DOI: 10.1002/chem.201404499
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Ratiometric Detection of Adenosine Triphosphate (ATP) in Water and Real-Time Monitoring of Apyrase Activity with a Tripodal Zinc Complex Stephen J. Butler*[a] Abstract: Two tripodal fluorescent probes Zn·L1,2 have been synthesised, and their anion-binding capabilities were examined by using fluorescence spectroscopy. Probe Zn·L1 allows the selective and ratiometric detection of adenosine triphosphate (ATP) at physiological pH, even in the presence of sev-
Introduction
eral competing anions, such as ADP, phosphate and bicarbonate. The probe was applied to the real-time monitoring of the apyrase-catalysed hydrolysis of ATP, in a medium that mimics an extracellular fluid.
lectivity is seldom assessed in a biologically relevant medium. This is critically important, because biofluids, such as serum and urine, contain a number of interfering species, including a high salt concentration and several competing anions (e.g., bicarbonate and phosphate), which will certainly influence the probe’s affinity and selectivity profile.[4, 21] Finally, probes capable of ratiometric (wavelength shift) detection of ATP are rare;[9, 22] most rely upon an “off/on” fluorescent signal change, which limits their application in cellulo, because uneven distribution of the probe may lead to unreliable readouts. Herein, we report a new water-soluble probe Zn·L1 that functions under physiological conditions and exhibits high selectivity for ATP over other polyphosphate anions. The zinc complex is based on a tripodal quinoline ligand, in which the
The design of probes that can detect changes in the presence and concentration of bioactive species in water is an active area of research.[1–3] Probes, which signal analyte binding by modulation of fluorescence emission, are most useful, because they provide spatial and temporal information rapidly and with high sensitivity.[4–6] Such systems have enormous potential to elucidate the crucial roles that certain bioactive substrates have in dynamic cellular processes. Adenosine triphosphate (ATP) is arguably one of the most important anions in living systems; not only does it serve as the chemicalenergy source for most biological functions, but also it plays key roles in extracellular signalling and DNA replication. Fluorescent molecules that signal the presence of ATP reversibly and selectively in aqueous solution could be used to monitor kinase activity in real time or quantify the energy supplied by ATP in different cellular compartments.[7, 8] There are very few synthetic probes for ATP, which can operate under physiological conditions with the required affinity and selectivity, notwithstanding some notable exceptions.[9–11] Most reported ATP-selective probes are limited in utility either because they cannot effectively discriminate between polyphosphate species (e.g., ADP and pyrophosphate may induce a similar or even greater fluorescent response compared to that of ATP), or the detection range of the probe is too low (0.1–10 mm) and does not match the concentration levels of ATP present in Figure 1. Structures of ligands L1,2. Complex Zn·L1 is expected to have one water molecells (1–5 mm).[12–20] Moreover, anion affinity and se- cule coordinated to ZnII in aqueous solution. [a] Dr. S. J. Butler Department of Chemistry, Durham University South Road, Durham, DH1 3LE (UK) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404499. Chem. Eur. J. 2014, 20, 15768 – 15774
fifth coordination site is likely to be occupied by a water molecule (Figure 1). Such a complex is capable of forming strong metal–ligand interactions with ATP. The central ZnII ion pre-organizes the three quinoline groups, each functionalised at the seventh position with aryl urea units to engage in hydrogen15768
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Full Paper bonding interactions with the P O oxygen atoms of ATP. Synthetic anion receptors that rely solely on hydrogen bonding are generally ineffective in water; however, in conjunction with metal–ligand interactions, cooperative binding sites capable of hydrogen bonding or p–p stacking with a target anion can substantially enhance binding affinity and selectivity.[21, 24–26] It was envisaged that selectivity for ATP over non-nucleoside phosphates, such as HPO42 and P2O74 (PPi), could be enhanced through p–p stacking interactions between the terminal aromatic groups of Zn·L1 and the adenosine moiety of ATP. The terminal aryl groups possess p-substituted sulphonate moieties to increase water solubility. As a comparison, the structural analogue Zn·L2 was synthesised to investigate the effects that the position and geometry of the aryl urea binding sites would have on anion affinity, selectivity and fluorescence response.
quenching of the PET mechanism, leading to a CHEF effect.[27] Zinc titrations performed at pH 7.4 revealed that one equivalent of Zn(ClO4)2 was sufficient to obtain 100 % of the zinc complexes of L1,2 (Figure S3 in the Supporting Information). The binding stoichiometry between L1,2 and Zn in aqueous solution (25 mm 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer, pH 7.4) was determined to be 1:1 by using the method of continuous variation (Job’s Plot, Figure S4 in the Supporting Information). Further evidence for a 1:1 binding was provided by high-resolution (ESI) mass spectrometric data, which revealed major signals for the singly charged species [L1 + Zn] and [L2 + Zn] (Figure S21 in the Supporting Information). The UV/Vis spectra of Zn·L1,2 are very similar to one another, presenting an intense band at l = 270 nm and a broad featureless band at 344 nm (e = 8200 m 1 cm 1, Figure S5 in the Supporting Information).
Results and Discussion
Anion-binding studies at physiological pH
Synthesis and characterisation A modular approach was adopted for the syntheses of Zn·L1,2. A representative synthesis of L1 is given in Scheme 1. First, the C3-symmetric scaffold, tris(7-amino-2-quinolinyl)amine (3) was prepared by reacting the appropriately functionalised 2-quinolinyl amine (1) with two equivalents of the 2-methylquinoline mesylate ester 2 in the presence of K2CO3. The tert-butoxycarbonyl (Boc) protecting groups of 3 were removed by using 4 m HCl in dioxane, followed by a basic work-up procedure to give the tris(amine) 4. Next, the aryl urea groups were introduced by reacting tris-amine 4 with 4-(chlorosulfonyl)phenyl isocyanate in CH3CN/DMF at room temperature, followed by basic hydrolysis of the chlorosulfonyl groups, to give ligand L1 in good yield following RP-HPLC purification. A similar procedure was adopted for the synthesis of L2. Subsequent addition of one equivalent of Zn(ClO4)2 in water at pH 7.4 gave the water soluble zinc(II) complexes Zn·L1,2. The experimental details and characterisation data are provided in the Supporting Information. In the presence of one equivalent of Zn(ClO4)2, L1,2 exhibited large enhancements in fluorescence emission centred at l = 510 nm (ten-fold and eight-fold increase, respectively, lexc = 344 nm; Figure S1–2 in the Supporting Information) due to
