A Highly Selective and Sensitive Turn-On Fluorescent Chemosensor Based on Rhodamine 6G for Iron(III).

Recently, more and more rhodamine derivatives have been used as fluorophores to construct sensors due to their excellent spectroscopic properties. A rhodamine-based fluorescent and colorimetric Fe(3+) chemosensor 3',6'-bis(ethylamino)-2-acetoxyl-2',7'-dimethyl-spiro[1H-isoindole-1,9'-[9H]xanthen]-3(2H)-one (RAE) was designed and synthesized. Upon the addition of Fe(3+), the dramatic enhancement of both fluorescence and absorbance intensity, as well as the color change of the solution, could be observed. The detection limit of RAE for Fe(3+) was around 7.98 ppb. Common coexistent metal ions showed little or no interference in the detection of Fe(3+). Moreover, the addition of CN(-) could quench the fluorescence of the acetonitrile solution of RAE and Fe(3+), indicating the regeneration of the chemosensor RAE. The robust nature of the sensor was shown by the detection of Fe(3+) even after repeated rounds of quenching. As iron is a ubiquitous metal in cells and plays vital roles in many biological processes, this chemosensor could be developed to have applications in biological studies.

Introduction

Fluorescent sensors for detection of transition metal ions, such as Cu2+, Hg2+, and so on, have attracted a great deal of attention in the last decades.[1, 2] Some of the more important among them are selective and sensitive fluorescent sensors for Fe3+.[3, 4] Iron, which is a ubiquitous metal in cells, plays vital roles in many biological processes.[5] However, deficiencies or excesses in iron are toxic or can lead to disturbances in glucose levels and lipid metabolism.[6] Though the human body can regulate iron to some extent, detection and analysis of bioactive iron remains an important healthcare challenge for chemists. Most literature reports use fluorescence quenching as the readout mechanism for the sensor response,[3] but very few involve a fluorescence “turn-on” response.[4] Moreover, most turn-on fluorescence sensors for Fe3+ are not selective over Cr3+ and Cu2+.[4a–c] Therefore, new chemosensors that show high selectivity for iron and involve a fluorescence turn-on response appear to be particularly attractive because of the simplicity, high sensitivity, and low detection limit of the fluorescence.

Recently, more and more rhodamine derivatives have been successfully utilized as fluorophores to construct sensors due to their excellent spectroscopic properties: namely, a large molar extinction coefficient, high fluorescence quantum yield, visible light excitation, and long wavelength emission.[2, 7] As a result, fluorescent chemosensors for Pb2+,[8] Hg2+,[9] Cr3+,[4a, 10] Ag+,[11] Cu2+[12], and so on, have been developed. Rhodamine derivatives are nonfluorescent and colorless, whereas addition of targeted metal ions leads to ring opening of the corresponding spirolactam, giving rise to a strong fluorescence emission and a color change from colorless to pink. Based on the understanding of the sensing mechanism of rhodamine-based molecular sensors, herein, we report the design and synthesis of a new rhodamine-based chemosensor RAE (Scheme 1), which shows a highly selective and sensitive fluorescence enhancement in response to Fe3+ in acetonitrile solution. Moreover, the addition of CN− could quench the fluorescence of the RAE–Fe3+ complex, indicating the regeneration of the chemosensor RAE. The color response allows the rapid and accurate recognition of Fe3+ with the naked eye, making this new chemosensor a very promising alternative for the detection of Fe3+.

Scheme 1

Synthetic route to RAE. Reagents and conditions: a) EtOH, NaOH, H2O, reflux, 2 h, 85 %; b) CH2Cl2, POCl3, reflux, 3 h, 91 %; c) CH2Cl2, NH2OH⋅HCl, Et3N, rt, 6 h, 31 %; d) 1. CH3CN, NaH, CH3COCl, 0–5 °C, 30 min, 41 %.

Results and Discussion

The chemosensor RAE was synthesized as shown in Scheme 1. Compounds 2[13] and 3[14] were synthesized according to literature methods. The reaction of acetyl chloride 3 and hydroxylamine hydrochloride afforded rhodamine derivative 4 with triethylamine as the base. Subsequently, the chemosensor RAE was obtained through the condensation of 4 and acetyl chloride with a yield of 41 %. The structure of RAE was characterized by 1H NMR and 13C NMR spectroscopy. With RAE in hand, we investigated its fluorescence properties by fluorescence measurements. After conducting a preliminary survey with various solvent systems, we chose acetonitrile for possible application of the system in metal ion analysis.

Upon the addition of different amounts of Fe3+, the absorbance intensity of RAE in acetonitrile became enhanced, and a new absorbance peak at ∼520 nm was observed (Figure S2 in the Supporting Information). Therefore, we chose 520 nm as the excitation wavelength in the fluorescence experiments. When the colorless solution containing RAE was subjected to fluorescence measurement, the solution (1.0×10−6 m) exhibited a very weak emission as shown in Figure 1. At the same time, the pink solution consisting of RAE (1.0×10−6 m) and Fe3+(10 equiv) (Figure S3 in the Supporting Information) showed relatively strong fluorescence intensity. Similar to some reported rhodamine-based fluorescent sensors for Fe3+,[4f, g] the fluorescence enhancement of RAE solution in the presence of Fe3+ is also attributed to the formation of the spirolactam-ring-opened form of rhodamine induced by Fe3+. Other tested metal ions did not induce any distinct fluorescence enhancement (Figure 1). Thus, sensor RAE is capable of fluorescence recognition of Fe3+ in acetonitrile.

