The dicyclen–TPE zinc complex as a novel fluorescent ensemble for nanomolar pyrophosphate sensing in 100% aqueous solution

Abstract

A dicyclen–TPE Zn(II) complex was developed as a fluorescent ensemble for pyrophosphate (PPi) in water. The complex was characterized by X-ray single crystal diffraction, which could selectively respond to PPi over other phosphates with a large enhancement of fluorescence intensity, and the detection limit was determined to be 22.8 nM.

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Pyrophosphate (PPi) plays important roles in a wide range of chemical and biological processes of human life. Therefore, the development of fluorescence probes for PPi is very essential and necessary.¹ In the past few decades, a number of zinc-complex based fluorescent chemosensors have been developed.² Several classic groups such as bis(2-pyridylmethyl) amine (DPA),³ terpyridine (tpy),⁴ and amide⁵ have been reported. Nevertheless, these probes are faced with some drawbacks: (a) low sensitivity (usually in the μM range); (b) lack of selectivity (the interference of certain nucleoside polyphosphates);⁶ and (c) poor water-solubility. To the best of our knowledge, there is only one example reported that showed a lower detection limit towards PPi.⁷ Therefore, developing water-soluble probes toward PPi possessing high selectivity and sensitivity, especially those which could respond in the nM range, is increasingly important.

1,4,7,10-Tetraazacyclododecane (cyclen) is one kind of cyclic organic compound, which is a versatile metal chelator with excellent water-solubility,⁸ and its Zn(II) complexes have been developed for biological polyphosphate anion detection.⁹ However, their selectivity is not good and they could not discriminate PPi over other anions, especially ATP and ADP. It is possible to improve this situation by introducing a suitable fluorophore. Tetraphenyl-ethene (TPE), a kind of propeller-shaped fluorogen, could affect the fluorescence intensity according to the restriction of intramolecular rotation (RIR) mechanism.¹⁰ A number of TPE-based chemical sensors have been presented.¹¹ It is non-emissive when the probe dissolves in solution, because energy is dissipated by the intramolecular rotation, while through aggregation or somehow else, the intramolecular rotation could be restricted. According to this mechanism, Hong and coworkers reported a DPA–TPE based sensor for PPi with a detection limit of 0.9 μM.¹² Inspired by this work, we examined whether a good chemosensor for PPi could be achieved by introducing cyclen units. Herein, we present a dicyclen–TPE zinc complex (Scheme 1) and investigate its ability for biologically important phosphate anion detections in aqueous solution.

Scheme 1 The structures of dCT and dCT·Zn.

The target compound and the intermediates were successfully synthesized and characterized by NMR and ESI-MS. The conjugate (dCT) and its zinc complex (dCT·Zn) had good water solubility and suitable lipophilicity, which is in conformity with our previous design. The fluorescence response of dCT·Zn at different pH was first investigated (Fig. S1, ESI†). The results indicated that the fluorescence intensity of dCT·Zn was stable in the pH range of 2.0–13.0 and the coordination of PPi with the Zn²⁺ centre could result in a better enhancement of the probe fluorescence emission in the pH range of 6.0–9.0. Considering the potential biological applications of the probe, HEPES buffer (pH = 7.4) was used as the solvent for spectroscopic experiments.

Then, the fluorescence responses of dCT to a range of metal ions were observed. As shown in Fig. S2 (ESI†), except for Cu²⁺ that caused a little fluorescence quenching, all other ions including Zn²⁺ did not cause any obvious changes in the fluorescence spectrum, which indicated that the solubility did not change significantly after the metal ions coordinate with dCT. Excellent water solubility made the benzene ring of TPE rotate freely. However, upon addition of 2.0 equiv. of Zn²⁺ to dCT followed by 0.5 equiv. of PPi, a 15-fold enhancement of the fluorescence intensity was observed (Fig. 1). This might be because the water solubility of the probe decreased after it coordinated with PPi, which restricted the intramolecular rotation and changed the intensity of fluorescence emission. In order to prove that it is the Zn²⁺ centre coordinating with PPi that leads to the enhancement, we added 0.5 equiv. of PPi to dCT and found no obvious fluorescence change. While adding another 2.0 equiv. of Zn²⁺ to the mixture above, a great change of the fluorescence spectrum came out again, indicating that the Zn(II)-cyclen complex could coordinate with PPi efficiently. Meanwhile, the responding abilities of dCT·Zn toward other biologically important phosphate anions and common anions were also investigated (Fig. 2). The results indicated that dCT·Zn showed good selectivity towards PPi over other anions, although ATP could result in a slight fluorescence enhancement.