To evaluate the fluorescence response of Zn·L1,2 for different anions, the emission spectra of Zn·L1,2 (20 mm) were recorded in water (25 mm HEPES, pH 7.4, lexc = 344 nm) in the presence of a 500-fold excess of selected anions (Figure 2). When ATP was added to Zn·L1, a 2.5-fold increase in fluorescence emission intensity was observed, whereas ADP caused only a 1.4fold fluorescence enhancement. In sharp contrast, PPi, HPO42 and chloride resulted in quenching of the fluorescence emission centred at lem = 510 nm (up to 50 % in the case of PPi). AMP and cAMP also caused quenching of emission by approximately 20 %. Thus, Zn·L1 is selective for nucleoside polyphosphate anions, especially for ATP, over PPi and monophosphate anions. The only other anion tested that induced an enhancement in fluorescence emission was citrate (two-fold increase, Figure S13 in the Supporting Information). Notably, the addition of bicarbonate resulted in a large blueshift in the emission maximum from l = 510 to 445 nm, as well as quenching of the emission by 50 % (Figure 2). A similar response was observed in the presence of sulfate, although the spectrum consisted of a pair of bands with lem = 445 and 500 nm (Figure S14 in the Supporting Information). The appearance of two spectral bands centred at 445 and 510 nm in the presence of different anions, such as bicarbonate and ATP, can be ascribed to emission from the quinoline
Scheme 1. Synthesis of the tripodal quinoline ligand L1. Chem. Eur. J. 2014, 20, 15768 – 15774
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Full Paper For Zn·L2, in which the aryl urea groups are located at the quinoline 6-position, no significant change in emission spectral form or intensity was observed for every anion tested (Figure S5 in the Supporting Information). This clearly indicates that the position and geometry of the urea binding sites relative to the central ZnII ion has a profound influence on the anion binding and signalling properties of these systems. Specifically, in the case of Zn·L1, the urea functionalities are suitably positioned to allow cooperative hydrogen-bonding interactions with multiple P O atoms of ATP (Scheme 2). Titrations were carried out for Zn·L1 with various anions by adding aqueous solutions of the anion in 1–10 mL aliquots to a solution of the complex in water (25 mm HEPES, pH 7.4). Association constants were determined for those anions able to induce a significant change in the emission spectrum of Zn·L1 (Table 1). For ATP and ADP, the emission increase ratio (F/F0 at lem = 510 nm) was measured as a function of added anion concentration, whilst for bicarbonate, citrate and sulfate, changes in the ratio of monomer and excimer emission bands were used. The titration data was analysed by using a non-linear, least squares curve-fitting procedure based on a 1:1 binding model. Probe Zn·L1 showed strong binding to ATP in water (log Ka = 5.21), approximately 20 times stronger than to ADP (log Ka = 3.86), and 100 times stronger than citrate (log Ka = Figure 2. Top: change in fluorescence emission spectra of Zn·L1 (20 mm) in the presence of different anions (10 mm, sodium salts). Bottom: change in fluorescence emission of Zn·L1 (20 mm) as a function of added ATP (up to 1.6 mm). The inset shows the fit (line) to the titration data, for log Ka = 5.21 ( 0.10) Conditions: aqueous solution of HEPES buffer (25 mm, pH 7.4), lexc = 344 nm, 25 8C.
Table 1. Association constants (log Ka) determined for Zn·L1 and various anions in aqueous solution (25 mm HEPES, pH 7.4, 295 K). Each value represents the average of two duplicate titration experiments.[a] Anion
ATP
ADP
AMP
PPi
Citrate
HCO3
Log Ka
5.21 (2.94)[b]
3.86 (2.07)[b]
n/a[c]
n/a[c]
3.19
1.75
monomer and intramolecular excimer of Zn·L1, respectively.[28, 29] Given that there is no significant change in the UV ab[a] Typical errors in measurement were 0.10 with the statistical error assorption or excitation spectra when bicarbonate or ATP is sociated with the fit of the data < 0.05. [b] Values in parenthesis refer to added, a ground-state charge-transfer interaction can be ruled measurements in an aqueous solution that simulates an extracellular fluid out. Complexation of small anions, such as bicarbonate and (see the main text for details). [c] Affinity constants could not be determined, because only minor changes in fluorescence emission were obsulphate, tend to favour monomer formation (lem = 445 nm), served. n/a = not analysed. V whilst P oxyanions favour the excimer form (lem = 510 nm). It is hypothesised that ATP and ADP stabilise the excimer form, possibly through secondary interactions between the aromatic adenosine and quinoline groups, leading to an increase in emission intensity. ESI mass spectrometric data supports intramolecular excimer formation and a 1:1 binding mode for certain hostanion complexes. Major signals were observed for the singly charged ternary complexes Zn·L1 + ATP, Zn·L1 + ADP, Zn·L1 + PPi and Zn·L1 + perchlorate, which were in close agreement with the calculated isotopic distributions (Figure S23–26 in the Scheme 2. Proposed binding mode of the ternary complexes of Zn·L1 with ATP and bicarbonate. Selectivity for Supporting Information). ATP is attributed to the synergistic effect between metal–ligand and hydrogen-bonding interactions. Chem. Eur. J. 2014, 20, 15768 – 15774
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Full Paper 3.19). Bicarbonate and sulfate were found to bind to Zn·L1 rather weakly (log Ka = 1.75 and 0.98, respectively). Binding constants for AMP, HPO42 , and PPi could not be estimated with any confidence, because only minor changes in fluorescence emission took place (Figure S8–14 in the Supporting Information). The mode of binding of Zn·L1 to ATP in D2O was studied by NMR spectroscopy. 1H NMR spectra of Zn·L1 in the absence and presence of ATP showed broadened signals in the aromatic region due to exchange processes occurring (Figure S27 in the Supporting Information). This prohibited a detailed structural characterization of the host-anion interaction. The 31 P NMR spectral data was more informative; distinct 31P signals were observed for the a-, b- and g-phosphorus atoms of ATP (pD 7.4, Figure 3). Subsequent addition of one equivalent
ATP detection in a biorelevant medium In many biological fluids, such as serum or cerebrospinal fluid, the most abundant anions are chloride and bicarbonate, which are present in concentrations 10–100 times greater than those of other anions.[4] To determine the practical utility of these systems in biological assays, we examined the ability of Zn·L1 to detect ATP in an aqueous solution that mimics an extracellular fluid.[31] The solution contains the sodium salts of chloride (110 mm), bicarbonate (30 mm), lactate (2.3 mm), hydrogenphosphate (0.9 mm) and citrate (0.13 mm), buffered to pH 7.4 (10 mm HEPES). Under these conditions, the emission spectral form of Zn·L1 was very similar to that observed in the presence of excess bicarbonate only (lem = 445 nm). Titration of Zn·L1 (20 mm) with ATP resulted in a large redshift in the emission maximum from 445 to 510 nm and a substantial enhancement in emission intensity (Figure 4), consistent with displacement of the bound bicarbonate anion and concomitant excimer formation. ADP and PPi caused similar shifts in emission maximum, but with only small enhancements in emission intensity, signifying a closer competition between these anions and bicarbonate. Meanwhile, the addition of millimolar concentrations of AMP, cAMP, HPO42, phosphotyrosine, citrate and lactate did not elicit a significant spectral response (Figures S15–S18 in the Supporting Information).
