A Simple Benzimidazole Based Fluorescent Sensor for Ratiometric Recognition of Zn²⁺ in Water

Abstract

A phenylbenzimidazole derivatized sensor (L) that behaves as a ratiometric fluorescent receptor for $Zn^{2+}$ in water has been described. In HEPES buffer at pH 7.4, sensor L displays a weak fluorescence emission band at 367 nm. Upon addition of $Zn^{2+}$, the emission intensity at 367 nm is decreased, concomitantly, a new emission band centered at 426 nm is developed, thus facilitates a ratiometric $Zn^{2+}$ sensing behavior. Sensor L binds $Zn^{2+}$ through a 1:1 binding stoichiometry with high selectivity over other metal cations. Sensor L displays a linear response to $Zn^{2+}$ concentration from 0 to $6.0{\times}10^{-5}M$, sensor L also exhibits high sensitivity to $Zn^{2+}$ with a detection limit of $3.31{\times}10^{-7}M$.

Introduction

The design and synthesis of metal ion selective fluorescent probes has been an important field in supramolecular chemistry and coordination chemistry in the past two decades.1 As the second most-abundant transition metal ion in human body, Zn2+ plays vital roles in physiological and pathological processes such as brain activity, neural signal transmitters or modulators, regulators of gene transcription and immune function.2 Both its deficiency and excess can cause some health problems, including acrodermatitis enteropathica, prostate cancer, diabetes, Alzheimer’s and Parkinson’s diseases.2b,3 Although some analytical techniques including UV-vis spectroscopy,4 flame atomic absorption spectrometry5 and potentiometry6 have been applied to Zn2+ detection in various samples, considerable attention has been paid to the development of fluorescent Zn2+ sensors owing to the simplicity, low cost and high sensitivity of fluorescence techniques.1c,7

To date, a large amount of Zn2+ selective fluorescent sensors have been reported. However, a majority of them could not perform in pure water. Thus, the design and synthesis of Zn2+ sensors that can be applied in absolute water solution is imperative. Ratiometric fluorescent sensors permit dual wavelength signals to detect target species by measuring the ratio of fluorescence intensities at two different wavelengths, which would increase the dynamic range of fluorescence measurements due to its built-in correction property.8 Consequently, it is of great significance to design ratiometric fluorescent sensors.9 To the best of our knowledge, only a few ratiometric fluorescent sensors that can detect Zn2+ in absolute water solution have been documented,10 however, these reported sensors requires intricate synthetic procedures, simple and water soluble fluorescent Zn2+ sensor based on benzimidazole fluorophore is still rare.

We herein report the synthesis and Zn2+ recognition property of a new 2-(2'-aminophenyl)benzimidazole derivatized fluorescent sensor L (Scheme 1). Sensor L exhibits ratiometric emission responses to Zn2+ in water solution (HEPES 10 mM, pH 7.4) with high selectivity and sensitivity.

 

Experimental

Instruments and Materials. Unless mentioned otherwise, all of the solvents and reagents used were purchased from commercial suppliers and used without further purification. Compound 1 was prepared according to the literature method.11 Nuclear magnetic resonance (NMR) spectra were recorded on Agilent 400-MR NMR spectrometer. Chemical shifts (δ) were expressed in parts per million (ppm) and coupling constants (J) in Hertz. High resolution mass spectroscopy (HRMS) was measured on an Agilent 1200 time-offlight mass spectrometer (Bruker, micrOTOF-Q). Fluorescence measurements were performed on a Sanco 970-CRT spectrofluorometer (Shanghai, China). The pH measurements were made with a Model PHS-25B meter (Shanghai, China).

General Methods. All titration experiments were carried out at (298.2 ± 0.1) K. Fluorescence spectra were measured with 10 μM solution of sensor L in buffered water solution (HEPES 10 mM, pH = 7.4) and the solutions of metal salts were prepared in distilled water. All of the metal salts were used in water-soluble sulfate, nitrate or chloride salts. These solutions were used for all spectroscopic studies after appropriate dilution. Double-distilled water was used throughout the experiments. Moreover, the fluorescence spectra were measured 3 minutes after metal ion addition.

