. Fig. 2a. The binding constant of 1/ANS is
(1.7 0.1) ¥ 104 M-1 cf. Fig. 1a. b Determined by competitive fluorescence
titration using 2/HPTS in 10 mM phosphate buffer, pH 7.4. The binding
constant of 2/HPTS was determined as (7 3) ¥ 106 M-1, cf. Fig. 1b.
Successive addition of macrocycle to dye solutions resulted
in a strong fluorescence enhancement by a factor of 17 (lexc
=
318 nm, lobs = 462 nm, for 1/ANS) or quenching by a factor
of more than 2000 (lexc = 425 nm, lobs = 512 nm, for 2/HPTS),
from which dye binding constants of 1.7 ¥ 104 and 7 ¥ 106 M-1
were determined (Fig. 1). Addition of different nucleotides
reversed these fluorescence changes due to competitive binding
(Fig. 2 and Fig. 3). Both systems showed a charge-dependent
differentiation of nucleotides (e.g., between ATP, ADP, AMP, and
orthophosphate),10 which is the sufficient and necessary criterion
for our presently explored application (cf . binding constants in
Table 1). Specifically, we implemented the 1/ANS and 2/HPTS
Fig. 2 Changes in fluorescence intensity and spectra of the 1/ANS
reporter pair (10 mM 1 and 25 mM ANS, lexc = 318 nm) upon displacement
by a) varying concentrations of ATP, ADP, AMP, and orthophosphate
(lobs = 462 nm) and b) different nucleotide triphosphates (16 mM), in
NaOAc buffer, pH 5.5.
Fig. 1 Plots of the fluorescence intensity of a) 10 mM ANS with increasing
concentrations of 1 in 50 mM NaOAc buffer, pH 5.5 (lexc = 318 nm, lobs
=
462 nm) and b) 6.3 mM HPTS with increasing concentrations of 2 in 10 mM
phosphate buffer, pH 7.4 (lexc = 425 nm, lobs = 512 nm). The nonlinear
fittings were made by assuming the formation of a 1 : 1 complexation
model. It should be noted that the fluorescence of HPTS increased again
slightly at higher macrocycle (2) concentrations, suggesting the formation
of higher-order complexes. An equimolar mixture of 2 and HPTS was
consequently employed as reporter pair.
Fig. 3 Changes in fluorescence spectra of the 2/HPTS reporter pair (both
6.3 mM) upon addition of a) ATP, ADP, and AMP (each 4 mM) and
b) different nucleotide triphosphates (4 mM), in succinate buffer, pH 6.5;
lexc = 403 nm.
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reporter pairs into supramolecular tandem assays for potato
apyrase, an ectoenzyme of the class of ATP-diphosphohydrolases
(E.C. 3.6.1.5) which convert ATP to AMP through a stepwise
dephosphorylation with ADP as an intermediate.29–32 Apyrases
regulate extracellular nucleotide levels (in animal tissues) and the
biosynthesis of starch and cell walls (in plants).
The working principle is illustrated in Scheme 1. Our assay is
based on the propensity of the highly positively charged hosts 1
and 2 to form self-assembled complexes with negatively charged
guest molecules, particularly ANS and HPTS, as well as ATP. The
dye ANS is weakly fluorescent in its uncomplexed form, but expe-
riences an efficient fluorescence enhancement upon complexation
with 1, due to its sensitivity towards environmental polarity.33 In
contrast, the dye HPTS is highly fluorescent in its free form, but
suffers from a strong fluorescence quenching when complexed with
2, presumably by charge-transfer-induced quenching.10
Behaving as a strong competitor, ATP displaces the dyes from
the complexes, which results in a low or high initial fluorescence
intensity, respectively (Fig. 2 and Fig. 3). Upon enzymatic
dephosphorylation, AMP and orthophosphate are formed as final
products, which behave as weak competitors (due to their lower
net negative charge) and thereby allow a successive complexation
of the fluorescent dyes with the macrocycles. This results in a
switch-ON (for 1/ANS) or switch-OFF (for 2/HPTS) fluorescence
response,20 which allows a highly sensitive monitoring of the
enzymatic activity. Moreover, since the guest-macrocycle exchange
equilibria are much faster than the enzymatic reactions, the
response of the system to concentration changes is immediate
and allows for real-time continuous kinetic monitoring. It is
thus possible to follow the ATP depletion as a result of an
enzymatic reaction or, conversely, the enzymatic reaction through
the depletion of ATP. Conceptually, the resulting tandem assays
can be qualified as substrate-selective assays because the substrate
binds more strongly than the product.22
pre-formed reporter pair partially recovered the fluorescence of
the uncomplexed dyes through competitive displacement and
an approximately 10-fold fluorescence reduction (for 1/ANS) or
more than 100-fold enhancement (for 2/HPTS) was observed.