Figure 3. Partial 31P NMR spectra (700 MHz) in D2O (pD 7.4) of: a) ATP (5 mm); and b) ATP (5 mm) and Zn·L1 (5 mm). The spectra have been normalised for comparison.
of Zn·L1 resulted in exchange-broadened signals of the b- and g-P atoms into the baseline, as well as clear shifts to higher frequency from d = 22.8 and 9.6 ppm to 22.1 and 9.0 ppm for b-P and g-P, respectively. In contrast, the chemical shift of the a-P atom did not change in the presence of Zn·L1, suggesting that binding of ATP occurs predominantly at the band g-P atoms. Taken together, this data is consistent with the binding mode illustrated for ATP and Zn·L1 in Scheme 2, in which one negatively charged g-P-O atom coordinates to the zinc metal and both g- and b-P-O atoms engage in hydrogenbonding interactions with the urea NH groups. A molecular model (optimized at HF/3-21G* with the Gaussian 09 package,[30] Figure S28 in the Supporting Information) of the ternary complex of Zn·L1 with a model triphosphate anion suggests that the urea functionalities are suitably positioned to form hydrogen bonds with the oxygen atoms of the g- and b-phosphorus, whereas the negatively charged oxygen atom of the a-P is too distant for a sufficient interaction to occur. Chem. Eur. J. 2014, 20, 15768 – 15774
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Figure 4. Ratiometric fluorescence detection of ATP (top) and ADP (bottom) in a simulated extracellular fluid, by monitoring the emission spectral change (510/445 nm) of Zn·L1 (20 mm) as a function of added anion. The inset shows the fit (line) to the titration data. Conditions: aqueous solution of HEPES buffer (10 mm, pH 7.4) containing NaCl (110 mm), NaHCO3 (30 mm), sodium lactate (2.3 mm), Na2HPO4 (0.9 mm) and sodium citrate (0.13 mm); lexc = 344 nm, 25 8C.
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Full Paper Association constants were determined for ATP and ADP by plotting the changes in the ratio of the monomer (445 nm) and excimer (510 nm) emission bands as a function of added anion concentration, and fitting the data to a 1:1 binding model. ATP was found to bind to Zn·L1 with log Ka = 2.94, approximately eight times greater than the binding to ADP (log Ka = 2.07).
Monitoring apyrase activity in real time Having established that Zn·L1 can distinguish ATP from ADP, PPi and AMP, the probe was applied to real-time monitoring of the enzymatic activity of apyrase. Apyrase is a hydrolytic enzyme that converts ATP to ADP, AMP and HPO42 . To a buffered aqueous solution (25 mm HEPES, pH 7.4) containing the Zn·L1 (20 mm) and ATP (2 mm) was added various concentrations of apyrase (150–300 mU), and the decrease in emission intensity at 510 nm was monitored as a function of time (Figure 5). The time-trace plots reveal that the reaction rate in-
Figure 6. Real-time monitoring of the apyrase-catalysed hydrolysis of ATP (5 mm) under mimicked physiological conditions, by monitoring the emission ratio 505/450 nm of Zn·L1. Conditions: aqueous solution of HEPES buffer (10 mm, pH 7.4) containing NaCl (110 mm), NaHCO3 (30 mm), sodium lactate (2.3 mm), Na2HPO4 (0.9 mm) and sodium citrate (0.13 mm); 300 mm apyrase; lexc = 344 nm, 25 8C.
serves as a simple and effective means for monitoring apyrase activity in a biorelevant medium, auguring well for its implementation in real biological systems.
Conclusion
Figure 5. Time-trace plots of the hydrolysis of ATP (2 mm) by apyrase (150– 300 mU) monitored by the change in fluorescence emission (510 nm) of Zn·L1. Conditions: buffered aqueous solution (25 mm HEPES, pH 7.4); lexc = 344 nm, 25 8C.
creases in proportion to enzyme concentration. The complete hydrolysis of ATP was verified by 31P NMR spectroscopy. The reaction mixture containing 300 mU apyrase was quenched after 20 min by the addition of EDTA (100 mm). The 31P NMR spectrum of the quenched mixture revealed signals corresponding to ADP, and AMP in the approximate ratio 2:1 (Figure S20 in the Supporting Information). Next, apyrase activity was monitored in a simulated extracellular fluid, by using 5 mm ATP and 300 mU of apyrase (Figure 6). Under these conditions, the enzyme-catalysed hydrolysis of ATP resulted in a decrease in the ratio of the excimer/monomer emission. A plot of the emission ratio, F505/F450, versus time revealed a slower reaction rate compared to that observed in water alone, consistent with a higher starting concentration of ATP. After 20 min, the reaction reached a plateau; the spectral form at this point was almost identical to that observed for Zn·L1 in the presence of 5 mm ADP (Figure 4), indicating quantitative hydrolysis of ATP after 20 min. Thus, Zn·L1 Chem. Eur. J. 2014, 20, 15768 – 15774
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In summary, an ATP-selective fluorescent probe Zn·L1 that functions at physiological pH has been created. The integration of several cooperative binding sites (i.e., metal–ligand interactions, hydrogen bonding and p–p stacking) within the probe afforded tight binding to ATP in water (log Ka = 5.21), with a useful level of selectivity over other polyphosphate species including ADP and PPi. In comparison, no anion-induced fluorescence signalling was achieved by Zn·L2 due to its lack of suitably positioned aryl urea groups. In an aqueous medium that simulates extracellular fluid selectively of Zn·L1 for ATP is tuned to the millimolar range. This permits its ratiometric detection in the metabolic concentration range (1–5 mm), even in the presence of a number of competing anions, including bicarbonate, phosphate and citrate. Finally, it has been demonstrated that Zn·L1 can operate in a simulated biological fluid to monitor the apyrase-catalysed hydrolysis of ATP in real time. Future work will be directed towards the creation of cell-permeable analogues that can report on changes in local ATP concentrations in cellulo. The modular approach to synthesis will enable factors, such as cytotoxicity, cellular uptake and pH effects, to be addressed; this work is underway.
Experimental Section Details of instrumentation, ligand synthesis, methods of purification, characterisation data and spectral titrations with added anions are given in the Supporting Information.
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Full Paper Ligand 1 (L1)
Tris[7-(tert-butoxycarbonylamino)-2-quinolinyl]amine (3) To a solution of the free amine (1; 32 mg, 0.117 mmol) and the mesylate ester (2; 70 mg, 0.234 mmol) in anhydrous CH3CN (3 mL) was added K2CO3 (48 mg, 0.351 mmol), and the mixture was stirred under argon at 60 8C. The progress of the reaction was monitored by LC-MS analysis at regular intervals, which revealed complete consumption of starting material after 12 h. At this time, a yellow precipitate had formed. The solvent was removed under reduced pressure, and the residue was dissolved in a mixture of CH2Cl2/ CH3OH (8:2, 3 mL). The solution was decanted from excess potassium salts and the solvent was removed under reduced pressure. The residue was triturated with CH3CN (3 5 mL) to afford tris[7(tert-butoxycarbonylamino)-2-quinolinyl]amine 3 as a colourless solid (70 mg, 76 %); m.p. 175–178 8C; 1H NMR (700 MHz, CDCl3): d = 9.75 (3 H, br s, CONH), 8.35 (3 H, s, H8), 8.28 (3 H, d, 3JH H 8.3 Hz, H4), 7.89 (3 H, d, 3JH H 8.8 Hz, H5), 7.78 (3 H, d, 3JH H 8.3 Hz, H3), 7.76 (3 H, d, 3JH H 8.8 Hz, H6), 4.09 (6 H, s, H9), 1.53 ppm (27 H, s, H12); 13C NMR (176 MHz, CDCl3): d = 160.9 (C2), 153.5 (C10), 148.9 (C2’), 141.4 (C7), 136.3 (C4), 128.5 (C5), 123.6 (C3’), 119.6(4) (C6), 119.5(7) (C3), 114.9 (C8), 79.8 (C11), 61.1 (C9), 28.0 ppm (C12); LRMS (ESI): m/z 786 [M + H] + ; (HRMS +) m/z 786.3960 [M + H] + (C45H52N7O6 requires 786.3979); Rf = 0.25 (silica gel; hexane/EtOAc 1:1).
