Making pyrophosphate visible: the first precipitable and real-time fluorescent sensor for pyrophosphate in aqueous solution

Abstract

An in situ generated BINOL–DPA–Zn(II) complex is presented as a chemo-sensing ensemble for the recognition of phosphate-based molecules. The ensemble showed high sensitivity and selectivity for pyrophosphates (PPi), and it could be successfully applied in imaging PPi in living cells. Notably, the R1–Zn(ii) ensemble exhibited a very low detection limit (95 nM) for PPi and could realize the real time detection of PPi by the naked eye through precipitate experiments. The ensemble also showed good selectivity towards ATP, and the selectivity coefficient for PPi and ATP was calculated to be 4.1/2.8.

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Introduction

Anions such as phosphate-based inorganic as well as organic molecules play important roles in a wide range of chemical and biological processes of human life,^1–3 hence, considerable attention has been given to the development of synthetic receptors for the recognition and sensing of such anions. Among the phosphates, adenosine triphosphate (ATP) and inorganic pyrophosphates (PPi) have been of particular interest because they are involved in energy transduction in organisms, and they control metabolic processes through participation in various enzymatic reactions. These processes play important roles in biological processes such as energy storage, signal transduction and DNA sequencing.⁴ Therefore, developing phosphate-based probes that could be applied in bioimaging with high selectivity and sensitivity are highly needed.

In a classical chemical analysis, color change and precipitate formation are the two most convenient visual detection methods. In the past decades, significant progress has been made in the recognition of phosphate-based bioactive anions, and many examples of visuals that rely only on color change have been reported.^5,6 Analyte treatment with suitable probes or sensors often involves the formation of an acid–base or a host–guest complex that may precipitate from solution. However, such precipitation has not been utilized to date for the development of PPi sensors. Meanwhile, compared to the traditional chemosensors, precipitable sensors could realize the real-time detection of the analytes, which would give a corresponding signal immediately and provide the information in a short time.

On the other hand, it is challenging to selectively recognize bioactive anions in an aqueous system because of the strong hydration effects of anions. In this regard, metal–ligand receptors that have a stronger affinity for anions in competitive water are widely used in designing anion sensors within the past decade. Among them, the di-(2-picolyl) amine (DPA) unit has been widely exploited as a motif within metal complex-based anion sensor designs, particularly for the detection of phosphate-based molecules.^7,8 Most notably, the Zn(II) complex of DPA exhibits an affinity for phosphate-bearing moieties, which can lead to either an increase in the fluorescence or quenching of a tethered fluorophore and the binding mode adopted by the interacting anion.⁹ So far, the fluorescent sensors for phosphate-based anions based on the Zn(II) complex of DPA mainly employed anthracene,¹⁰ naphthalimide,¹¹ acedan,¹² perylene,¹³ naphthalene diimide,^14,15 pyrene,¹⁶ naphthalene,¹⁷ NBD,¹⁸etc., as the fluorophores.

Based on the number of DPA units incorporated, the DPA–fluorophore conjugate could be divided into two classes: mono- and di-DPA sensors. Two mono-DPA–Zn(II) complexes could coordinate with one PPi by forming an excimer, which causes fluorescent changes. Compared with mono-DPA–Zn(II) sensors, the interaction modes between di-DPA–Zn(II) sensors and PPi are complicated. It might form excimers between the two sensors after coordinating with two PPi ions; it is also possible to form supermolecules via one PPi coordinating with two DPA–Zn(II) units at two different sensors; additionally, two DPA–Zn(II) units in one sensor could also collaboratively coordinate with one PPi, if the distance between the units is allowable. Obviously, the location and the distance of the binding site are important to the interaction mode. Two binding sites could supply a higher binding energy, therefore, di-DPA–Zn(II) sensors have attracted much attention. It's worth noting that most di-DPA based sensors used the same skeleton. Since the binding mode is closely related to the framework, developing a novel skeleton for di-DPA units are highly need. Lippard et al. reported the first fluorescein platform with two DPA–Zn(II) units, which was recently proven to be coordinated with one PPi.¹⁹ Yoon reported a 8-naphthalenetetracarboxylic dianhydride based di-DPA–Zn(II) complex, however, due to the planar rigidity, the sensor formed a 2 + 2 type excimer with PPi rather than a monomer coordination.²⁰ These results indicated that the distance and the rigidity of the fluorophore are important to the binding mode with PPi (Scheme 1).

