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Cite this: DOI: 10.1039/c4cc08044k Received 11th October 2014, Accepted 21st October 2014
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A ratiometric fluorescent probe with unexpected high selectivity for ATP and its application in cell imaging† Jia-Liang Tang,a Chun-Yan Li,*a Yong-Fei Lib and Chun-Xiang Zoua
DOI: 10.1039/c4cc08044k www.rsc.org/chemcomm
Naphthalimide–rhodamine compound (NR) is developed as a ratiometric fluorescent probe for ATP detection based on the FRET mechanism. It shows an unexpected high selectivity for ATP over other anions, especially organic phosphate anions, due to simultaneous interactions of two recognition sites, which benefits fluorescence imaging in living cells.
The development of fluorescent probes for selective recognition and detection of organic phosphate anions such as ATP, ADP, AMP, CTP, GTP, UTP and TTP (Fig. S1, ESI†) is an important research field due to their biological significance.1 Of all the organic phosphate anions, adenosine triphosphate (ATP) is particularly significant since it is perceived as the ‘‘molecular unit of currency’’ for intracellular energy transfer in the living cells and plays pivotal roles in various cellular events.2 So the detection of ATP is of considerable significance and has become the subject of current research. In recent years, there have been considerable efforts to develop fluorescent probes for ATP.3 However, most of the probes are based on zinc complexes and thus lack selectivity for a certain organic phosphate anion as they only contain one recognition site for the phosphate group.3a–i Therefore, it is still a challenging task to discriminate a certain organic phosphate anion among various organic phosphate anions. On the other hand, the probes reported for ATP are usually based on a single emission intensity change,3a–l which tends to be affected by a variety of factors such as instrumental efficiency, probe molecule concentration, and the micro-environment. These limitations can be circumvented by the use of ratiometric probes, which are beneficial for applications in biological systems. To the best of our knowledge, only two fluorescent probes have a
Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan, 411105, P. R. China. E-mail: [email protected]; Fax: +86 731 58292477; Tel: +86 731 58292205 b College of Chemical Engineering, Xiangtan University, Xiangtan, 411105, P. R. China † Electronic supplementary information (ESI) available: Detailed experimental procedures and analytical data. See DOI: 10.1039/c4cc08044k
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been reported for ratiometric detection of ATP with moderate success, to date.3m,n One is based on the fluorescence resonance energy transfer (FRET) mechanism and lacks selectivity among various organic phosphate anions.3m The other is based on excimer emission of pyrene, which is not favorable for ratiometric cell imaging, since the two emission wavelengths are both rather short (below 500 nm).3n Thus development of probes for ratiometric detection of ATP with high selectivity and a long emission wavelength is urgently required. Rhodamine spirolactam derivatives with long wavelengths and zero-background signals are ideal fluorophores for fluorescent probes. In recent years, great attention has been paid to the development of rhodamine-based probes for detection of ions through naked-eye observation and/or a fluorescence method because of their particular structural properties.4 To date, many rhodamine spirolactam-based probes have been designed for metal cations.5 However, the probes for anions are rare and mainly focus on conventional anions such as F , CN , P2O74 and CH3COO .6 To the best of our knowledge, no rhodamine-based ratiometric fluorescent probe has been reported for the detection of organic phosphate anions. Herein, a novel naphthalimide–rhodamine compound (NR) was designed and used as a probe for colorimetric and ratiometric fluorescent detection of ATP based on the FRET mechanism. In this sensing system, a highly efficient ring-opening reaction of the rhodamine spirolactam induced by ATP generates longwavelength emission from rhodamine which can act as the energy acceptor. Naphthalimide is chosen as the energy donor because its fluorescence spectrum can match well with the absorption spectrum of rhodamine.7 Probe NR (Fig. 1) was synthesized following the synthetic route shown in Fig. S2 (ESI†) and its chemical structure was well confirmed by 1H NMR, 13C NMR, MS and elemental analysis (Fig. S3–S5, ESI†). Detailed synthetic procedures and structural characterization are described in the ESI.† Initially, colorimetric responses of probe NR to a range of anions were investigated. In the colorimetric test, a large color change from light yellow to red was observed upon addition of ATP (Fig. 2a). In contrast, the other organic phosphate anions
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Fig. 1
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Chemical structure of compound NR.
Fig. 2 Change in color (a) and fluorescence (b) of NR (10 mM) with various anions (1 mM) in HEPES buffer solution (pH = 7.2).