Addition of apyrase to a solution containing ATP as substrate
along with one of the reporter pairs led to a time-resolved rise
(factor of 4 for 1/ANS) or decay (factor of 12 for 2/HPTS) of the
fluorescence intensity with a plateau being reached within 1–2 h
(black traces, Fig. 4). In accordance with Scheme 1, we assigned the
time-resolved fluorescence response to the enzymatic conversion,
thereby establishing a convenient supramolecular tandem assay for
apyrases, and generally, for the conversion of ATP. The occurrence
of the enzymatic reaction on the same time scale was independently
monitored by 31P NMR (Fig. S1 in ESI†). The assay could also
be conducted with ADP as substrate (see Fig. S2 in ESI†), but
for AMP no significant fluorescence change was noticed, as no
dephosphorylation is possible (negative control, data not shown).
Enzyme assays are employed in pharmaceutical-industrial high-
throughput screening. Previously, we have demonstrated the
potential of tandem assays in the screening of inhibitors,22 and the
apyrase assay offered the complementary opportunity to screen
for activators, namely, divalent metal ions (Ni2+, Mg2+, Mn2+, and
Ca2+). In all cases, we observed rate enhancements between a factor
of 1.3 to 17, with Ca2+ and Mn2+ being the most potent activators
of potato apyrase (Fig. 4),34 consistent with literature findings
obtained by more laborious and complex multi-step colorimetric
and radioisotope-based assays.30–32 Consequently, all enzymatic
reactions were subsequently performed with Mn2+ (for 1/ANS)
or Ca2+ (for 2/HPTS), which reduced the assay times typically to
10–20 min.
One could argue that the 1/ANS pair is 2 orders of magnitude
more “sensitive” since it allows the conversion of mM instead
of mM amounts of ATP to be followed. However, one has to
recall that the substrate (or cofactor) concentration range is not
the limiting factor for an assay, as it could be for a sensor.
Rather, the substrate concentration can be adjusted at will to the
desired time and the specific activity of the enzyme, such that
even assays with mM substrate concentrations are common.26
For example, the reported KM values of potato apyrase ranges
The two reporter pairs are complementary in the sense that they
show mM or mM affinities to the different nucleotides, respectively
(Fig. 2 and Fig. 3). This determines the substrate concentration
range of the tandem assays, which were run with 25 mM ATP
for 1/ANS and 2.3 mM for 2/HPTS. Addition of ATP to the
Scheme 1 Working principle of a supramolecular tandem assay for monitoring dephosphorylation of ATP.
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Fig. 5 a) Evolution of normalized fluorescence intensity monitoring
potato apyrase activity (25 mg ml-1) at different substrate (ATP) concentra-
tions with the 1/ANS reporter pair (both 25 mM, in NaOAc buffer, pH 5.5,
with 1.8 mM Mn2+, lexc = 318 nm, lobs = 462 nm). ATP concentrations
(in mM) are, from bottom to top trace: 6, 10, 12.5, 15, 20, 25. b) Plot of the
initial rates, v0, versus substrate concentration and linear correlation line.
The initial rates were obtained by linear fits of the normalized intensities
(assuming a conversion linear with the fluorescence intensity and full
conversion at the plateau region).
Fig. 4 Evolution of normalized fluorescence intensity monitoring potato
apyrase activity with and without activating metal ions (1.7 mM). Assays
were initiated by addition of a) 25 mg ml-1 enzyme to ATP (25 mM) with
the 1/ANS reporter pair (25 mM both) and b) 100 mg ml-1 enzyme to ATP
(2.3 mM) with the 2/HPTS reporter pair (6.3 mM both).
from 24 to 200 mM,31,32 suggesting that the 1/ANS and 2/HPTS
reporter pairs operate either below or above the KM value. For
the 1/ANS reporter pair, the initial rates do in fact increase
linearly with the substrate concentration (Fig. 5). A more detailed
analysis of the enzyme kinetics is, however, not possible because
the complexation of the substrate by the host lowers the effective
substrate concentration, which presents a peculiarity of substrate-
selective assays.22 This was possible, however, for the 2/HPTS
reporter pair, for which a large excess of substrate was used
and where the enzyme kinetics was expected to be zero-order
with respect to the substrate. Indeed, determination of the initial
reaction rates from the fluorescence decays at a fixed enzyme
concentration (50 mg ml-1) and 4 varying substrate concentra-
tions (0.5–3.2 mM) afforded the same rate, within 10% error
(0.17 mmol min-1, expected from reported commercial activity:
0.19 mmol min-1). This rate was assigned to the vmax value when
working far above the KM value. Finally, as shown for both reporter
pairs (Fig. S3 in ESI†), the initial rates of ATP dephosphorylation
increased linearly with the enzyme concentration.
Important to note, all nucleotide triphosphates caused a flu-
orescence decrease for the 1/ANS reporter pair and a fluores-
cence enhancement of the 2/HPTS reporter pair, by competitive
displacement (see Fig. 2b and Fig. 3b). This shows that hosts
1 and 2 are actually unselective or at best moderately selective
anion receptors, at least in the investigated concentration range.