Scheme 1 The structures of chemosensing probes and their proposed binding modes with PPi.

1,1′-Bi-2-naphthol (BINOL) and its derivatives have attracted particular attention in fluorescence-based molecular recognition. Previously, we presented the first example of an enantioselective gel collapsing, as a new means of visual chiral sensing utilizing a BINOL–terpyridine-based Cu(II) complex.²¹ More recently, we reported a BINOL-based fluorescent sensor for Zn²⁺ and a ratiometric fluorescent sensor for Cu²⁺ in aqueous solution.²³ The two 2-naphthol units in BINOL are not on the same plane. The introduction of a DPA unit into the BINOL skeleton could affect the axial chirality of the BINOL, which might provide better conditions for PPi after coordination with Zn(II). In fact, Ahn reported a naphthol based mono-DPA–Zn(II) complex which could selectively respond to PPi, however, it needed a large quantity of PPi (200 equiv.) and a longer time (6 h).²² We doubt whether the reactivity and selectivity could be improved by introducing two-DPA units at the 3,3′position of BINOL. In connection with our continuing research on fluorescent sensors for biologically and environmentally important ions, herein, we present a BINOL–DPA–Zn(II) complex as the first precipitable and real-time fluorescent sensor for PPi.

In this manuscript, we report R1, R2, R3 (Scheme 2) conjugates which employ the BINOL skeleton as the fluorophores and DPA–Zn(II) as the binding unit for the selective detection of phosphate-based anions in aqueous solution. Delightfully, the in situ generated R1–Zn(ii) ensemble could serve as the first precipitable and real-time fluorescent sensor for PPi, and it could further successfully be applied in imaging PPi in living cells. Moreover, the R1–Zn(ii) ensemble also exhibited a very low detection limit for PPi.

Scheme 2 Synthesis route for the sensors R1/R2/R3.

Results and discussion

The sensor R1 was synthesized from (R)-(2,2-bis(methoxymethoxy)-1,1′-binaphthyl-3,3′-diyl)dimethol (Scheme 2). The intermediate 1 was obtained by the reaction of (R)-(2,2-bis(methoxymethoxy)-1,1′-binaphthyl-3,3′-diyl)dimethol with methylsulfonyl chloride and lithium bromide, which was further transformed into the R1 reaction with di(2-picolyl) amine. R2 was synthesized by similar procedures to R1, while R3 was synthesized by removing the MOM group (–CH₂OCH₃) of R1 using concentrated HCl in a CH₂Cl₂/C₂H₅OH solution. The precursors and final products were characterized by analytical and spectral techniques (see the Experimental section and ESI†).

The recognition of Zn²⁺ by sensors R1, R2, R3 were investigated by fluorescence titration. The sensor R1 was excited at 316 nm, and its fluorescence emission was collected in the range of 336–612 nm. The experiments were carried out in HEPES buffer by maintaining the sensor concentration of 10 μM throughout the experiment and varying the mole ratio of the added Zn²⁺; R1 is a weak emitter as its fluorescence is quenched due to the photoelectron transfer (PET) process from the lone pair of the nitrogen of the DPA group on BINOL. During the titration with Zn²⁺, the fluorescence intensity of sensor R1 was monitored as a function of the increase in the mole ratio of [Zn²⁺]/[R1] (Fig. 1). The reason for the resulting fluorescence enhancement upon the addition of Zn²⁺ is due to the suppression of the photoelectron transfer effect (PET) from the lone pair of electrons on the DPA group. Upon the titration of Zn²⁺, a 1 : 2 stoichiometry between R1 and Zn²⁺ was confirmed by the Job's plot analysis (Fig. 1, inset) and mass spectral analysis (Fig. S1, ESI†). The constant between R1 and Zn²⁺, 5.06 × 10¹⁰ M⁻¹, was calculated by the Benesi–Hildebrand equation (Fig. S2, ESI†). The proposed binding mode of R1 and Zn²⁺ is shown in Fig. 2. Moreover, the responses for Zn²⁺ of the sensors R2 and R3 were also investigated by the fluorescence study. As shown in Fig. S3,† when Zn²⁺ was added into the solution of R2, a little red shift in the maximal emission was found, and the emission intensity increased gradually with the increase of Zn²⁺. These changes in the fluorescence spectrum stopped when the concentration of Zn²⁺ reached 1.2 equiv. of R2 (Fig. S3, ESI†). However, there was no change of R3 under the same conditions (Fig. S4, ESI†).