(AMP, GTP, UTP and TTP), inorganic phosphate anions (PO43 , HPO42 , H2PO4 , P2O74 and P3O105 ) and common anions (SO42 , NO3 , CO32 , AcO and citrate), even at a high concentration (1 mM), did not display significant color changes. Only two selected organic phosphate anions (ADP and CTP) induced small color changes when added. These observations clearly indicate that NR exhibits an unexpectedly high selectivity for naked-eye detection of ATP over other various anions in aqueous solution. The corresponding absorption spectra of probe NR in various anions are shown in Fig. S6 (ESI†). Fluorescence responses of probe NR to different anions were also studied. Visual features are shown in Fig. 2b. Mx1 is a mixture of PO43 , HPO42 , H2PO4 , P2O74 and P3O105 and Mx2 is a mixture of SO42 , NO3 , CO32 , AcO and citrate. Fluorescence spectra are shown in Fig. S7 (ESI†). ATP induced the most significant fluorescence change unexpectedly and the other anions showed very small or almost no effects on the fluorescence of probe NR. Competition experiments were also carried out by adding an excess amount of organic phosphate anions such as ADP, AMP, CTP, GTP, UTP and TTP (1 mM) to 10 mM ATP solutions. The fluorescence response (I580/I530) of the probe was detected and then compared with that of the buffer solution containing only 10 mM ATP (Fig. S8, ESI†). Our probe showed almost unchanged response to ATP before and after the addition of the competing organic phosphate anions. Moreover, the presence of common cations like Na+, K+, Mg2+, Ca2+, Zn2+, Fe3+, Cu2+, Mn2+, Co2+, Ni2+, Ag+, Pb2+, Cr3+ and Cd2+ (1 mM) created small or no interference with the detection of ATP (Fig. S9, ESI†). To investigate this observed unique fluorescence response to ATP at certain concentrations, titration experiments were performed, from which more information about the binding could be obtained (Fig. 3a). We can see that probe NR itself displayed fluorescence emission of the naphthalimide moiety at 530 nm. The addition of ATP led the naphthalimide emission at 530 nm to decrease and the rhodamine emission at 580 nm to appear and increase remarkably. The emission intensity ratio, I580/I530, was gradually increased with the increasing ATP concentration and varied from 0.34 to 10.8 with the
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Fig. 3 (a) The fluorescence emission spectra of compound NR (10 mM) in the presence of different concentrations of ATP (0, 0.1, 0.2, 0.5, 0.8, 1.0, 2.0, 4.0, 5.0, 10.0, 50.0 mM) in HEPES buffer solution (pH = 7.2). The excitation wavelength is 420 nm. (b) The curve is plotted with the fluorescence intensity ratio (I580/I530) versus ATP concentrations.
concentration of ATP ranging from 0 to 50 mM, corresponding to a signal-to-background ratio of 31.8. The linear range of probe NR for ATP detection was found to be 0.1 to 10 mM (Fig. 3b) with a detection limit (LOD) of 0.1 mM (3s/slope). pH fluorescence titration experiments of NR in the presence and absence of ATP were also carried out (Fig S10, ESI†). The results showed that NR possesses good fluorescence response toward ATP in the neutral pH range from 6.0 to 8.0. The binding pattern of NR with ATP was investigated using the method of continuous variations (Job’s plot), which showed that NR binds ATP in a 1 : 1 stoichiometry (Fig. S11, ESI†). In addition, the peak at m/z = 1384.4 corresponding to [NR + ATP + H]+ in the mass spectra indicated the formation of the 1 : 1 complex of ATP–NR (Fig. S12, ESI†). The association constant (Ks) between NR and ATP was determined to be 1.2 106 M 1 (ESI†), which indicates that ATP binds to NR fairly strongly. The kinetics of the fluorescence of NR upon the addition of ATP with fluorescence intensity at 580 nm and 530 nm were also studied by time-dependent fluorescence spectroscopy, respectively (Fig. S13, ESI†). Once a plateau is reached, the fluorescence intensity at 580 nm and 530 nm stays relatively stable for the rest of the measurement, indicating that the probe is photostable. A comparison of the performance of this probe with previously reported ATP probes is shown in Table S1 (ESI†). All these results further prove that the probe has the ability to sense ATP.