Tris(7-amino-2-quinolinyl)amine (4; 23 mg, 0.047 mmol) was dissolved in a mixture of CH3CN/DMF (1:1, 1 mL). A solution of 4(chlorosulfonyl)phenyl isocyanate (41 mg, 0.190 mmol) in a mixture of CH3CN/DMF (1:1, 1 mL) was added dropwise over a period of 30 min, causing a precipitate to form. The resultant mixture was stirred at RT for 12 h under argon. The solvent was removed under reduced pressure to give a cream coloured solid. The crude material was suspended in water (3 mL), and the pH was adjusted to ten by the dropwise addition of NaOH (1 m). The mixture was stirred at RT for 30 min at which point the solid had completely dissolved. The pH of the solution was adjusted to 7.5 by the addition of HCl (1 m), and the aqueous mixture was purified by preparative RPHPLC (gradient: 10–100 % acetonitrile in 25 mm triethylammonium acetate over 10 min; tR = 7.3 min, Figure 7) to give L1 as a colourless oil (28 mg, 55 %); 1H NMR (400 MHz, D2O): d = 7.94 (6 H, complex m), 7.59 (12 H, br m), 7.42 (9 H, br m), 3.97 (6 H, br s); N H and O H signals were not observed; LRMS (ESI) m/z 1081 [M H] ; (C51H41N10O12S3 requires (HRMS + ) m/z 1081.2045 [M H] 1081.2068).
Figure 7. Analytical RP-HPLC of L1: tR 7.3 min (gradient: 10 to 100 % acetonitrile in 25 mm triethylammonium acetate over 10 min).
Acknowledgements S.J.B. wishes to thank the Ramsay Memorial Fellowship Trust and Durham University for support and funding. Thanks to Dr. Fernanda Duarte and Dr. Mark Fox for assisting with molecular modelling studies.
Tris(7-amino-2-quinolinyl)amine (4) Tris[7-(tert-butoxycarbonylamino)-2-quinolinyl]amine (3; 56 mg, 0.071 mmol) was suspended in anhydrous CH2Cl2 (1.5 mL) and cooled to 0 8C. HCl in dioxane (4 m, 1.5 mL) was added slowly causing the mixture to fully dissolve. The yellow solution was allowed to warm to RT and stirred for 1 h under argon. The solvent was removed under reduced pressure to give the tris hydrochloride salt as a yellow oil, which was partitioned between CHCl3/iPrOH (3:1, 20 mL) and saturated aqueous NaHCO3 solution (20 mL). The aqueous layer was extracted with CHCl3/iPrOH (3:1, 3 20 mL) and the combined organic fractions were dried (MgSO4) and concentrated under reduced pressure to afford the tris(amine) 4 as a colourless oil (23 mg, 75 %); 1H NMR (700 MHz, CD3OD:) d = 8.30 (3 H, d, 3JH H 8.0 Hz, H4), 7.63 (3 H, d, 3JH H 8.9 Hz, H5), 7.38 (3 H, d, 3JH H 8.0 Hz, H3), 7.13 (3 H, dd, 3JH H 8.9 Hz, 4JH H 2.0 Hz, H6), 6.95 (3 H, d, 4JH H 2.0 Hz, H8), 4.39 ppm (6 H, s, H9), N H signals were not observed; 13 C NMR (176 MHz, CD3OD): d = 157.4 (C2), 152.8 (C7), 145.3 (C2’), 143.3 (C4), 131.4 (C5), 123.6 (C3’), 123.0 (C6), 116.8 (C3), 96.5 (C8), 58.8 ppm (C9); LRMS (ESI): m/z 486 [M + H] + ; (HRMS + ) m/z 486.2406 [M + H] + (C30H28N7 requires 486.2403); Rf = 0.10 (silica gel; CH2Cl2/CH3OH, 80:20). Chem. Eur. J. 2014, 20, 15768 – 15774
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Keywords: anions · ATP · enzymes · fluorescent probes · quinolines
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Full Paper [8] Y. Kurishita, T. Kohira, A. Ojida, I. Hamachi, J. Am. Chem. Soc. 2010, 132, 13290 – 13299. [9] Z. Xu, N. J. Singh, J. Lim, J. Pan, H. N. Kim, S. Park, K. S. Kim, J. Yoon, J. Am. Chem. Soc. 2009, 131, 15528 – 15533. [10] H. Fenniri, M. W. Hosseini, J. M. Lehn, Helv. Chim. Acta 1997, 80, 786 – 803. [11] S. E. Schneider, S. N. O’Nei, E. V. Anslyn, J. Am. Chem. Soc. 2000, 122, 542 – 543. [12] H. T. Ngo, X. Liu, K. A. Jolliffe, Chem. Soc. Rev. 2012, 41, 4928 – 4965; Y. Zhou, Z. Xu, J. Yoon, Chem. Soc. Rev. 2011, 40, 2222 – 2235. [13] K. M. K. Swamy, S. K. Kwon, H. N. Lee, S. M. S. Kumar, J. S. Kim, J. Yoon, Tetrahedron Lett. 2007, 48, 8683 – 8686. [14] M. Schferling, O. S. Wolfbeis, Chem. Eur. J. 2007, 13, 4342 – 4349. [15] A. Ojida, S.-k. Park, Y. Mito-oka, I. Hamachi, Tetrahedron Lett. 2002, 43, 6193 – 6195. [16] A. Ojida, I. Takashima, T. Kohira, H. Nonaka, I. Hamachi, J. Am. Chem. Soc. 2008, 130, 12095 – 12101. [17] A. Sreenivasa Rao, D. Kim, H. Nam, H. Jo, K. H. Kim, C. Ban, K. H. Ahn, Chem. Commun. 2012, 48, 3206 – 3208. [18] A. J. Moro, P. J. Cywinski, S. Korsten, G. J. Mohr, Chem. Commun. 2010, 46, 1085 – 1087. [19] E. Kataev, R. Arnold, T. Rffer, H. Lang, Inorg. Chem. 2012, 51, 7948 – 7950. [20] H. H. Jang, S. Yi, M. H. Kim, S. Kim, N. H. Lee, M. S. Han, Tetrahedron Lett. 2009, 50, 6241 – 6243. [21] S. J. Butler, K. A. Jolliffe, Chem. Asian J. 2012, 7, 2621 – 2628. [22] A. Ojida, H. Nanaka, Y. Miyahara, S.-i. Tamaru, K. Sada, I. Hamachi, Angew. Chem. Int. Ed. 2006, 45, 5518 – 5521; Angew. Chem. 2006, 118, 5644 – 5647. [23] A number of CuII-based tripodal quinoline complexes have been reported: N. Wei, N. N. Murthy, K. D. Karlin, Inorg. Chem. 1994, 33, 6093 – 6100; N. Wei, N. N. Murthy, Q. Chen, J. Zubieta, K. D. Karlin, Inorg. Chem. 1994, 33, 1953 – 1965; L. A. Joyce, M. S. Maynor, J. M. Dragna, G. M. da Cruz, V. M. Lynch, J. W. Canary, E. V. Anslyn, J. Am. Chem. Soc. 2011, 133, 13746 – 13752; L. A. Joyce, J. W. Canary, E. V. Anslyn, Chem. Eur. J. 2012, 18, 8064 – 8069; S. Zahn, J. W. Canary, J. Am. Chem. Soc. 2002, 124, 9204 – 9211; S. Zahn, J. W. Canary, Angew. Chem. Int. Ed. 1998, 37, 305 – 307; Angew. Chem. 1998, 110, 321 – 323. [24] S. L. Tobey, B. D. Jones, E. V. Anslyn, J. Am. Chem. Soc. 2003, 125, 4026 – 4027; S. L. Tobey, E. V. Anslyn, J. Am. Chem. Soc. 2003, 125, 14807 – 14815.