Fig. 1 Fluorescence emission spectra of R1 upon addition of Zn²⁺ in HEPES (10 mM, pH = 7.4) [R1] = 10 μM, [Zn²⁺] = (0 μM, 1 μM, 2 μM, 3 μM, 4 μM, 6 μM, 8 μM, 10 μM, 15 μM, 20 μM, 25 μM). λ_ex = 316 nm. Inset: Job's plot obtained from the fluorescent titration of R1 with Zn²⁺ in HEPES, the total concentration is 10 μM.

Fig. 2 Proposed binding mode of R1 and Zn²⁺.

To further understand the coordination properties of R1 towards Zn²⁺, DFT calculations with the B3LYP exchange functional were performed using the Gaussian 03 package. The geometries were optimized and energies were estimated at the B3LYP/6-31G(d)&LANL2DZ level. The optimized configuration is shown in Fig. 3, which indicates that the two Zn²⁺ were well chelated by the N1, N2, N3, and N3, N4, N5 and N6 of R1. The Zn1–N1 bond length is 2.008 Å, the Zn1–N2 bond length is 2.018 Å, the Zn1–N3 bond length is 2.022 Å, the Zn2–N4 bond length is 2.082 Å, the Zn2–N5 bond length is 2.054 Å, and the Zn2–N6 bond length is 2.118 Å. After the calculation, we found the binding energy of R1 and Zn²⁺ was −74.3 kcal mol⁻¹, which was in agreement with the other experimental results.

Fig. 3 Optimized geometries of the R1–Zn(ii) complex.

The anion recognition studies were carried out by titrating an in situ generated complex R1–Zn(ii), R2–Zn(ii), R3–Zn(ii) by different anions, viz., PPi, F⁻, SCN⁻, H₂PO₄⁻, HCO₃⁻, Br⁻, PO₄³⁻, NO₃⁻, AcO⁻, citrate, I⁻, N₃⁻, SO₄²⁻. As shown in Fig. 4, for the sensor R1–Zn(ii), all the anion titrations exhibited no significant change in the fluorescence intensity except for PPi (Fig. 4, green and Fig. 5). The fluorescence of the in situ generated R1–Zn(ii) was increased gradually with the addition of PPi, wherein the intensity was saturated at 3 μM (0.3 equiv.) (Fig. 6). This clearly suggests that R1–Zn(ii) shows a stronger affinity with phosphates than other anions (F⁻, SCN⁻, H₂PO₄⁻, HCO₃⁻, Br⁻, PO₄³⁻, NO₃⁻, AcO⁻, citrate, I⁻, N₃⁻, SO₄²⁻) and it is a sensitive chemo-sensing ensemble for PPi among the thirteen anions in aqueous buffer solution. Under the same conditions, almost no fluorescence intensity changes of the in situ generated complexes R2–Zn(ii) and R3–Zn(ii) were observed in the presence of various anions (Fig. 4 yellow and red). These results suggest that the number of DPA–Zn(II) units and the MOM group is important for the binding ability.