In tandem assays, such a “sloppy” molecular recognition24,27
presents actually an advantage, because dephosphorylation of
both nucleotide di- and triphosphates can be monitored quite
generally (for example also GTP, TTP and CTP, Fig. 6), which
opens screening possibilities in many directions.
Conclusions
In conclusion, we have introduced convenient supramolecular
tandem assays for monitoring biocatalytic ATP dephosphoryl-
ation. The assay principle relies on the addition of an anion-
receptor macrocycle and a fluorescent dye setting up a reporter
pair. It is the high specificity of the enzyme itself which reduces
selectivity considerations in the choice of a receptor to a minimum,
bypasses the necessity to utilize designed chemosensors, but
instead allows the use of simple, even if moderately selective,
macrocycles. We demonstrated applications in the screening of
activators and a broader applicability for other nucleotides and
complementary reporter pairs. Our dephosphorylation assays
should be transferable to other nucleotide-dependent enzymes.
This work was supported by the Deutsche Forschungsgemein-
schaft (DFG grant NA-686/5-1), the Fonds der Chemischen
Industrie, and the BIG Bremer Investitions-Gesellschaft mbH
¨
(Okologiefonds des Landes Bremen). The authors also thank Dr
A. Hennig for helpful discussions.
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Instrumentation
Fluorescence spectra, intensities, and time courses were recorded
on a Varian Eclipse fluorometer at ambient temperature (for
the titrations) or at 25.0
0.1 ◦C (using an external Peltier
thermostat, only for the enzyme assays). UV measurements were
performed with a Varian Cary 4000 UV-Vis spectrophotometer.
The dephosphorylation of nucleotides was additionally monitored
by 31P NMR using a Jeol JNM-ECX 400 spectrometer.
Fluorescence titrations
All fluorescence titrations were performed in 1.4 mL quartz
cuvettes (Starna, Typ 29-F/Q/10 mm). The titration of host
with fluorophore was performed by keeping the dye concen-
tration constant. Accordingly, the titrant contained the same
concentration of dye, and a much higher host concentration
(approximately 10–100 times higher). The competitive titrations
using different nucleotides were carried out similarly to the host-
guest titrations, ensuring a constant host and dye concentration
during the titration. Therefore, the titrant contained, besides a high
concentration of competitor, host and dye in concentrations equal
to those in the cuvette solution. All titrations were performed in
50 mM sodium acetate buffer, pH 5.5 when the 1/ANS reporter
pair was employed, and in 10 mM sodium phosphate buffer,
pH 7.4, or 10 mM sodium succinate buffer, pH 6.5, when using
the 2/HPTS reporter pair.
Fig. 6 Evolution of normalized fluorescence intensity monitoring de-
phosphorylation of various nucleotide triphosphates. Assays were initiated
by addition of a) 12.5 mg ml-1 potato apyrase in the presence of 1/ANS
(25 mM both) to nucleotides (25 mM), and b) 100 mg ml-1 potato apyrase
in the presence of 2/HPTS (6.3 mM both) to nucleotides (4 mM).
Supramolecular tandem assays
Stock solutions of substrate (nucleotides), host (1 or 2), fluorescent
dye (ANS or HPTS), and enzyme were required for performing
the enzyme assays. The stock solutions were prepared in 50 mM
sodium acetate buffer, pH 5.5, with 1.6–1.8 mM MnCl2 (when
using 1/ANS reporter pair) and in 10 mM sodium succinate
buffer, pH 6.5, with 1.6–1.8 mM CaCl2 (when 2/HPTS was
employed). Careful control of the pH was required to obtain
reproducible fluorescence intensities. The required volumes of all
stock solutions were calculated to afford the final concentrations
in 1 mL total assay volume. All assays were conducted using
equimolar mixtures of host and fluorescent dye (25 mM for 1/ANS
and 6.3 mM for 2/HPTS). The volumes of nucleotide stock
solutions were calculated to obtain the respective concentrations.
The enzymatic reactions were initiated by adding an aliquot from
a stock solution of 1 mg mL-1 enzyme.
Experimental
Materials
The macrocycles and dyes employed in the initial screening
are shown in Charts 1 and 2. 2-Anilinonaphtalene-6-sulfonic
acid (ANS), and Dapoxyl were obtained from Molecular
Probes. 8-Anilinonaphtalene-1-sulfonic acid (1,8-ANS), 8-
hydroxy-1,3,6-pyrene trisulfonic acid (HPTS), 6-(p-toluidino)-
2-naphtalene-sulfonic acid (2,6-TNS), fluorescein sodium,
GTP, and ADP were purchased from Fluka. The naphtalimide
biscarboxylate, 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO), and
DBO carboxylate were synthesized according to the literature.35–37
ATP, AMP, CTP, TTP, and apyrase (grade I, from potato,
7.7 U/mg) were from Sigma-Aldrich. The cyclodextrin samples
octakis(6-amino-6-deoxy)-g-cyclodextrin (1), heptakis(6-deoxy-
6-N-(2-hydroxy)ethylamino)-b-cyclodextrin (4), 6-monoamino-
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