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[25] H. Lee Jae, J. Park, S. Lah Myoung, J. Chin, J.-I. Hong, Org. Lett. 2007, 9, 3729 – 3731. [26] C. M. G. dos Santos, P. B. Fernandez, S. E. Plush, J. P. Leonard, T. Gunnlaugsson, Chem. Commun. 2007, 3389 – 3391. [27] The ZnII complex of a basic tripodal quinoline ligand,TQA, has been reported. The X-ray crystal strucure reveals a 5-coordinate Zn with 4 Ndonors and a single bound water molecule: N. J. Williams, W. Gan, J. H. Reibenspies, R. D. Hancock, Inorg. Chem. 2009, 48, 1407 – 1415. [28] For examples of quinoline-based systems that undergo anion-induced excimer emission, see: K. Ghosh, S. Adhikari, Tetrahedron Lett. 2008, 49, 658 – 663; A. Pramanik, G. Das, Tetrahedron 2009, 65, 2196 – 2200; K. Ghosh, S. Adhikari, Tetrahedron Lett. 2006, 47, 3577 – 3581. [29] For examples of metal-based quinoline complexes capable of excimer emission, see: Y. Mikata, A. Ugai, R. Ohnishi, H. Konno, Inorg. Chem. 2013, 52, 10223 – 10225; S. Patra, R. Gunupuru, R. Lo, E. Suresh, B. Ganguly, P. Paul, New J. Chem. 2012, 36, 988 – 1002; L. J. Govenlock, J. A. K. Howard, J. M. Moloney, D. Parker, R. D. Peacock, G. Siligardi, J. Chem. Soc. Perkin Trans. 2 1999, 2415 – 2418. [30] Gaussian 09, Revision A.02, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, Jr., J. A. Montgomery, J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox, Gaussian, Inc., Wallingford CT, 2009. [31] S. J. Butler, B. K. McMahon, R. Pal, D. Parker, J. W. Walton, Chem. Eur. J. 2013, 19, 9511 – 9517.
Received: July 21, 2014 Published online on October 9, 2014
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& Fluorescent Probes | Hot Paper |
Ratiometric Detection of Adenosine Triphosphate (ATP) in Water and Real-Time Monitoring of Apyrase Activity with a Tripodal Zinc Complex Stephen J. Butler*[a] Abstract: Two tripodal fluorescent probes Zn·L1,2 have been synthesised, and their anion-binding capabilities were examined by using fluorescence spectroscopy. Probe Zn·L1 allows the selective and ratiometric detection of adenosine triphosphate (ATP) at physiological pH, even in the presence of sev-
Introduction
eral competing anions, such as ADP, phosphate and bicarbonate. The probe was applied to the real-time monitoring of the apyrase-catalysed hydrolysis of ATP, in a medium that mimics an extracellular fluid.
lectivity is seldom assessed in a biologically relevant medium. This is critically important, because biofluids, such as serum and urine, contain a number of interfering species, including a high salt concentration and several competing anions (e.g., bicarbonate and phosphate), which will certainly influence the probe’s affinity and selectivity profile.[4, 21] Finally, probes capable of ratiometric (wavelength shift) detection of ATP are rare;[9, 22] most rely upon an “off/on” fluorescent signal change, which limits their application in cellulo, because uneven distribution of the probe may lead to unreliable readouts. Herein, we report a new water-soluble probe Zn·L1 that functions under physiological conditions and exhibits high selectivity for ATP over other polyphosphate anions. The zinc complex is based on a tripodal quinoline ligand, in which the
The design of probes that can detect changes in the presence and concentration of bioactive species in water is an active area of research.[1–3] Probes, which signal analyte binding by modulation of fluorescence emission, are most useful, because they provide spatial and temporal information rapidly and with high sensitivity.[4–6] Such systems have enormous potential to elucidate the crucial roles that certain bioactive substrates have in dynamic cellular processes. Adenosine triphosphate (ATP) is arguably one of the most important anions in living systems; not only does it serve as the chemicalenergy source for most biological functions, but also it plays key roles in extracellular signalling and DNA replication. Fluorescent molecules that signal the presence of ATP reversibly and selectively in aqueous solution could be used to monitor kinase activity in real time or quantify the energy supplied by ATP in different cellular compartments.[7, 8] There are very few synthetic probes for ATP, which can operate under physiological conditions with the required affinity and selectivity, notwithstanding some notable exceptions.[9–11] Most reported ATP-selective probes are limited in utility either because they cannot effectively discriminate between polyphosphate species (e.g., ADP and pyrophosphate may induce a similar or even greater fluorescent response compared to that of ATP), or the detection range of the probe is too low (0.1–10 mm) and does not match the concentration levels of ATP present in Figure 1. Structures of ligands L1,2. Complex Zn·L1 is expected to have one water molecells (1–5 mm).[12–20] Moreover, anion affinity and se- cule coordinated to ZnII in aqueous solution. [a] Dr. S. J. Butler Department of Chemistry, Durham University South Road, Durham, DH1 3LE (UK) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404499. Chem. Eur. J. 2014, 20, 15768 – 15774
fifth coordination site is likely to be occupied by a water molecule (Figure 1). Such a complex is capable of forming strong metal–ligand interactions with ATP. The central ZnII ion pre-organizes the three quinoline groups, each functionalised at the seventh position with aryl urea units to engage in hydrogen15768
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Full Paper bonding interactions with the P O oxygen atoms of ATP. Synthetic anion receptors that rely solely on hydrogen bonding are generally ineffective in water; however, in conjunction with metal–ligand interactions, cooperative binding sites capable of hydrogen bonding or p–p stacking with a target anion can substantially enhance binding affinity and selectivity.[21, 24–26] It was envisaged that selectivity for ATP over non-nucleoside phosphates, such as HPO42 and P2O74 (PPi), could be enhanced through p–p stacking interactions between the terminal aromatic groups of Zn·L1 and the adenosine moiety of ATP. The terminal aryl groups possess p-substituted sulphonate moieties to increase water solubility. As a comparison, the structural analogue Zn·L2 was synthesised to investigate the effects that the position and geometry of the aryl urea binding sites would have on anion affinity, selectivity and fluorescence response.
quenching of the PET mechanism, leading to a CHEF effect.[27] Zinc titrations performed at pH 7.4 revealed that one equivalent of Zn(ClO4)2 was sufficient to obtain 100 % of the zinc complexes of L1,2 (Figure S3 in the Supporting Information). The binding stoichiometry between L1,2 and Zn in aqueous solution (25 mm 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer, pH 7.4) was determined to be 1:1 by using the method of continuous variation (Job’s Plot, Figure S4 in the Supporting Information). Further evidence for a 1:1 binding was provided by high-resolution (ESI) mass spectrometric data, which revealed major signals for the singly charged species [L1 + Zn] and [L2 + Zn] (Figure S21 in the Supporting Information). The UV/Vis spectra of Zn·L1,2 are very similar to one another, presenting an intense band at l = 270 nm and a broad featureless band at 344 nm (e = 8200 m 1 cm 1, Figure S5 in the Supporting Information).
