2+ in Aqueous Solution"> A Simple Carbazole-based Schiff Base as Fluorescence "off-on" Probe for Highly Selective Recognition of Cu²⁺ in Aqueous Solution

Abstract

A carbazole-based Schiff base CB2 was synthesized and applied as a highly selective and sensitive fluorescent probe for $Cu^{2+}$ in $H_2O$-DMSO (8/2, v/v, pH = 7.4) solution. CB2 exhibits an excellent selectivity to $Cu^{2+}$ over other examined metal ions with a prominent fluorescence "turn-on" at 475 nm. CB2 and $Cu^{2+}$ forms a 1:2 binding ratio complex with detection limit of $9.5{\mu}M$. In addition, the $Cu^{2+}$ recognition process is hardly interfered by other examined metal ions.

Introduction

Selective detection of Cu2+ has received considerable attention because it is not only an environmental pollutant at high concentrations1 but also an essential trace element for many biological processes and systems.2 Cu2+ is the third most abundant metal after iron and zinc in human body,3 it played pivotal biological roles as cofactor of many proteins, or as catalyst in oxido-reduction reactions.4 However, excess Cu2+ in human body has been reported to cause serious disease such as prion disease,5 several neurodegenerative diseases including Menkes and Wilson diseases,6 Alzheimer’s and Parkinson’s diseases.7 In our daily lives, Cu2+ is generated and accumulated in the environment and food chain with the development of various industries such as electroplating, wood, painting and paper industries. The limit of copper in drinking water is 1.3 ppm (~20 μM) set by US Environmental Protection Agency (EPA). Therefore, the development of fluorescence emission “off-on” probes for Cu2+ sensing in aqueous media with high selectivity and sensitivity is still imperative.

To date, many excellent works associated with colorimetric or fluorescent probes for Cu2+ sensing have been documented.8 However, most of them displayed fluorescence “on-off” response to Cu2+ owing to the paramagnetic nature of Cu2+. Recently, a number of fluorescence “off-on” Cu2+ probes based on fluorophores such as rhodamine,9 coumarin,10 and 1,8-naphthalimide11 have been reported. It is noteworthy that effective fluorescent probes derived from carbazole fluorophore are still rare.

Carbazole is a conjugated unit with interesting optical and electronic properties. A number of carbazole derivatives have been synthesized and employed as fluorescent probes for the recognition of Cd2+,12 Hg2+,13 and Pb2+14 ions. Very recently, we have reported a carbazole-based fluorescent probe CB1 for Cu2+ sensing in CH3CN-H2O (9:1, v/v) solution.15 However, the Cu2+ recognition by CB1 suffers from the influence of Co2+. Inspired by the previous work, in this work, we designed and synthesized a new carbazole-based fluorescent probe CB2, which contains two picolinohydrazide imines as Cu2+ chelators. Probe CB2 exhibits fluorescence “off-on” response to Cu2+ in H2O-DMSO (8:2, v/v, pH = 7.4) solution with high selectivity and sensitivity.

Scheme 1.Synthesis of probe CB2 and the structures of CB1 and CB3.

 

Expermental

Reagents and Instruments. Unless otherwise stated, solvents and reagents were analytical grade and used without further purification. Doubly distilled water was used for spectral detection. 9-Ethyl-9H-carbazole-3,6-dicarboxaldehyde (1)16 was prepared by the literature method. 1H NMR and 13C NMR spectra were recorded on Agilent 400-MR spectrometer, chemical shifts (δ) were expressed in ppm and coupling constants (J) in Hertz. High-resolution mass spectroscopy (HRMS) was measured on a Bruker micrOTOF-Q mass spectrometer (Bruker Daltonik, Bremen, Germany). Low-resolution mass spectroscopy (LRMS) was measured on an Agilent 1100 series LC/MSD mass spectrometer. Fluorescence measurements were performed on a Sanco 970-CRT spectrofluorometer (Shanghai, China). The pH values were measured with a Model PHS-25B meter (Shanghai Dapu instruments Co., Ltd., China)

The salts used in stock solutions of metal ions are Ni(NO3)2·6H2O, Hg(NO3)2, Ba(NO3)2, Mg(NO3)2·6H2O, AgNO3, FeSO4·7H2O, KNO3, NaCl, CaCl2·6H2O, Al(NO3)3·9H2O, Mn(NO3)2, Pb(NO3)2, Sr(NO3)2, Cu(NO3)2·3H2O, Co(NO3)2 ·6H2O, Zn(NO3)2·6H2O, Cd(NO3)2, CrCl3·6H2O, Fe(NO3)3 ·9H2O, respectively.