Synthesis of Sensor L. At room temperature, diethanolamine (498 mg, 4.74 mmol) was added to a stirred solution of N-[2-(1H-benzoimidazol-2-yl) phenyl]-2-chloroacetamide (1) (450 mg, 1.58 mmol) in 10 mL of N,N-dimethylformamide (DMF). The reaction mixture was stirred overnight at room temperature, then poured into 300 mL of distilled water and led up to pH = 7 by diluted HCl solution. The mixture was extracted with CH2Cl2 (3 × 100 mL). The combined organic phase was dried over anhydrous MgSO4, followed by filtration and rotary evaporation. The residue was purified by a silica gel column chromatography with CH2Cl2/CH3OH (6:1, v/v) as eluent to give 442 mg of L as brown solid. Yield: 79%. mp 179.5-180 ℃. 1H NMR (400 MHz, DMSO-d6) δ 13.10 (s, 2H), 8.85 (d, J = 8.8 Hz, 1H), 8.05 (d, J = 7.2 Hz, 1H), 7.73 (s, 1H), 7.59 (s, 1H), 7.49 (t, J = 7.2 Hz, 1H), 7.29-7.25 (m, 3H), 4.58 (s, 2H), 3.57 (d, J = 4.4 Hz, 4H), 3.43 (s, 2H), 2.79 (t, J = 4.4 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ 172.08, 150.88, 138.18, 131.06, 128.35, 123.34, 120.24, 116.62, 60.95, 59.59, 58.27. HRMS(+). Calcd for C19H22N4O3Na [M+Na+]+: m/z 377.1590. Found: m/z 377.1582.

Scheme 1.Synthesis of sensor L.

 

Results and Discussion

Metal Ion Recognition in Water. Sensor L was facilely synthesized by the reaction of 1 with diethanolamine (Scheme 1) and was characterized by 1H NMR, 13C NMR and HRMS. The good water solubility of L makes it has a potential application for metal ion detection in water solution. Based on this, we select the HEPES buffered water solution at pH 7.4 (HEPES 10 mM) as the working moiety. The fluorescence spectra were obtained with the excitation at 310 nm. The fluorescence properties of sensor L (10 μM) were investigated with various metal ions. As shown in Figure 1, sensor L exhibits a very weak fluorescence at 367 nm, which is attributed to the photoinduced electron transfer (PET) from diethanolamine nitrogen atom to the photoexcited phenylbenzimidazole moiety.9f However, a significant fluorescence enhancement at 426 nm with a remarkable 59 nm red-shift was observed upon addition of 80 μM of Zn2+. A fluorescence quenching was detected on addition of Cu2+, Co2+, Ni2+ and Hg2+, implying that these metal ions could also bind with L but lead to fluorescence quenching due to their paramagnetic nature. The addition of Cd2+ elicits a slight fluorescence enhancement at 426 nm. By contrast, other metal ions such as Ag+, Pb2+, Sr2+, Ba2+, Fe2+, Mn2+, Fe3+, Al3+, Cr3+, Mg2+, K+, Ca2+ and Na+ do not cause any significant fluorescence spectrum changes. These results indicate that sensor L has a specific selectivity to Zn2+ through emission enhancement and red-shift. In addition, a time course study reveals that the Zn2+-induced fluorescence enhancement can complete within 1 minute (Figure 2), indicating the rapid response of sensor L to Zn2+.

Figure 1.Fluorescence spectra changes of L (10 μM) in H2O (HEPES 10 mM, pH = 7.4) in the presence of different metal ions (80 μM of each, λex = 310 nm).

Figure 2.Fluorescence intensity changes of L (10 μM) in H2O (HEPES 10 mM, pH = 7.4) in the presence of Zn2+ (80μM) against time. λex = 310 nm, λem = 426 nm.

Fluorescence Titration Studies. To further understand the sensing properties of L to Zn2+, the fluorescence titration experiments were performed. As shown in Figure 3, the emission intensity at 367 nm of L solution progressively decreased on incremental increasing the added Zn2+ concentration (0 to 80 μM), meanwhile, the emission intensity at 426 nm increased impressively. The observed two wavelength variations behave ratiometric feature with an isoemissive point at 376 nm. Under the present conditions, the fluorescence ratio at 426 nm and 367 nm (F426/F367) increased linearly against Zn2+ concentration (0 to 60 μM, R = 0.9977) (Figure 3, inset), which allowed the detection of Zn2+ by the ratiometric fluorescence method. The detection limit of sensor L to Zn2+ was determined to be 3.31 × 10−7 M.12

Figure 3.Fluorescence spectra of sensor L solution (10 μM) in water (HEPES 10 mM, pH = 7.4) on addition of different amounts of Zn2+ (0 to 8 equiv.). Inset: Fluorescence ratio (F426/F367) changes on addition of Zn2+ (0 to 6 equiv.) in water.