Results and Discussion
Anion-binding studies at physiological pH
Synthesis and characterisation A modular approach was adopted for the syntheses of Zn·L1,2. A representative synthesis of L1 is given in Scheme 1. First, the C3-symmetric scaffold, tris(7-amino-2-quinolinyl)amine (3) was prepared by reacting the appropriately functionalised 2-quinolinyl amine (1) with two equivalents of the 2-methylquinoline mesylate ester 2 in the presence of K2CO3. The tert-butoxycarbonyl (Boc) protecting groups of 3 were removed by using 4 m HCl in dioxane, followed by a basic work-up procedure to give the tris(amine) 4. Next, the aryl urea groups were introduced by reacting tris-amine 4 with 4-(chlorosulfonyl)phenyl isocyanate in CH3CN/DMF at room temperature, followed by basic hydrolysis of the chlorosulfonyl groups, to give ligand L1 in good yield following RP-HPLC purification. A similar procedure was adopted for the synthesis of L2. Subsequent addition of one equivalent of Zn(ClO4)2 in water at pH 7.4 gave the water soluble zinc(II) complexes Zn·L1,2. The experimental details and characterisation data are provided in the Supporting Information. In the presence of one equivalent of Zn(ClO4)2, L1,2 exhibited large enhancements in fluorescence emission centred at l = 510 nm (ten-fold and eight-fold increase, respectively, lexc = 344 nm; Figure S1–2 in the Supporting Information) due to
To evaluate the fluorescence response of Zn·L1,2 for different anions, the emission spectra of Zn·L1,2 (20 mm) were recorded in water (25 mm HEPES, pH 7.4, lexc = 344 nm) in the presence of a 500-fold excess of selected anions (Figure 2). When ATP was added to Zn·L1, a 2.5-fold increase in fluorescence emission intensity was observed, whereas ADP caused only a 1.4fold fluorescence enhancement. In sharp contrast, PPi, HPO42 and chloride resulted in quenching of the fluorescence emission centred at lem = 510 nm (up to 50 % in the case of PPi). AMP and cAMP also caused quenching of emission by approximately 20 %. Thus, Zn·L1 is selective for nucleoside polyphosphate anions, especially for ATP, over PPi and monophosphate anions. The only other anion tested that induced an enhancement in fluorescence emission was citrate (two-fold increase, Figure S13 in the Supporting Information). Notably, the addition of bicarbonate resulted in a large blueshift in the emission maximum from l = 510 to 445 nm, as well as quenching of the emission by 50 % (Figure 2). A similar response was observed in the presence of sulfate, although the spectrum consisted of a pair of bands with lem = 445 and 500 nm (Figure S14 in the Supporting Information). The appearance of two spectral bands centred at 445 and 510 nm in the presence of different anions, such as bicarbonate and ATP, can be ascribed to emission from the quinoline
Scheme 1. Synthesis of the tripodal quinoline ligand L1. Chem. Eur. J. 2014, 20, 15768 – 15774
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Full Paper For Zn·L2, in which the aryl urea groups are located at the quinoline 6-position, no significant change in emission spectral form or intensity was observed for every anion tested (Figure S5 in the Supporting Information). This clearly indicates that the position and geometry of the urea binding sites relative to the central ZnII ion has a profound influence on the anion binding and signalling properties of these systems. Specifically, in the case of Zn·L1, the urea functionalities are suitably positioned to allow cooperative hydrogen-bonding interactions with multiple P O atoms of ATP (Scheme 2). Titrations were carried out for Zn·L1 with various anions by adding aqueous solutions of the anion in 1–10 mL aliquots to a solution of the complex in water (25 mm HEPES, pH 7.4). Association constants were determined for those anions able to induce a significant change in the emission spectrum of Zn·L1 (Table 1). For ATP and ADP, the emission increase ratio (F/F0 at lem = 510 nm) was measured as a function of added anion concentration, whilst for bicarbonate, citrate and sulfate, changes in the ratio of monomer and excimer emission bands were used. The titration data was analysed by using a non-linear, least squares curve-fitting procedure based on a 1:1 binding model. Probe Zn·L1 showed strong binding to ATP in water (log Ka = 5.21), approximately 20 times stronger than to ADP (log Ka = 3.86), and 100 times stronger than citrate (log Ka = Figure 2. Top: change in fluorescence emission spectra of Zn·L1 (20 mm) in the presence of different anions (10 mm, sodium salts). Bottom: change in fluorescence emission of Zn·L1 (20 mm) as a function of added ATP (up to 1.6 mm). The inset shows the fit (line) to the titration data, for log Ka = 5.21 ( 0.10) Conditions: aqueous solution of HEPES buffer (25 mm, pH 7.4), lexc = 344 nm, 25 8C.