Fig. 4 Fluorescence responses of the in situ prepared R1–Zn(ii)/R2–Zn(ii)/R3–Zn(ii) towards 100 μM (10 equiv.) various anions at 383 nm in HEPES(10 mM, pH = 7.4, 1% CH₃OH as the assistant solvent). Excitation at 316 nm: (1) R1–Zn(ii), R2–Zn(ii), R3–Zn(ii) (2) F⁻ (3) SCN⁻ (4) H₂PO₄⁻ (5) HCO₃⁻ (6) Br⁻(7) PO₄³⁻ (8) NO₃⁻ (9) AcO⁻ (10) citrate (11) I⁻ (12) Cl⁻ (13) N₃⁻ (14) SO₄²⁻ (15) PPi; λ_ex = 316 nm.

Fig. 5 Fluorescence responses of the in situ prepared R1–Zn(ii) complex towards 100 μM (10 equiv.) of various anions in HEPES (pH = 7.4, 10 mM, 1% CH₃OH as the assistant solvent), λ_ex = 316 nm.

Fig. 6 Fluorescence emission spectra of the in situ prepared R1–Zn(ii) complex upon addition of PPi in HEPES (10 mM, pH = 7.4) [R1–Zn(ii)] =10 μM, [PPi] = (0 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1.0 μM, 1.5 μM, 2.0 μM, 3.0 μM, 4.0 μM). λ_ex = 316 nm.

To better investigate the practical applicability of the in situ prepared R1–Zn(ii) complex, competitive experiments were carried out in the presence of PPi (10 μM) mixed with F⁻, SCN⁻, H₂PO₄⁻, HCO₃⁻, Br⁻, PO₄³⁻, NO₃⁻, AcO⁻, citrate, I⁻, N₃⁻, and SO₄²⁻ (50 μM), as shown in Fig. 7. There was no significant change in the fluorescence intensity when R1–Zn(ii) was placed in the mixture of PPi (10 μM) and other test anions (Fig. S5, ESI†), therefore there was almost no interference of the R1–Zn(ii) complex recognition of PPi in the presence of other anions. The results of the competitive experiments suggest that the in situ prepared R1–Zn(ii) complex shows good selectivity for PPi among the other tested anions. The quantitative analytical behaviour of the in situ prepared R1–Zn(ii) complex for the analysis of PPi was examined by fluorescence titration. As displayed in Fig. 7, the in situ prepared R1–Zn(ii) complex displayed a weak fluorescence emission at 383 nm, and upon gradual addition of PPi to the solution of R1–Zn(ii), the emission intensity at 383 nm increased significantly. There was almost a 4-fold enhancement of fluorescence intensity compared to the intensity of the in situ prepared R1–Zn(ii) complex itself. The Job's plot of the data revealed a 1 : 1 complex formation of R1–Zn(ii) and PPi (Fig. S6, ESI†), which was also supported by the HRMS experiment (Fig. S7, ESI†). The association constant and detection limit of R1–Zn(ii) for PPi was calculated to be 6.7 × 10⁵ M⁻¹ and 95 nM, respectively (Fig. S8 and S9, ESI†).²⁵ The UV-vis absorption spectra of R1–Zn(ii) in the presence of various concentrations of PPi also further proved the complexation between R1–Zn(ii) and PPi (Fig. S10, ESI†).

Fig. 7 Emissions of the in situ prepared R1–Zn(ii) complex with various anions (100 μM) in the absence and presence of PPi (10 μM) at 383 nm in HEPES(10 mM, pH = 7.4), the red bar: from left to right: R1–Zn²⁺ (10 μM); R1–Zn²⁺ (10 μM) + PPi (10 μM); R1–Zn²⁺ (10 μM) + PPi (10 μM) + AcO⁻ (50 μM); R1–Zn²⁺ (10 μM) + PPi (10 μM) + Cl⁻ (50 μM); R1–Zn²⁺ (10 μM) + PPi (10 μM) + HCO₃⁻ (50 μM); R1–Zn²⁺ (10 μM) + PPi (10 μM) + SO₄²⁻ (50 μM); R1–Zn²⁺ (10 μM) + PPi (10 μM) + Br⁻ (50 μM); R1–Zn²⁺ (10 μM) + PPi (10 μM) + F⁻(50 μM); R1–Zn²⁺ (10 μM) + PPi (10 μM) + N₃⁻(50 μM); R1–Zn²⁺ (10 μM) + PPi (10 μM) + SCN⁻ (50 μM); R1–Zn²⁺ (10 μM) + PPi (10 μM) + HPO₄²⁻ (50 μM); R1–Zn²⁺ (10 μM) + PPi (10 μM) + PO₄³⁻ (50 μM); R1–Zn²⁺ (10 μM)+ PPi (10 μM) + NO₃⁻ (50 μM); R1–Zn²⁺(10 μM) + PPi (10 μM) + I⁻ (50 μM). λ_ex = 316 nm.