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Based on the fluorescence and absorption spectra evidence, we proposed a possible binding mechanism (Fig. S14, ESI†). Owing to the fact that the rhodamine moiety in NR is in a spirolactam ring form, the probe only exhibits emission of the naphthalimide moiety. Upon the addition of ATP, the formation of the NR–ATP complex causes the configuration of the rhodamine moiety to transform from a spirolactam ring to a ring-opened amide. So the FRET process can occur from the naphthalimide moiety (energy donor) to the rhodamine moiety (energy acceptor). And then, naphthalimide green emission decreases and rhodamine red emission appears. The insights into the nature of the interactions between ATP and NR are provided by 13P NMR and 1H NMR experiments (Fig. S15 and S16, ESI†). After mixing with ATP, all the 13P signals of ATP shifted, indicating that the hydrogen bonds may exist between the amino groups of NR and the triphosphate anion of ATP. Moreover, the proton signals of the rhodamine moiety of NR and the proton signals of the adenine of ATP shifted, while the proton signals of the naphthalimide moiety of NR did not shift, demonstrating that p–p stacking interactions may be between the rhodamine moiety and ATP. In addition, two control compounds (compound 2 and 3) were synthesized and their fluorescence properties were investigated. Compound 2 exhibited no change in fluorescence (Fig. S17, ESI†), while compound 3 showed fluorescence enhancement due to the opening of spirolactam induced by ATP (Fig. S18, ESI†). More details on the interaction between compound 3 and ATP were studied by comparing the 1H NMR and mass spectra of 3 and 3 + ATP, respectively (Fig. S19–S21, ESI†). These results display that ATP opens the spirolactam of rhodamine in probe NR and the naphthalimide moiety seems to have no contribution to the binding of ATP. From these results, we can conclude that there are two main factors (the hydrogen bonds and p–p stacking interactions) that trigger the opening up of the spirolactam cooperatively. It was indicative that the observed binding preference of NR towards ATP could be attributed to the difference in the number of phosphate groups contained in nucleotides, and then the ability of ATP to bind to NR is stronger than that of ADP and AMP. And ATP is better than CTP, GTP, UTP and TTP owing to the fact that the rhodamine moiety of NR can interact most strongly with adenine by effective p stacking interactions, whereas there is little evidence for interaction between the rhodamine moiety and guanine, uracil or thymine due to their mismatched spatial orientations Therefore, the selectivity of NR for ATP over other organic phosphate anions is an outcome of the combination of the hydrogen bonds with the p–p stacking interactions that is unique for ATP. To further understand the interactions between NR and ATP, density functional theory (DFT) calculations were employed by using the Gaussian 03 package. The NR–ATP complex was subjected to energy optimization by using the PBE/LANL2DZ/6-31G* method and the most stable geometry is depicted in Fig. 4. On the one hand, the O atoms of the triphosphate anion in ATP interact with the H atoms of amino groups in NR by hydrogen bonds at the distance of 1.8 and 2.3 Å. On the other hand, the adenine base group of ATP forms p–p stacking interactions with the rhodamine group of NR at a distance of 3.9 Å owing to the fact that the well matched distance between the molecular size of ATP and NR allows the adenine to stand closely and parallel to rhodamine. In contrast, the binding patterns
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Fig. 4 Plot views of DFT calculated interactions between NR and ATP. Green dashed lines demonstrate the hydrogen bonds interactions. Red dashed lines demonstrate p–p stacking interactions.
calculated between NR and GTP (the most structurally similar nucleotide to ATP) only show the hydrogen bond interactions for both of them, and no p–p stacking interactions between the guanine group in GTP and the rhodamine group of NR were found due to their mismatched spatial orientations (Fig. S22, ESI†). Therefore, the NR probe shows excellent selectivity for ATP over other anions. Taking advantage of the selective sensing ability of NR toward ATP, the biological application of NR in HeLa cells was studied. As we all know, ATP is the most abundant phosphate anion in cells among various organic phosphate anions and the average concentration of ATP is reported to be B2–3 mM in mammalian cells.8 The cytotoxicity tests of HeLa cells were studied by an MTT assay with the concentration of NR ranging from 0 mM to 10 mM (Fig. S23, ESI†). The results showed that more than 97% cells were viable, which indicated the non-cytotoxicity of NR to cells under our experimental conditions. Fig. 5 shows fluorescence images at both red (580 10 nm) and green (520 10 nm) channels. One group of HeLa cells incubated with NR (10 mM) for 30 min at 37 1C showed a strong red intracellular fluorescence (Fig. 5b) and almost no green one (Fig. 5c), which suggested that the cells contain a certain amount of ATP. For comparison, another group of cells was treated with apyrase from 0 to 60 min and then incubated with NR (10 mM) for further 30 min at
Fig. 5 Images of HeLa cells incubated with probe NR in the absence or presence of apyrase. (a) The bright field image of HeLa cells incubated with NR; (b) The fluorescence image of (a) from the red channel; (c) the fluorescence image of (a) from the green channel; (d) the overlay image of frames (a) and (b); (e) the bright field image of another group of HeLa cells treated with apyrase and then incubated with NR. (f) The fluorescence image of (e) from the red channel. (g) The fluorescence image of (e) from the green channel; (h) the overlay image of frames (e) and (g). Excitation was set at 458 nm.