Fig. 1 Fluorescence titration of dCT·Zn with various anions (10 μM) in HEPES (pH = 7.4, 10 mM).

Fig. 2 Fluorescence titration of dCT·Zn (10 μM) with PPi (0–10 eq.) in HEPES (pH = 7.4, 10 mM). The inset shows the fluorescence changes at 468 nm as a function of the amount of PPi.

Due to the nucleoside existence, ATP could not form the same structure as PPi with dCT·Zn. Thus, the aggregation degree of dCT-Zn-ATP was lower than that of dCT-Zn-PPi, which leads to the weaker fluorescence emission.

A Job's plot of dCT·Zn was used to prove the 2 : 1 stoichiometry with PPi (Fig. S3, ESI†). The binding constant was calculated as K_dCT·Zn = 170.94 M⁻² (Fig. S4, ESI†). The quantum yield of dCT·Zn increased from 0.426% to 1.757% (fluorescence of the sulphate solution as the reference, Qr = 0.560), and the detection limit of dCT·Zn was 22.8 nM. To inspect the excellent detection limit, titration of dCT·Zn with trace quantities of PPi (100 nM) was performed (Fig. S5, ESI†). A good linear relationship was achieved, which evidenced that dCT·Zn could detect PPi in the nM range. Furthermore, a kinetic study indicated that the speed of dCT·Zn interacting with PPi was fast and the enhancement of the fluorescence intensity could be stable for a long time (Fig. S6, ESI†).

The interference experiment was conducted to determine the fluorescence emission of dCT·Zn (10 μM) containing 0.5 equivalent of P₂O₇⁴⁻, H₂PO₄⁻, HPO₄²⁻, PO₄³⁻, Cl⁻, Br⁻, F⁻, I⁻, NO₃⁻, Ci³⁻, AcO⁻, N₃⁻, CN⁻, SO₄²⁻, SO₃²⁻, S²⁻, HCO₃⁻, CO₃²⁻, ATP, ADP, AMP, Glu, Asp, oxalic acid and malonic acid (Fig. 3). It was clearly seen that dCT·Zn was highly sensitive to PPi. The emission intensity of the dCT·Zn ensemble increased upon addition of PPi; only ATP could cause a slight disturbance to a certain extent. We could also directly see a fluorescence enhancement under UV lamp illumination (Fig. S7, ESI†). Moreover, the addition of 0.5 equivalent of inorganic anions and other phosphate anions would not interfere with PPi binding to dCT·Zn (Fig. 3).

Fig. 3 Fluorescence responses of dCT·Zn (10 μM) in HEPES buffer (10 mM, pH = 7.4) to various anions (5 μM) together with PPi (5 μM). Green bars represent the selectivity of the dCT·Zn complex upon addition of different anions. Red bars represent the competitive selectivity of the receptor dCT·Zn complex towards PPi in the presence of other anions. 1, PPi; 2, ATP; 3, ADP; 4, AMP; 5, PO₄³⁻; 6, HPO₄²⁻; 7, H₂PO₄⁻; 8, Asp; 9, Glu; 10, malonic acid; 11, oxalic acid; 12, HCO₃⁻; 13, SO₄²⁻; 14, Ci³⁻; 15, Br⁻; 16, N₃⁻; 17, F⁻; 18, AcO⁻; 19, I⁻; 20, Cl⁻; 21, SCN⁻; 22, NO₃⁻; 23, S²⁻; 24, SO₃²⁻; 25, CO₃²⁻.

To further understand the fluorescence sensing mechanism of dCT·Zn with PPi, the absorption spectrum of the receptor was explored. The addition of Zn²⁺ ions to the solution of dCT did not change the ultraviolet absorption (Fig. S8, ESI†). With further addition of PPi, there were still no obvious changes in the spectrum (Fig. S9, ESI†), which confirmed that the RIR mechanism would not cause significant absorption changes.