As the in situ prepared R1–Zn(ii) exhibited good selectivity to PPi among general anions, the studies were further extended to organic biorelevant molecules bearing a phosphate moiety, viz., ATP, ADP, GTP, CTP, dUTP and dTTP. As displayed in Fig. 8, when 100 μM (10 equiv.) ATP and ADP was added to the R1–Zn(ii) (10 μM) solution, fluorescence enhancement was observed, and the enhancement in the presence of ATP is larger than that of ADP, while GTP, CTP, dUTP and dTTP didn't induce any change of the fluorescence intensity of R1–Zn(ii). We also found that the fluorescence enhancement caused by PPi is larger than that caused by ATP, and the selectivity coefficient for PPi and ATP is calculated to be 4.1/2.8, suggesting that the enhancement is dependent on both the bulkiness of the organic moiety and the number of phosphates. Therefore, we suspect that this selectivity for binding with anions is connected with the distance and the rigidity of the two DPA–Zn(II) units of the sensor. In order to assess whether the in situ prepared R1–Zn(ii) complex can be of any use in the recognition of anions by naked eye detection, R1–Zn(ii) (2.5 mM, when the concentrations of the sensor and analytes are μM, they cannot form precipitates, so we choose the concentration of the sensor as 2.5 mM) was titrated with 0.15 equiv. various anions, viz., PPi, ATP, ADP, GTP, CTP, dUTP and dTTP in HEPES. It was observed that in the presence of PPi, and ATP, the R1–Zn(ii) complex solution formed a precipitate. However, in the presence of GTP, CTP, dTTP, dUTP, ADP, the R1–Zn(ii) complex solution didn't form a precipitate; this result suggests that the in situ prepared R1–Zn(ii) complex can show selectivity for PPi and ATP over other anions, which is observable by the naked eye. In addition, we investigated how the different concentrations of PPi influence the precipitate formation. As shown in Fig. 9, we can see that with the increasing concentration of PPi, the quantity of the precipitate increased. This precipitate formation experiment further explained R1–Zn(ii) and PPi formed complex. Furthermore, this precipitation could be formed at the interface of the organic solvents and water (video S1†). This is the visual precipitate detection for PPi.

Fig. 8 Fluorescence responses of the in situ prepared R1–Zn(ii) complex (10 μM) towards 100 μM (10 equiv.) of phosphate-based organic molecules at 383 nm in HEPES (pH = 7.4, 10 mM, 1% CH₃OH as the assistant solvent), λ_ex = 316 nm.

Fig. 9 (a) The responses of the R1–Zn(ii) ensemble (2.5 mM) in the absence and presence of anions, from left to right: absence of anions, presence of PPi, ATP, ADP, CTP, GTP, dUTP, dTTP (0.275 mM); (b) the responses of the R1–Zn(ii) ensemble in the presence of different concentrations of PPi.

To better understand this precipitation, scanning electron microscopy (SEM) was used to investigate the process (Fig. 10). The results indicated that R1 was uniformly dispersed in the solution by forming small particles (0.8–1.3 μm), due to its suitable solubility in the HEPES solution. We believed the MOM group and the two DPA units were responsible for the formation of these small particles, which could not be observed by the naked eye in the solution. However, upon the addition of Zn²⁺ to the system, the solution became clear and no small particles were found, which suggested that R1–Zn(ii) was totally soluble in the solution. Once PPi was added, rough and scaly solids with large enormous surfaces were observed. This result also suggests that the interaction occurred between R1–Zn(ii) and PPi.

Fig. 10 SEM micrographs, (a) for R1 (b) for R1–Zn(ii), (c) for R1-Zn(ii)–PPi.