Table 1. Association constants (log Ka) determined for Zn·L1 and various anions in aqueous solution (25 mm HEPES, pH 7.4, 295 K). Each value represents the average of two duplicate titration experiments.[a] Anion
ATP
ADP
AMP
PPi
Citrate
HCO3
Log Ka
5.21 (2.94)[b]
3.86 (2.07)[b]
n/a[c]
n/a[c]
3.19
1.75
monomer and intramolecular excimer of Zn·L1, respectively.[28, 29] Given that there is no significant change in the UV ab[a] Typical errors in measurement were 0.10 with the statistical error assorption or excitation spectra when bicarbonate or ATP is sociated with the fit of the data < 0.05. [b] Values in parenthesis refer to added, a ground-state charge-transfer interaction can be ruled measurements in an aqueous solution that simulates an extracellular fluid out. Complexation of small anions, such as bicarbonate and (see the main text for details). [c] Affinity constants could not be determined, because only minor changes in fluorescence emission were obsulphate, tend to favour monomer formation (lem = 445 nm), served. n/a = not analysed. V whilst P oxyanions favour the excimer form (lem = 510 nm). It is hypothesised that ATP and ADP stabilise the excimer form, possibly through secondary interactions between the aromatic adenosine and quinoline groups, leading to an increase in emission intensity. ESI mass spectrometric data supports intramolecular excimer formation and a 1:1 binding mode for certain hostanion complexes. Major signals were observed for the singly charged ternary complexes Zn·L1 + ATP, Zn·L1 + ADP, Zn·L1 + PPi and Zn·L1 + perchlorate, which were in close agreement with the calculated isotopic distributions (Figure S23–26 in the Scheme 2. Proposed binding mode of the ternary complexes of Zn·L1 with ATP and bicarbonate. Selectivity for Supporting Information). ATP is attributed to the synergistic effect between metal–ligand and hydrogen-bonding interactions. Chem. Eur. J. 2014, 20, 15768 – 15774
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Full Paper 3.19). Bicarbonate and sulfate were found to bind to Zn·L1 rather weakly (log Ka = 1.75 and 0.98, respectively). Binding constants for AMP, HPO42 , and PPi could not be estimated with any confidence, because only minor changes in fluorescence emission took place (Figure S8–14 in the Supporting Information). The mode of binding of Zn·L1 to ATP in D2O was studied by NMR spectroscopy. 1H NMR spectra of Zn·L1 in the absence and presence of ATP showed broadened signals in the aromatic region due to exchange processes occurring (Figure S27 in the Supporting Information). This prohibited a detailed structural characterization of the host-anion interaction. The 31 P NMR spectral data was more informative; distinct 31P signals were observed for the a-, b- and g-phosphorus atoms of ATP (pD 7.4, Figure 3). Subsequent addition of one equivalent
ATP detection in a biorelevant medium In many biological fluids, such as serum or cerebrospinal fluid, the most abundant anions are chloride and bicarbonate, which are present in concentrations 10–100 times greater than those of other anions.[4] To determine the practical utility of these systems in biological assays, we examined the ability of Zn·L1 to detect ATP in an aqueous solution that mimics an extracellular fluid.[31] The solution contains the sodium salts of chloride (110 mm), bicarbonate (30 mm), lactate (2.3 mm), hydrogenphosphate (0.9 mm) and citrate (0.13 mm), buffered to pH 7.4 (10 mm HEPES). Under these conditions, the emission spectral form of Zn·L1 was very similar to that observed in the presence of excess bicarbonate only (lem = 445 nm). Titration of Zn·L1 (20 mm) with ATP resulted in a large redshift in the emission maximum from 445 to 510 nm and a substantial enhancement in emission intensity (Figure 4), consistent with displacement of the bound bicarbonate anion and concomitant excimer formation. ADP and PPi caused similar shifts in emission maximum, but with only small enhancements in emission intensity, signifying a closer competition between these anions and bicarbonate. Meanwhile, the addition of millimolar concentrations of AMP, cAMP, HPO42, phosphotyrosine, citrate and lactate did not elicit a significant spectral response (Figures S15–S18 in the Supporting Information).
Figure 3. Partial 31P NMR spectra (700 MHz) in D2O (pD 7.4) of: a) ATP (5 mm); and b) ATP (5 mm) and Zn·L1 (5 mm). The spectra have been normalised for comparison.
of Zn·L1 resulted in exchange-broadened signals of the b- and g-P atoms into the baseline, as well as clear shifts to higher frequency from d = 22.8 and 9.6 ppm to 22.1 and 9.0 ppm for b-P and g-P, respectively. In contrast, the chemical shift of the a-P atom did not change in the presence of Zn·L1, suggesting that binding of ATP occurs predominantly at the band g-P atoms. Taken together, this data is consistent with the binding mode illustrated for ATP and Zn·L1 in Scheme 2, in which one negatively charged g-P-O atom coordinates to the zinc metal and both g- and b-P-O atoms engage in hydrogenbonding interactions with the urea NH groups. A molecular model (optimized at HF/3-21G* with the Gaussian 09 package,[30] Figure S28 in the Supporting Information) of the ternary complex of Zn·L1 with a model triphosphate anion suggests that the urea functionalities are suitably positioned to form hydrogen bonds with the oxygen atoms of the g- and b-phosphorus, whereas the negatively charged oxygen atom of the a-P is too distant for a sufficient interaction to occur. Chem. Eur. J. 2014, 20, 15768 – 15774
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Figure 4. Ratiometric fluorescence detection of ATP (top) and ADP (bottom) in a simulated extracellular fluid, by monitoring the emission spectral change (510/445 nm) of Zn·L1 (20 mm) as a function of added anion. The inset shows the fit (line) to the titration data. Conditions: aqueous solution of HEPES buffer (10 mm, pH 7.4) containing NaCl (110 mm), NaHCO3 (30 mm), sodium lactate (2.3 mm), Na2HPO4 (0.9 mm) and sodium citrate (0.13 mm); lexc = 344 nm, 25 8C.
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Full Paper Association constants were determined for ATP and ADP by plotting the changes in the ratio of the monomer (445 nm) and excimer (510 nm) emission bands as a function of added anion concentration, and fitting the data to a 1:1 binding model. ATP was found to bind to Zn·L1 with log Ka = 2.94, approximately eight times greater than the binding to ADP (log Ka = 2.07).
Monitoring apyrase activity in real time Having established that Zn·L1 can distinguish ATP from ADP, PPi and AMP, the probe was applied to real-time monitoring of the enzymatic activity of apyrase. Apyrase is a hydrolytic enzyme that converts ATP to ADP, AMP and HPO42 . To a buffered aqueous solution (25 mm HEPES, pH 7.4) containing the Zn·L1 (20 mm) and ATP (2 mm) was added various concentrations of apyrase (150–300 mU), and the decrease in emission intensity at 510 nm was monitored as a function of time (Figure 5). The time-trace plots reveal that the reaction rate in-
Figure 6. Real-time monitoring of the apyrase-catalysed hydrolysis of ATP (5 mm) under mimicked physiological conditions, by monitoring the emission ratio 505/450 nm of Zn·L1. Conditions: aqueous solution of HEPES buffer (10 mm, pH 7.4) containing NaCl (110 mm), NaHCO3 (30 mm), sodium lactate (2.3 mm), Na2HPO4 (0.9 mm) and sodium citrate (0.13 mm); 300 mm apyrase; lexc = 344 nm, 25 8C.
serves as a simple and effective means for monitoring apyrase activity in a biorelevant medium, auguring well for its implementation in real biological systems.
Conclusion
Figure 5. Time-trace plots of the hydrolysis of ATP (2 mm) by apyrase (150– 300 mU) monitored by the change in fluorescence emission (510 nm) of Zn·L1. Conditions: buffered aqueous solution (25 mm HEPES, pH 7.4); lexc = 344 nm, 25 8C.
creases in proportion to enzyme concentration. The complete hydrolysis of ATP was verified by 31P NMR spectroscopy. The reaction mixture containing 300 mU apyrase was quenched after 20 min by the addition of EDTA (100 mm). The 31P NMR spectrum of the quenched mixture revealed signals corresponding to ADP, and AMP in the approximate ratio 2:1 (Figure S20 in the Supporting Information). Next, apyrase activity was monitored in a simulated extracellular fluid, by using 5 mm ATP and 300 mU of apyrase (Figure 6). Under these conditions, the enzyme-catalysed hydrolysis of ATP resulted in a decrease in the ratio of the excimer/monomer emission. A plot of the emission ratio, F505/F450, versus time revealed a slower reaction rate compared to that observed in water alone, consistent with a higher starting concentration of ATP. After 20 min, the reaction reached a plateau; the spectral form at this point was almost identical to that observed for Zn·L1 in the presence of 5 mm ADP (Figure 4), indicating quantitative hydrolysis of ATP after 20 min. Thus, Zn·L1 Chem. Eur. J. 2014, 20, 15768 – 15774
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In summary, an ATP-selective fluorescent probe Zn·L1 that functions at physiological pH has been created. The integration of several cooperative binding sites (i.e., metal–ligand interactions, hydrogen bonding and p–p stacking) within the probe afforded tight binding to ATP in water (log Ka = 5.21), with a useful level of selectivity over other polyphosphate species including ADP and PPi. In comparison, no anion-induced fluorescence signalling was achieved by Zn·L2 due to its lack of suitably positioned aryl urea groups. In an aqueous medium that simulates extracellular fluid selectively of Zn·L1 for ATP is tuned to the millimolar range. This permits its ratiometric detection in the metabolic concentration range (1–5 mm), even in the presence of a number of competing anions, including bicarbonate, phosphate and citrate. Finally, it has been demonstrated that Zn·L1 can operate in a simulated biological fluid to monitor the apyrase-catalysed hydrolysis of ATP in real time. Future work will be directed towards the creation of cell-permeable analogues that can report on changes in local ATP concentrations in cellulo. The modular approach to synthesis will enable factors, such as cytotoxicity, cellular uptake and pH effects, to be addressed; this work is underway.