³¹P NMR spectroscopy was also undertaken to explain the detailed mechanism (Fig. 4). There was only one signal of ³¹P, suggesting that the two P atoms in PPi were equal. When PPi bound with dCT·Zn, obvious upfield shifts were observed for the ³¹P signal. The presence of a double ³¹P signal demonstrated that the two P atoms in the metal bound PPi were not magnetically equivalent, which meant the PPi bound to the binuclear zinc complex but not equally. ¹H NMR titration experiments were carried out (Fig. S10, ESI†). Upon the addition of Zn²⁺ to the solution of dCT, the protons of methylene and ethylene groups in cyclen displayed large downfield shifts. With further addition of PPi to the dCT·Zn complex, no significant change was found. This indicated that the conformation of dCT was transformed greatly when dCT coordinated with Zn²⁺, but almost no further change followed PPi addition.

Fig. 4 Partial ³¹P NMR (D₂O) spectra for PPi in the absence and presence of 2.0 equiv. of dCT·Zn.

Luckily, we achieved the single crystal of dCT·Zn, and investigated it using X-ray crystallographic analysis. As shown in Fig. 5, the structure of dCT·Zn did not show complete symmetry as two Zn²⁺ were towards the same side. This special structure of dCT·Zn provided the possibility of a 2 : 1 binding mode with PPi where four cyclen-Zn(II) centers towards the same side are non-covalently binding with one PPi. The details of the data collection and the structure refinement are given in the ESI (Table S1†).

Fig. 5 X-ray single crystal diffraction of the dCT·Zn complex.¹³

To further understand the coordination properties of dCT·Zn towards PPi, DFT calculations with the B3LYP exchange functional were performed using the Gaussian 03 package. Geometries were optimized and energies were estimated at the B3LYP/6-31G(d) & LANL2DZ level. In the optimized structure of dCT·Zn·PPi (Fig. 6), two molecules of dCT·Zn coordinated with one PPi. The guest molecule is wrapped by four zinc centers, which leads to greater distortion in the geometry. These four zincs are all five-coordinated, containing four N atoms from cyclen and one O⁻ atom from PPi. It should be noted that there are three hydrogen bonds between the PPi and dCT·Zn. The N–H in the cyclen unit serves as a hydrogen donor and the uncomplexed oxygens (O=P) as a hydrogen receptor.

Fig. 6 Optimized geometries of dCT·Zn with PPi calculated at the B2LYP/6-31G(d) level of theory. The dotted line represents the hydrogen bond. Bond lengths (Å): Zn1–O1 = 1.998, Zn2–O2 = 1.987, Zn3–O4 = 1.964, Zn4–O5 = 1.976, O3–H1 = 1.834, O3–H2 = 1.947, O6–H3 = 1.914.

The formation of hydrogen bonds could provide extra stability that is favourable for the coordination. As labelled in Fig. 6, O1 could form two hydrogen bonds, while O2 only forms a single hydrogen bond, which results in the different signal of PPi in ³¹P NMR spectra. Meanwhile, because of the compactness of four cyclen-Zn(II) centers, ATP and other molecules could not be inserted into the cavity suitably. As all zincs in dCT·Zn toward the internal cavity, the water solubility of the whole molecule becomes much worse than before; this restricts TPE's intramolecular rotation and causes a large fluorescence intensity enhancement.

Conclusions

In summary, we have designed a simple but efficient dicyclen–TPE based fluorescent sensor, which was characterized by X-ray single crystal diffraction. dCT·Zn showed high selectivity and sensitivity towards PPi in 100% aqueous medium. The results demonstrated that dCT·Zn coordinated with PPi in a 2 : 1 ratio rather than 1 : 1. Notably, it is one of the few probes with the detection limit in the nm range, and is the first example of a TPE based chemosensor with single crystal structure.

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: General experimental procedures and spectroscopic data (¹H-NMR, ¹³C-NMR and HRMS) for the corresponding products. CCDC 998114. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4qo00243a

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