To further understand the in situR1–Zn(ii) complex, DFT calculations with the B3LYP exchange functional were performed by using the Gaussian 03 package. The geometries were optimized and the energies were estimated by the B3LYP/6-31G(d) & LANL2DZ level. The optimized configuration is shown in Fig. 11, which indicates that the O1, O2, O3 and O4 were well chelated by the Zn1 and Zn2 of the R1–Zn(ii) complex. The Zn1–O1 bond length is 2.066 Å, the Zn1–O2 bond length is 1.989 Å, the Zn1–N2 bond length is 2.126 Å, the Zn2–O3 bond length is 2.034 Å, the Zn2–O4 bond length is 1.997 Å, the Zn2–N4 bond length is 2.114 Å, the Zn2–N5 bond length is 2.159 Å, but the Zn1–N3 bond and the Zn2–N6 bond were broken, the results of the bonds breaking were supported by the Hong groups,²⁴ after the calculation, we found the binding energy of R1-Zn(ii) and PPi was −173.8 kcal mol⁻¹, which was also in agreement with the other experimental results.

Fig. 11 Optimized geometries of R1–Zn(ii) and PPi complex.

In order to show the practical utility of R1–Zn(ii) for the detection of PPi in a biological medium, fluorescence microscopy studies were carried out using Hela cells. Hela cells were incubated in PBS buffer (pH = 7.4) containing 10 μM of R1 for 30 min at 37 °C, followed by washing the cells with the same buffer, at this stage, the Hela cells displayed almost no fluorescence (Fig. 12d), upon the addition of Zn²⁺/pyrithione to the cells exogenously via incubation for another 30 min at 37 °C, the cells exhibited a weak blue fluorescence. However, when R1 and Zn²⁺ were preincubated, and the Hela cells were further treated with PPi for 30 min at 37 °C, the cells exhibited highly intense blue fluorescence, therefore, the cellular studies clearly indicate that R1 exhibits good cell permeability and shows effective intracellular fluorescence emission by forming an in situ complex, which in turn increases its fluorescence intensity upon treatment with PPi.

Fig. 12 Fluorescence from images obtained from Hela cells upon treatment in PBS buffer at pH = 7.4: (a) bright field image when treated with R1 (10 μM); (b) bright field image when treated with R1 followed by 20 μM of Zn²⁺/pyrithione (1 : 1) solution; (c) bright field image of the Hela cells treated with R1–Zn(ii) followed by 20 μM of PPi; (d) fluorescence image of that given under (a); (e) fluorescence image of that given under (b); (f) fluorescence image of that given under (c).

Experimental

General

Mass spectrometry (ESI-MS) and High Resolution Mass Spectrometry (HRMS) data were recorded on a Finnigan LCQDECA and a Bruker Daltonics Bio TOF mass spectrometer, respectively. The ¹H NMR and ¹³C NMR spectra were measured on a Bruker AM400 NMR spectrometer and the δ scale in ppm was referenced to residual solvent peaks or internal tetramethylsilane (TMS). Absorption spectra were recorded on a Hitachi U1900 spectrophotometer at 298 K. Fluorescence emission spectra were obtained using a FluoroMax-4 Spectrofluorophotometer (HORIBA JobinYvon) at 298 K. The MDA-MB-231 cells were analyzed in an Array Scan VTI HCS Reader (Thermo Fisher Scientific, Inc.). Unless otherwise indicated, all syntheses and manipulations were carried out under N₂ atmosphere. All of the solvents were dried according to the standard methods prior to use. All of the solvents were either HPLC or spectroscopic grade in the optical spectroscopic studies.