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37 1C (Fig. S24, ESI†). After 60 min, cells displayed a strong quenching of the red fluorescence intensity (Fig. 5f) and a remarkable enhancement in the green fluorescence intensity (Fig. 5g). This is because apyrase, a hydrolytic enzyme, can convert ATP into AMP and inorganic phosphate (Pi).9 The overlay of fluorescence and bright-field images (Fig. 5d and h) indicated that the fluorescence signals are localized in the intracellular area and showed good cellmembrane permeability of probe NR. All these experimental results demonstrate that probe NR can be used for ratiometric imaging of ATP in biological samples with high selectivity. In conclusion, we have developed a novel ratiometric fluorescent probe (NR) for ATP detection in aqueous solution and living cells. The probe exhibits an unexpected high selectivity for ATP over other organic phosphate anions due to simultaneous interactions with both the polyphosphate chain and the nucleic base group. To the best of our knowledge, this is the first example of a rhodamine compound showing high selectivity for ATP based on the FRET mechanism. The ratiometric probe for ATP detection provides a novel strategy for the design of selective probes for other target organic phosphate anions. This work was supported by the National Natural Science Foundation of China (21005068), the China Postdoctoral Science Foundation funded project (2014M552140) and the Hunan Provincial Natural Science Foundation of China (11JJ3023, 13JJ6039, 2015JJ6104 and 12JJ7002).
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Cite this: DOI: 10.1039/c4cc08044k Received 11th October 2014, Accepted 21st October 2014
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A ratiometric fluorescent probe with unexpected high selectivity for ATP and its application in cell imaging† Jia-Liang Tang,a Chun-Yan Li,*a Yong-Fei Lib and Chun-Xiang Zoua
DOI: 10.1039/c4cc08044k www.rsc.org/chemcomm
Naphthalimide–rhodamine compound (NR) is developed as a ratiometric fluorescent probe for ATP detection based on the FRET mechanism. It shows an unexpected high selectivity for ATP over other anions, especially organic phosphate anions, due to simultaneous interactions of two recognition sites, which benefits fluorescence imaging in living cells.
The development of fluorescent probes for selective recognition and detection of organic phosphate anions such as ATP, ADP, AMP, CTP, GTP, UTP and TTP (Fig. S1, ESI†) is an important research field due to their biological significance.1 Of all the organic phosphate anions, adenosine triphosphate (ATP) is particularly significant since it is perceived as the ‘‘molecular unit of currency’’ for intracellular energy transfer in the living cells and plays pivotal roles in various cellular events.2 So the detection of ATP is of considerable significance and has become the subject of current research. In recent years, there have been considerable efforts to develop fluorescent probes for ATP.3 However, most of the probes are based on zinc complexes and thus lack selectivity for a certain organic phosphate anion as they only contain one recognition site for the phosphate group.3a–i Therefore, it is still a challenging task to discriminate a certain organic phosphate anion among various organic phosphate anions. On the other hand, the probes reported for ATP are usually based on a single emission intensity change,3a–l which tends to be affected by a variety of factors such as instrumental efficiency, probe molecule concentration, and the micro-environment. These limitations can be circumvented by the use of ratiometric probes, which are beneficial for applications in biological systems. To the best of our knowledge, only two fluorescent probes have a
Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan, 411105, P. R. China. E-mail: [email protected]; Fax: +86 731 58292477; Tel: +86 731 58292205 b College of Chemical Engineering, Xiangtan University, Xiangtan, 411105, P. R. China † Electronic supplementary information (ESI) available: Detailed experimental procedures and analytical data. See DOI: 10.1039/c4cc08044k