Sensing Mechanism. In order to explore the recognition mechanism of the sensor L, the 1H NMR spectra of L before and after addition of Zn2+ was compared (Figure 4). On addition of Zn2+, the intensity of NHs signal in free L at 13.10 ppm (Figure 4(a)) decreased to one NH signal intensity and became broadened (Figure 4(b)). Addition of Zn2+ led to down-field shift of tertiary N neighboring CH2 proton signals (Ha and Hb). However, the proton signals in CH2OH moiety (Hc and OH) did not show significant shifts, indicating the absence of interaction of OH with Zn2+. The signal at 8.85 ppm in free L could be assigned to Hd due to the possible hydrogen bonding between Hd and amide O atom, Zn2+ binding destroyed the hydrogen bonding and led to its up-field shift.13 These results indicate that sensor L may bind Zn2+ with an imidic acid form through tertiary N and imidic acid O atoms. This binding mode of sensor L with Zn2+ suppressed the PET process from diethanolamine nitrogen to phenylbenzimidazole moiety,14 which is responsible for the fluorescence enhancement. In addition, a chelation promoted amide NH deprotonation process (it was supported by HRMS analysis) exerted an internal charge transfer (ICT) from phenylbenzimidazole to Zn2+ ion (Figure 5), thus led to the observed emission red-shift.15

Binding Stoichiometry. Job’s plot analysis shows that the fluorescence intensity of the tested solutions reach maximum when the mole fraction of Zn2+ is 0.5 (Figure 6), which advocates the formation of 1:1 complex between L and Zn2+. The 1:1 interaction of L and Zn2+ was further supported by mass spectrometry analyses, in which the most prominent peak appeared at m/z 417.0896 is assignable to [L+Zn2+−H+]+ (calcd = 417.0894). Moreover, from the fluorescence titration profile, the association constant (Ka) of L and Zn2+ was calculated to be 1.4 × 105 M−1 by nonlinear least-squares fitting (R2 = 0.9988) based on the 1:1 binding equation (Figure 7).16

Figure 4.1H NMR spectrum (in DMSO-d6) of L before (a) and after (b) addition of Zn2+.

Figure 5.Proposed sensing mechanism of L to Zn2+.

Figure 6.Job’s plot for L-Zn2+ complex in HEPES-buffered H2O solution (pH = 7.4). The total concentration of Zn2+ and L was 20 μM.

Reversibility and pH Effect. As a sensor, the recognition process should be reversible. Upon addition 100 μM of ethylenediamine tetracetic acid disodium salts (EDTANa2) to L-Zn2+ solution, the fluorescence emission restored to the similar state of free L (Figure 8), indicative of the reversibility of Zn2+ recognition.

Figure 7.Nonlinear fitting of fluorescence intensity against Zn2+ concentration (λem= 426 nm).

Figure 8.Fluorescence changes of L solution by alternative addition of Zn2+ and EDTA (λex = 310 nm).

Figure 9.The fluorescence changes of L (λem = 367 nm) and L-Zn2+ (λem = 426 nm) solution at different pH values (λex = 310 nm).

Furthermore, the fluorescence changes of sensor L with and without Zn2+ at different pH values were also investigated (Figure 9). Acid-base titration results show that free sensor L displays weak emission at near neutral pH conditions, acidic (pH < 5) and basic (pH > 10) conditions can induce fluorescence enhancement. Therefore, sensor L can successfully detect Zn2+ in water in a wide pH range from 5 to 10. Moreover, the fluorescence intensity of L-Zn2+ complex is stable between pH 7 to 10. These results indicate that sensor L has a potential practical applicability for Zn2+ detection in physiological and environmental systems.

Competition Experiment Studies. The fluorescence changes of L solution to Zn2+ in the presence of potential competitive metal ions were investigated to evaluate the anti-interference of Zn2+ recognition (Figure 10). Significant fluorescence quenching of L-Zn2+ solution was observed in the presence of paramagnetic metal ions of Cu2+ and Hg2+, this phenomenon was usual in some previously reported metal ion sensors.17 However, other metal ions do not induce such dramatic fluorescence quenching effect. Thus, sensor L displays a good selectivity to Zn2+ over other metal ions except Cu2+ and Hg2+.

Figure 10.Fluorescence responses of L solution (10 μM) in the presence of 80 μM of miscellaneous metal ions (black bars) and upon further addition of 80 μM of Zn2+ (red bars) (λex = 310 nm).

 

Conclusion

In summary, we have developed a new ratiometric fluorescent sensor L for Zn2+ recognition in absolute water solution at pH 7.4. Free sensor L displays weak fluorescence emission, however, chelating with Zn2+ results in fluorescence enhancement with a remarkable emission red-shift, which leads to ratiometric output signal. Sensor L shows good selectivity and sensitivity for Zn2+ over other metal ions. Job’s plot and HRMS analyses indicate the formation of a 1:1 complex.