Preparation of (R)-3,3′-bis (bromomethyl)-2,2′-bis(methoxy-methyl)-1,1′binaphol

To an ice-cooled solution of (R)-(2,2′-bis(methoxymethoxy)–1,1′-binaphthyl-3,3′-diyl)dimethanol (1.0 g, 2.3 mmol) in toluene (30 mL) were successively added Et₃N (3.3 g, 23 mmol) and MsCl (2.62 g, 23 mmol). The mixture was stirred at 0 °C for 90 min and then LiBr (2.0 g, 23 mmol) in 30 mL DMF was added. The mixture was stirred at room temperature overnight. It was then diluted with ethyl acetate (120 mL) and washed with saturated brine and finally dried over MgSO₄ and condensed in vacuo to give crude product; purification by a silica column afforded the product as a yellow solid. ¹H NMR (400 Hz, CDCl₃) δ (ppm): 8.12 (s, 2H), 7.91 (d, 2H, J = 8.0 Hz), 7.44 (t, 2H, J = 8.0 Hz), 7.17–7.27 (m, 5H), 7.30 (t, 1H, J = 8.0 Hz), 7.36 (d, 1H, J = 12.0 Hz), 7.89 (t, 2H, J = 8.0 Hz), 7.29 (t, 2H, J = 8.0 Hz), 7.18 (d, 2H, J = 8.0 Hz),4.98 (q, 4H, J =12.0 Hz, 16.0 Hz), 4.64 (d, 2H, J = 4.0 Hz), 4.64 (d, 2H, J = 4.0 Hz), 2.98 (s, 6H). ¹³C NMR (100 MHz, CDCl₃), δ (ppm): 42.4, 56.9, 99.5, 125.5, 123.4, 125.6, 126.0, 127.2, 128.15, 130.6, 131.0, 131.2, 129.4, 134.2, 130.7, 152.2.

Preparation of sensors R1, R2, R3

R1 . Di-(2-picoly) amine (0.31 g, 1.4 mmol), K₂CO₃ (0.32 g, 3.0 mmol), and dry CH₃CN (5 mL) were placed in a round bottom flask under a N₂ atmosphere at room temperature. To this mixture, a CH₃CN solution of 1 (0.36 g, 0.6 mmol) was added dropwise under a N₂ atmosphere at room temperature over 30 min. After stirring for 44 h, the insoluble materials were removed by filtration, and washed with CH₂Cl₂ several times until the washing solvent became colourless. The combined filtrate was concentrated, and the resulting residue was purified by alumina column chromatography by using CH₂Cl₂ : CH₃OH =100 : 1 as the eluent to give a yellow oil in 82% yield. HRMS (m/z): calcd for C₅₀H₄₈N₆NaO₄, [M + Na⁺], 819.3635, found 819.3565, ¹H NMR (400 Hz, CDCl₃) δ (ppm): 8.54 (d, 4H, J = 4.0 Hz), 8.31 (s, 2H), 7.88 (d, 2H, J = 8.0 Hz), 7.61–7.68 (m, 8H), 7.37 (t, 1H, J = 8.0 Hz, 2H), 7.19 (d, 2H, J = 8.0 Hz), 7.16–7.12 (m, 6H), 3.92–4.06 (m, 12H), 4.46 (d, 2H, J = 4.0 Hz),4.35 (d, 2H, J = 4.0 Hz), 2.68 (s, 6H, J =4.0 Hz). ¹³C NMR (100 MHz, CDCl₃), δ (ppm): 53.8, 56.5, 60.3, 99.0, 122.0, 124.9, 125.4, 126.1, 127.8, 128.8, 129.3, 131.8, 132.6, 133.3, 136.5, 148.1, 153.0, 159.6.