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been reported for ratiometric detection of ATP with moderate success, to date.3m,n One is based on the fluorescence resonance energy transfer (FRET) mechanism and lacks selectivity among various organic phosphate anions.3m The other is based on excimer emission of pyrene, which is not favorable for ratiometric cell imaging, since the two emission wavelengths are both rather short (below 500 nm).3n Thus development of probes for ratiometric detection of ATP with high selectivity and a long emission wavelength is urgently required. Rhodamine spirolactam derivatives with long wavelengths and zero-background signals are ideal fluorophores for fluorescent probes. In recent years, great attention has been paid to the development of rhodamine-based probes for detection of ions through naked-eye observation and/or a fluorescence method because of their particular structural properties.4 To date, many rhodamine spirolactam-based probes have been designed for metal cations.5 However, the probes for anions are rare and mainly focus on conventional anions such as F , CN , P2O74 and CH3COO .6 To the best of our knowledge, no rhodamine-based ratiometric fluorescent probe has been reported for the detection of organic phosphate anions. Herein, a novel naphthalimide–rhodamine compound (NR) was designed and used as a probe for colorimetric and ratiometric fluorescent detection of ATP based on the FRET mechanism. In this sensing system, a highly efficient ring-opening reaction of the rhodamine spirolactam induced by ATP generates longwavelength emission from rhodamine which can act as the energy acceptor. Naphthalimide is chosen as the energy donor because its fluorescence spectrum can match well with the absorption spectrum of rhodamine.7 Probe NR (Fig. 1) was synthesized following the synthetic route shown in Fig. S2 (ESI†) and its chemical structure was well confirmed by 1H NMR, 13C NMR, MS and elemental analysis (Fig. S3–S5, ESI†). Detailed synthetic procedures and structural characterization are described in the ESI.† Initially, colorimetric responses of probe NR to a range of anions were investigated. In the colorimetric test, a large color change from light yellow to red was observed upon addition of ATP (Fig. 2a). In contrast, the other organic phosphate anions
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Fig. 1
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Chemical structure of compound NR.
Fig. 2 Change in color (a) and fluorescence (b) of NR (10 mM) with various anions (1 mM) in HEPES buffer solution (pH = 7.2).
(AMP, GTP, UTP and TTP), inorganic phosphate anions (PO43 , HPO42 , H2PO4 , P2O74 and P3O105 ) and common anions (SO42 , NO3 , CO32 , AcO and citrate), even at a high concentration (1 mM), did not display significant color changes. Only two selected organic phosphate anions (ADP and CTP) induced small color changes when added. These observations clearly indicate that NR exhibits an unexpectedly high selectivity for naked-eye detection of ATP over other various anions in aqueous solution. The corresponding absorption spectra of probe NR in various anions are shown in Fig. S6 (ESI†). Fluorescence responses of probe NR to different anions were also studied. Visual features are shown in Fig. 2b. Mx1 is a mixture of PO43 , HPO42 , H2PO4 , P2O74 and P3O105 and Mx2 is a mixture of SO42 , NO3 , CO32 , AcO and citrate. Fluorescence spectra are shown in Fig. S7 (ESI†). ATP induced the most significant fluorescence change unexpectedly and the other anions showed very small or almost no effects on the fluorescence of probe NR. Competition experiments were also carried out by adding an excess amount of organic phosphate anions such as ADP, AMP, CTP, GTP, UTP and TTP (1 mM) to 10 mM ATP solutions. The fluorescence response (I580/I530) of the probe was detected and then compared with that of the buffer solution containing only 10 mM ATP (Fig. S8, ESI†). Our probe showed almost unchanged response to ATP before and after the addition of the competing organic phosphate anions. Moreover, the presence of common cations like Na+, K+, Mg2+, Ca2+, Zn2+, Fe3+, Cu2+, Mn2+, Co2+, Ni2+, Ag+, Pb2+, Cr3+ and Cd2+ (1 mM) created small or no interference with the detection of ATP (Fig. S9, ESI†). To investigate this observed unique fluorescence response to ATP at certain concentrations, titration experiments were performed, from which more information about the binding could be obtained (Fig. 3a). We can see that probe NR itself displayed fluorescence emission of the naphthalimide moiety at 530 nm. The addition of ATP led the naphthalimide emission at 530 nm to decrease and the rhodamine emission at 580 nm to appear and increase remarkably. The emission intensity ratio, I580/I530, was gradually increased with the increasing ATP concentration and varied from 0.34 to 10.8 with the
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Fig. 3 (a) The fluorescence emission spectra of compound NR (10 mM) in the presence of different concentrations of ATP (0, 0.1, 0.2, 0.5, 0.8, 1.0, 2.0, 4.0, 5.0, 10.0, 50.0 mM) in HEPES buffer solution (pH = 7.2). The excitation wavelength is 420 nm. (b) The curve is plotted with the fluorescence intensity ratio (I580/I530) versus ATP concentrations.