Experimental Section Details of instrumentation, ligand synthesis, methods of purification, characterisation data and spectral titrations with added anions are given in the Supporting Information.
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Full Paper Ligand 1 (L1)
Tris[7-(tert-butoxycarbonylamino)-2-quinolinyl]amine (3) To a solution of the free amine (1; 32 mg, 0.117 mmol) and the mesylate ester (2; 70 mg, 0.234 mmol) in anhydrous CH3CN (3 mL) was added K2CO3 (48 mg, 0.351 mmol), and the mixture was stirred under argon at 60 8C. The progress of the reaction was monitored by LC-MS analysis at regular intervals, which revealed complete consumption of starting material after 12 h. At this time, a yellow precipitate had formed. The solvent was removed under reduced pressure, and the residue was dissolved in a mixture of CH2Cl2/ CH3OH (8:2, 3 mL). The solution was decanted from excess potassium salts and the solvent was removed under reduced pressure. The residue was triturated with CH3CN (3 5 mL) to afford tris[7(tert-butoxycarbonylamino)-2-quinolinyl]amine 3 as a colourless solid (70 mg, 76 %); m.p. 175–178 8C; 1H NMR (700 MHz, CDCl3): d = 9.75 (3 H, br s, CONH), 8.35 (3 H, s, H8), 8.28 (3 H, d, 3JH H 8.3 Hz, H4), 7.89 (3 H, d, 3JH H 8.8 Hz, H5), 7.78 (3 H, d, 3JH H 8.3 Hz, H3), 7.76 (3 H, d, 3JH H 8.8 Hz, H6), 4.09 (6 H, s, H9), 1.53 ppm (27 H, s, H12); 13C NMR (176 MHz, CDCl3): d = 160.9 (C2), 153.5 (C10), 148.9 (C2’), 141.4 (C7), 136.3 (C4), 128.5 (C5), 123.6 (C3’), 119.6(4) (C6), 119.5(7) (C3), 114.9 (C8), 79.8 (C11), 61.1 (C9), 28.0 ppm (C12); LRMS (ESI): m/z 786 [M + H] + ; (HRMS +) m/z 786.3960 [M + H] + (C45H52N7O6 requires 786.3979); Rf = 0.25 (silica gel; hexane/EtOAc 1:1).
Tris(7-amino-2-quinolinyl)amine (4; 23 mg, 0.047 mmol) was dissolved in a mixture of CH3CN/DMF (1:1, 1 mL). A solution of 4(chlorosulfonyl)phenyl isocyanate (41 mg, 0.190 mmol) in a mixture of CH3CN/DMF (1:1, 1 mL) was added dropwise over a period of 30 min, causing a precipitate to form. The resultant mixture was stirred at RT for 12 h under argon. The solvent was removed under reduced pressure to give a cream coloured solid. The crude material was suspended in water (3 mL), and the pH was adjusted to ten by the dropwise addition of NaOH (1 m). The mixture was stirred at RT for 30 min at which point the solid had completely dissolved. The pH of the solution was adjusted to 7.5 by the addition of HCl (1 m), and the aqueous mixture was purified by preparative RPHPLC (gradient: 10–100 % acetonitrile in 25 mm triethylammonium acetate over 10 min; tR = 7.3 min, Figure 7) to give L1 as a colourless oil (28 mg, 55 %); 1H NMR (400 MHz, D2O): d = 7.94 (6 H, complex m), 7.59 (12 H, br m), 7.42 (9 H, br m), 3.97 (6 H, br s); N H and O H signals were not observed; LRMS (ESI) m/z 1081 [M H] ; (C51H41N10O12S3 requires (HRMS + ) m/z 1081.2045 [M H] 1081.2068).
Figure 7. Analytical RP-HPLC of L1: tR 7.3 min (gradient: 10 to 100 % acetonitrile in 25 mm triethylammonium acetate over 10 min).
Acknowledgements S.J.B. wishes to thank the Ramsay Memorial Fellowship Trust and Durham University for support and funding. Thanks to Dr. Fernanda Duarte and Dr. Mark Fox for assisting with molecular modelling studies.
Tris(7-amino-2-quinolinyl)amine (4) Tris[7-(tert-butoxycarbonylamino)-2-quinolinyl]amine (3; 56 mg, 0.071 mmol) was suspended in anhydrous CH2Cl2 (1.5 mL) and cooled to 0 8C. HCl in dioxane (4 m, 1.5 mL) was added slowly causing the mixture to fully dissolve. The yellow solution was allowed to warm to RT and stirred for 1 h under argon. The solvent was removed under reduced pressure to give the tris hydrochloride salt as a yellow oil, which was partitioned between CHCl3/iPrOH (3:1, 20 mL) and saturated aqueous NaHCO3 solution (20 mL). The aqueous layer was extracted with CHCl3/iPrOH (3:1, 3 20 mL) and the combined organic fractions were dried (MgSO4) and concentrated under reduced pressure to afford the tris(amine) 4 as a colourless oil (23 mg, 75 %); 1H NMR (700 MHz, CD3OD:) d = 8.30 (3 H, d, 3JH H 8.0 Hz, H4), 7.63 (3 H, d, 3JH H 8.9 Hz, H5), 7.38 (3 H, d, 3JH H 8.0 Hz, H3), 7.13 (3 H, dd, 3JH H 8.9 Hz, 4JH H 2.0 Hz, H6), 6.95 (3 H, d, 4JH H 2.0 Hz, H8), 4.39 ppm (6 H, s, H9), N H signals were not observed; 13 C NMR (176 MHz, CD3OD): d = 157.4 (C2), 152.8 (C7), 145.3 (C2’), 143.3 (C4), 131.4 (C5), 123.6 (C3’), 123.0 (C6), 116.8 (C3), 96.5 (C8), 58.8 ppm (C9); LRMS (ESI): m/z 486 [M + H] + ; (HRMS + ) m/z 486.2406 [M + H] + (C30H28N7 requires 486.2403); Rf = 0.10 (silica gel; CH2Cl2/CH3OH, 80:20). Chem. Eur. J. 2014, 20, 15768 – 15774
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Keywords: anions · ATP · enzymes · fluorescent probes · quinolines
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Received: July 21, 2014 Published online on October 9, 2014
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