R2 . Di-(2-picoly) amine (0.16 g, 0.7 mmol), K₂CO₃ (0.16 g, 1.5 mmol), and dry CH₃CN (5 mL) were placed in a round bottom flask under a N₂ atmosphere at room temperature. To this mixture, a CH₃CN solution of 1 (0.33 g, 0.7 mmol) was added dropwise under a N₂ atmosphere at room temperature over 30 min. After stirring for 44 h, the insoluble materials were removed by filtration, and washed with CH₂Cl₂ several times until the washing solvent became colourless. The combined filtrate was concentrated, and the resulting residue was purified by alumina column chromatography by using CH₂Cl₂ : CH₃OH = 100 : 1 as the eluent to give a yellow oil in 85% yield. HRMS (m/z): calcd for C₃₇H₃₆N₃O₄, [M + H⁺], 586.2706, found 586.2708. ¹H NMR (400 Hz, CDCl₃) δ (ppm): 8.55 (d, 2H, J = 4.0 Hz), 8.30 (s, 1H), 7.96 (d, 1H, J = 12.0 Hz), 7.91 (d, 1H, J = 8.0 Hz), 7.86 (d, 1H, J = 8.0 Hz), 7.66–7.74 (m, 4H), 7.58 (d, 1H, J = 8.0 Hz), 7.32–7.40 (m, 2H), 7.13–7.25 (m, 6H), 5.09 (d, 1H, J = 4.0 Hz), 5.00 (d, 1H, J = 8.0 Hz), 4.44 (d, 1H, J = 8.0 Hz), 4.34 (d, 1H, J = 8.0 Hz) 3.99–4.08 (m, 6H), 3.15 (s, 3H),2.80 (s, 3H). ¹³C NMR (100 MHz, CDCl₃), δ (ppm): 53.7, 55.9, 55.6, 60.4, 94.9, 99.0, 116.5, 120.8, 122.0, 122.8, 124.1, 124.9, 125.3, 125.6, 125.7, 125.8, 126.6, 127.9, 129.1, 129.6, 129.8, 130.9, 132.4, 133.1, 134.0, 136.5, 149.0, 152.7, 152.9, 159.8.

R3 . To a CH₂Cl₂ : C₂H₅OH = 1 : 1 (v/v, 20 mL) of R1 (0.2 g, 0.25 mmol) was added con. HCl (5 mL). The mixture was refluxed for 4 h, after removal of the solvent, the resulting residue was dissolved in water and the aqueous solution was treated with saturated aqueous Na₂CO₃. The mixture was extracted with CH₂Cl₂ (3 × 30 mL), and the combined organic layer was washed with brine, and then dried over Na₂SO₄. After removal of the Na₂SO₄ by filtration, evaporation of the solvent gave the crude product, and then crude product was purified by alumina column chromatography by using CH₂Cl₂ as the eluent to give a white solid in 92% yield. HRMS (m/z): calcd for C₄₆H₄₁N₆O₂, [M + H⁺], 709.3291, found 709.3293. ¹H NMR (400 Hz, CDCl₃) δ (ppm): 10.79 (s, 2H, J = 4.0 Hz), 8.41 (d, 2H, J = 4.0 Hz), 7.81 (d, 2H, J = 8.0 Hz), 7.77 (s, 2H), 7.36–7.44 (m, 8H), 7.24 (t, 1H, J = 4.0 Hz), 7.23 (d, 1H, J = 4.0 Hz), 7.17 (s, 1H), 7.14 (s, 1H), 7.00–7.07 (m, 6H), 4.19 (d, 2H, J = 12.0 Hz), 4.06 (d, 2H, J = 12.0 Hz), 3.92–4.00 (m, 8H). ¹³C NMR (100 MHz, CDCl₃), δ (ppm): 58.1, 59.2, 117.4, 122.1, 122.8, 123.7, 125.1, 127.6, 128.2, 129.3, 134.3, 136.8, 148.8, 153.3, 158.1.

Conclusions

In conclusion, a series of in situ generated Zn²⁺ complexes of BINOL–DPA sensors R1–Zn(ii), R2–Zn(ii), R3–Zn(ii) have been presented in this paper. The fluorescence properties of these ensembles were investigated, only the R1–Zn(ii) ensemble showed high sensitivity and selectivity for PPi with a detection limit of 95 nM, and it can successfully be applied in the imaging of PPi in living cells. Moreover, the R1–Zn(ii) ensemble could also realize the real-time detection of PPi by the naked eye through precipitate experiments.

Acknowledgements

This work was financially supported by the National Program on Key Basic Research Project of China (973 Program, 2012CB720603 and 2013CB328900), the National Science Foundation of China (no. 21232005, 21321061, J1310008 and J1103315), and the Specialized Research Fund for the Doctoral Program of Higher Education in China (20120181130006).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: NMR and HRMS spectras. See DOI: 10.1039/c4an01615g

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