concentration of ATP ranging from 0 to 50 mM, corresponding to a signal-to-background ratio of 31.8. The linear range of probe NR for ATP detection was found to be 0.1 to 10 mM (Fig. 3b) with a detection limit (LOD) of 0.1 mM (3s/slope). pH fluorescence titration experiments of NR in the presence and absence of ATP were also carried out (Fig S10, ESI†). The results showed that NR possesses good fluorescence response toward ATP in the neutral pH range from 6.0 to 8.0. The binding pattern of NR with ATP was investigated using the method of continuous variations (Job’s plot), which showed that NR binds ATP in a 1 : 1 stoichiometry (Fig. S11, ESI†). In addition, the peak at m/z = 1384.4 corresponding to [NR + ATP + H]+ in the mass spectra indicated the formation of the 1 : 1 complex of ATP–NR (Fig. S12, ESI†). The association constant (Ks) between NR and ATP was determined to be 1.2 106 M 1 (ESI†), which indicates that ATP binds to NR fairly strongly. The kinetics of the fluorescence of NR upon the addition of ATP with fluorescence intensity at 580 nm and 530 nm were also studied by time-dependent fluorescence spectroscopy, respectively (Fig. S13, ESI†). Once a plateau is reached, the fluorescence intensity at 580 nm and 530 nm stays relatively stable for the rest of the measurement, indicating that the probe is photostable. A comparison of the performance of this probe with previously reported ATP probes is shown in Table S1 (ESI†). All these results further prove that the probe has the ability to sense ATP.
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Based on the fluorescence and absorption spectra evidence, we proposed a possible binding mechanism (Fig. S14, ESI†). Owing to the fact that the rhodamine moiety in NR is in a spirolactam ring form, the probe only exhibits emission of the naphthalimide moiety. Upon the addition of ATP, the formation of the NR–ATP complex causes the configuration of the rhodamine moiety to transform from a spirolactam ring to a ring-opened amide. So the FRET process can occur from the naphthalimide moiety (energy donor) to the rhodamine moiety (energy acceptor). And then, naphthalimide green emission decreases and rhodamine red emission appears. The insights into the nature of the interactions between ATP and NR are provided by 13P NMR and 1H NMR experiments (Fig. S15 and S16, ESI†). After mixing with ATP, all the 13P signals of ATP shifted, indicating that the hydrogen bonds may exist between the amino groups of NR and the triphosphate anion of ATP. Moreover, the proton signals of the rhodamine moiety of NR and the proton signals of the adenine of ATP shifted, while the proton signals of the naphthalimide moiety of NR did not shift, demonstrating that p–p stacking interactions may be between the rhodamine moiety and ATP. In addition, two control compounds (compound 2 and 3) were synthesized and their fluorescence properties were investigated. Compound 2 exhibited no change in fluorescence (Fig. S17, ESI†), while compound 3 showed fluorescence enhancement due to the opening of spirolactam induced by ATP (Fig. S18, ESI†). More details on the interaction between compound 3 and ATP were studied by comparing the 1H NMR and mass spectra of 3 and 3 + ATP, respectively (Fig. S19–S21, ESI†). These results display that ATP opens the spirolactam of rhodamine in probe NR and the naphthalimide moiety seems to have no contribution to the binding of ATP. From these results, we can conclude that there are two main factors (the hydrogen bonds and p–p stacking interactions) that trigger the opening up of the spirolactam cooperatively. It was indicative that the observed binding preference of NR towards ATP could be attributed to the difference in the number of phosphate groups contained in nucleotides, and then the ability of ATP to bind to NR is stronger than that of ADP and AMP. And ATP is better than CTP, GTP, UTP and TTP owing to the fact that the rhodamine moiety of NR can interact most strongly with adenine by effective p stacking interactions, whereas there is little evidence for interaction between the rhodamine moiety and guanine, uracil or thymine due to their mismatched spatial orientations Therefore, the selectivity of NR for ATP over other organic phosphate anions is an outcome of the combination of the hydrogen bonds with the p–p stacking interactions that is unique for ATP. To further understand the interactions between NR and ATP, density functional theory (DFT) calculations were employed by using the Gaussian 03 package. The NR–ATP complex was subjected to energy optimization by using the PBE/LANL2DZ/6-31G* method and the most stable geometry is depicted in Fig. 4. On the one hand, the O atoms of the triphosphate anion in ATP interact with the H atoms of amino groups in NR by hydrogen bonds at the distance of 1.8 and 2.3 Å. On the other hand, the adenine base group of ATP forms p–p stacking interactions with the rhodamine group of NR at a distance of 3.9 Å owing to the fact that the well matched distance between the molecular size of ATP and NR allows the adenine to stand closely and parallel to rhodamine. In contrast, the binding patterns
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Fig. 4 Plot views of DFT calculated interactions between NR and ATP. Green dashed lines demonstrate the hydrogen bonds interactions. Red dashed lines demonstrate p–p stacking interactions.