Synthetic Procedure.

Synthesis of CB2: A mixture of compound 1 (251 mg, 1 mmol) and picolinohydrazide (2, 240 mg, 2.5 mmol) in 50 mL ethanol was stirred and heated at reflux for 4 h. After cooling to room temperature, the precipitates formed were collected and purified by silica gel column chromatography to give CB2 as yellow solids. Yield: 75％. mp ＞ 250 ℃. 1H NMR (400 MHz, DMSO-d6) δ12.11 (s, 2H), 8.82 (s, 2H), 8.74 (d, J = 4.4 Hz, 2H), 8.55 (s, 2H), 8.16 (d, J = 7.6 Hz, 2H), 8.07 (t, J = 6.8 Hz, 2H), 7.98 (d, J = 8.0 Hz, 2H), 7.76 (d, J = 8.8 Hz, 2H), 7.68 (dd, J1 = 6.8 Hz, J2 = 4.8 Hz, 2H), 4.52 (dd, J = 7.2 Hz, 2H), 1.38 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ160.63, 150.59, 150.21, 148.97, 141.55, 138.45, 129.80, 127.33, 126.44, 125.17, 123.05, 122.73, 121.48, 110.56, 37.89, 14.32. HRMS (ESI+), calcd for C28H24N7O2 [M+H]+ 490.1991, found 490.1981.

Synthesis of CB3: Control compound CB3 was prepared following the similar procedures as depicted for CB2 with the exception of benzoylhydrazine was used. Yield: 80％. mp 159-160 ℃. 1H NMR (400 MHz, DMSO-d6) δ 11.90 (s, 2H), 8.62 (s, 2H), 8.53 (s, 2H), 7.94 (m, 6H), 7.70 (d, J = 8.8 Hz, 2H), 7.54 (m, 6H), 4.46 (dd, 7.2 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.48, 149.39, 141.55, 134.12, 132.06, 128.89, 128.14, 126.51, 125.20, 122.84, 121.34, 110.54, 14.33. LRMS (ESI+), calcd for C28H24N7O2 [M+H]+ 488, found 488.

General Spectroscopic Methods. Probe CB2 was dissolved in H2O-DMSO (8/2, v/v, pH = 7.4) to afford the test solution (10 μM). Titration experiments were carried out in 10-mm quartz curvettes at 25 ℃. Stock solutions of metal ions (as chloride or nitrate salts, 10 mM) were added to the host solution and used for the titration experiment. The excitation wavelength is 313 nm.

 

Results and Discussion

Probe CB2 was prepared by the condensation of 1 and 2 in absolute ethanol and was structurally characterized by 1H NMR, 13C NMR and HRMS spectroscopy. Then, the metal ion selectivity of CB2 was investigated in H2O-DMSO (8:2, v/v, pH = 7.4) solution (Fig. 1). Free CB2 solution (10 μM) exhibited a very weak fluorescence emission at 475 nm, this may attributed to the C=N isomerization in the excited state. On addition of 2.0 equiv. of Cu2+, a remarkable fluorescence enhancement at 475 nm was observed. This fluorescence enhancement can be mainly attributed to the inhibition of C=N isomerization after binding with Cu2+. The chelation of CB2 with Cu2+ also can induce increasing in rigidity of the receptor and leads to chelation-enhanced fluorescence (CHEF) of CB2. Whereas, addition of other transition and heavy metal ions including Ag+, Ba2+, Ca2+, Co2+, Cd2+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Sr2+, Zn2+, Hg2+, Fe3+, Cr3+, Al3+, and Fe2+ promoted no significant fluorescence enhancement, which led to greatly improved selectivity over the previous probe CB1. Additionally, the fluorescence color changes of CB2 before and after addition of Cu2+ was naked eye detectable (Fig. 1, inset). These results demonstrate that CB2 has an excellent selectivity toward Cu2+ over other interested metal ions.

Figure 1.Fluorescence spectra of CB2 (10 μM) in H2O-DMSO (8/ 2, v/v, pH = 7.4) on addition of various metal ions. Inset: Fluorescence color changes of CB2 before and after addition of Cu2+.