calculated between NR and GTP (the most structurally similar nucleotide to ATP) only show the hydrogen bond interactions for both of them, and no p–p stacking interactions between the guanine group in GTP and the rhodamine group of NR were found due to their mismatched spatial orientations (Fig. S22, ESI†). Therefore, the NR probe shows excellent selectivity for ATP over other anions. Taking advantage of the selective sensing ability of NR toward ATP, the biological application of NR in HeLa cells was studied. As we all know, ATP is the most abundant phosphate anion in cells among various organic phosphate anions and the average concentration of ATP is reported to be B2–3 mM in mammalian cells.8 The cytotoxicity tests of HeLa cells were studied by an MTT assay with the concentration of NR ranging from 0 mM to 10 mM (Fig. S23, ESI†). The results showed that more than 97% cells were viable, which indicated the non-cytotoxicity of NR to cells under our experimental conditions. Fig. 5 shows fluorescence images at both red (580 10 nm) and green (520 10 nm) channels. One group of HeLa cells incubated with NR (10 mM) for 30 min at 37 1C showed a strong red intracellular fluorescence (Fig. 5b) and almost no green one (Fig. 5c), which suggested that the cells contain a certain amount of ATP. For comparison, another group of cells was treated with apyrase from 0 to 60 min and then incubated with NR (10 mM) for further 30 min at
Fig. 5 Images of HeLa cells incubated with probe NR in the absence or presence of apyrase. (a) The bright field image of HeLa cells incubated with NR; (b) The fluorescence image of (a) from the red channel; (c) the fluorescence image of (a) from the green channel; (d) the overlay image of frames (a) and (b); (e) the bright field image of another group of HeLa cells treated with apyrase and then incubated with NR. (f) The fluorescence image of (e) from the red channel. (g) The fluorescence image of (e) from the green channel; (h) the overlay image of frames (e) and (g). Excitation was set at 458 nm.
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37 1C (Fig. S24, ESI†). After 60 min, cells displayed a strong quenching of the red fluorescence intensity (Fig. 5f) and a remarkable enhancement in the green fluorescence intensity (Fig. 5g). This is because apyrase, a hydrolytic enzyme, can convert ATP into AMP and inorganic phosphate (Pi).9 The overlay of fluorescence and bright-field images (Fig. 5d and h) indicated that the fluorescence signals are localized in the intracellular area and showed good cellmembrane permeability of probe NR. All these experimental results demonstrate that probe NR can be used for ratiometric imaging of ATP in biological samples with high selectivity. In conclusion, we have developed a novel ratiometric fluorescent probe (NR) for ATP detection in aqueous solution and living cells. The probe exhibits an unexpected high selectivity for ATP over other organic phosphate anions due to simultaneous interactions with both the polyphosphate chain and the nucleic base group. To the best of our knowledge, this is the first example of a rhodamine compound showing high selectivity for ATP based on the FRET mechanism. The ratiometric probe for ATP detection provides a novel strategy for the design of selective probes for other target organic phosphate anions. This work was supported by the National Natural Science Foundation of China (21005068), the China Postdoctoral Science Foundation funded project (2014M552140) and the Hunan Provincial Natural Science Foundation of China (11JJ3023, 13JJ6039, 2015JJ6104 and 12JJ7002).
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