Besides the high selectivity of probe to the target metal ion, its anti-interference ability to other potential competitive metal ions is also important. Thus, the competition experiments were subsequently carried out. As shown in Figure 2, except Cu2+, other tested metal ions (each cation was used as 2.0 equiv. to CB2) did not induce significant fluorescence changes. Nevertheless, a dramatic fluorescence emission enhancement was observed on further addition 2.0 equiv. of Cu2+ to the above competitive metal ion containing solutions. These results show that the Cu2+ recognition event by CB2 has a good anti-influence ability to other potential competitive metal ions.

Figure 2.Fluorescence intensity of CB2 (10 μM) in H2O-DMSO (8/2, v/v, pH = 7.4) at 475 nm. The black bars represent the emission intensity of CB2 in the presence of 2.0 equiv. of competing metal ion; the red bars represent the emission intensity of the above solution upon addition of 2.0 equiv. of Cu2+.

To further investigate the interaction of probe CB2 and Cu2+, fluorescence titration experiment was conducted (Fig. 3). Upon addition of increasing amounts of Cu2+ ions, the fluorescence emission band centered at 475 nm gradually increased. The maximum emission intensity was obtained when 2.0 equiv. of Cu2+ ions was employed. Based on the titration data, the detection limit of CB2 to Cu2+ was estimated to be 9.5 μM (Fig. 4),17 indicating that CB2 is sufficiently sensitive to detect Cu2+ concentration in some aqueous system.

Figure 3.Fluorescence spectra changes of CB2 (10 μM) in H2O-DMSO (8/2, v/v, pH = 7.4) upon addition of Cu2+ (0 to 2.0 equiv.).

Figure 4.Normalized fluorescence intensity of CB2 (10 μM) as a function of Log[Cu2+]. λem = 475 nm.

To get insight into the binding ratio of probe CB2 with Cu2+, the Job’s plot was examined with a total concentration of CB2 and Cu2+ as 20 μM. An emission turning point was observed when the molar fraction of Cu2+ is about 0.67, suggesting the formation of a 2:1 binding stoichiometry between Cu2+ and probe CB2 (Fig. 5). Linear fitting of the titration profiles using Benesi-Hildebrand plot based on a 2:1 binding mode results in a good linearity (correlation coefficient is over 0.99) (Fig. 6), which also strongly support the 2:1 binding stoicheiometry of Cu2+ and CB2, and the apparent association constant (Ka) is evaluated to be 3.7 × 109 M−2.

Figure 5.Job’s plot showing the 2:1 binding of Cu2+ to CB2.

Figure 6.Benesi-Hildebrand plot CB2 assuming 1:2 stoicheiometry with Cu2+. λem = 475 nm.

For practical application, the fluorescence intensity (at 475nm) of free probe CB2 at different pH values were explored (Fig. 7). The results showed that CB2 displayed weak fluorescence emission from pH 5.0 to 11.0. Under strong acidic (pH < 5.0) and strong basic (pH > 11) conditions, the solutions displayed greatly enhanced fluorescence emissions. These results demonstrate that CB2 can be used for Cu2+ recognition in a wide pH range.

Figure 7.Influences of pH on the fluorescence intensity (at 475 nm) of CB2 (10 μM) in H2O-DMSO (8/2, v/v) solution.

To validate the binding function of pyridine nitrogen atom in CB2, a carbazole based Schiff-base CB3 was prepared as a control compound, and its fluorescence responses to Cu2+ was also examined. Compound CB3 (10 μM) in H2O-DMSO (8/2, v/v, pH = 7.4) solution emitted a relative weak fluorescence. In the presence of Cu2+, the fluorescence emission was greatly quenched rather than increased (Fig. 8). This response of CB3 to Cu2+ is quite different from that of CB2, suggesting the pyridine moieties in CB2 are requisite for its fluorescence “off-on” Cu2+ selectivity. The proposed binding mode of CB2 and Cu2+ is illustrated in Scheme 2.

Figure 8.Different fluorescence responses of probe CB2 and control compound CB3 to Cu2+.

Scheme 2.roposed bind mode of CB2 with Cu2+.

 

Conclusions

In summary, we have developed a simple carbazole-based Schiff base CB2 as a fluorescence “turn-on” probe for Cu2+ recognition in aqueous solution. Probe CB2 displayed an extremely high selectivity toward Cu2+ over other interested metal ions with a 1:2 binding ratio. The low detection limit of CB2 to Cu2+ makes it have a potential applicability in real water sample Cu2+ detection.