Figure 1

Fluorescence spectra (λex=520 nm) of RAE (1 μm) in CH3CN with different metal ions (10 equiv) (Other ions=Cu2+, Ba2+,Ni3+, Mg2+, Na+, Ca2+, Pb2+, Ag+, Co3+, Zn2+, Hg2+, Cr3+, and Al3+).

Titration of RAE solution (1 μm) in acetonitrile by addition of 0–110 equiv Fe3+ was subsequently carried out. Upon incremental addition of Fe3+, the fluorescence intensity of RAE solution at 548 nm increases gradually and reaches the saturation point when 110 equiv of Fe3+ is added (Figure 2). The inset picture shows relative intensity (I/I0) versus the concentration of Fe3+ in the low concentration region up to 7.98 ppb.

Figure 2

Fluorescence titration of RAE (1 μm) in CH3CN with increasing Fe3+ concentration (λex=520 nm). Inset: the fluorescence of RAE (1 μm) (λem=548 nm) as a function of the Fe3+ concentration (0–8×10−9 m).

Achieving a highly selective response to the target analyte over a complex background of potentially competitive species is an important requirement for a chemosensor. Thus, the competition experiments in the presence of potentially competitive metal ions were conducted, and the results are shown in Figure 3. The results of the competitive-metal-ion binding studies clearly suggest a lack of interference by the other metal ions (100 equiv) on the selective detection of Fe3+ (10 equiv) by RAE.

Figure 3

Metal-ion selectivity of RAE in CH3CN. Dark bars represent the fluorescence intensity of a solution of RAE (1 μm) and 100 equiv of other metal ions (M=Na+, K+, Mg2+, Ce3+). Red bars show the fluorescence intensity after addition of Fe3+ (10 equiv) to the solution of RAE (1 μm) and different metal ions (100 equiv).

We also found that RAE could complex with Fe3+ in a 1:1 ratio, confirmed by the Job plot. A maximum emission intensity is seen when the molecular fraction of Fe3+ is ∼0.50, which indicates the formation of a 1:1 complex between RAE and Fe3+ with a total concentration of 10 μm (Figure 4). The binding ratio of RAE and Fe3+ was also confirmed by using the Benesi–Hidebrand method (Figure 5).

Figure 4

Job plot according to the method of continuous variations, indicating the 1:1 stoichiometry for RAE–Fe3+ (the total concentration of RAE and Fe3+ is 10.0 μm).

Figure 5

Benesi–Hildebrand plot of RAE (1 μm in CH3CN, 548 nm) assuming 1:1 stoichiometry between RAE and Fe3+.

Regeneration of the probe is a prerequisite in developing novel chemosensors for practical applications. The regeneration of the receptor RAE was performed by the addition of the Fe3+-binding agent CN−. As shown in Figure 6, addition of CN− to the solution of receptor RAE and Fe3+ results in diminution of the fluorescence intensity at 548 nm, which indicates the regeneration of the free receptor RAE. Furthermore, the fluorescence of the solution of RAE and Fe3+ can be recovered even after four cycles of Fe3+ addition followed by CN−-induced quenching (Figure 7). Such a regeneration process is important for the fabrication of Fe3+ sensors.

Figure 6

Fluorescence spectra of the solution of RAE (1 μm, CH3CN) and Fe3+ (10 equiv) with addition of different amount of CN− (increasing concentrations of CN− from 0 to 46 equiv) in CH3CN, λex=520 nm.

Figure 7

Regeneration of RAE (1 μm, CH3CN) upon repeated addition of Fe3+ (10 equiv) followed by CN− (25 equiv), λex=520 nm. Four cycles of Fe3+ and CN− addition are shown. The bars show the fluorescence intensity after addition of Fe3+(black) and after the addition of CN−(red).

Conclusions

A novel fluorescent sensor RAE was designed and synthesized. In acetonitrile, RAE exhibits highly selective and sensitive detection of Fe3+ over other metal ions with a fluorescence turn-on effect, and the detection limit is 7.98 ppb. Moreover, the addition of CN− could quench the fluorescence of the RAE–Fe3+ complex, indicating the regeneration of chemosensor RAE. Further efforts will be focused on the structure modification of the sensor so that it could also be operated in aqueous solution for possible biological applications.

Experimental Section

Instruments and materials: The fluorescence spectra were recorded on a Hitachi F-4500 spectrofluorometer. A 1.0 cm quartz cuvette with a volume of 3.0 mL was used for all spectra collection. Thin-layer chromatography (TLC) was performed on glass plates coated with SiO2 GF254. The plates were inspected by UV light or in I2 vapor. Column chromatography was performed on silica gel (200–300 mesh). 1H and 13C NMR spectra were recorded on a Bruker AV 500 NMR (500 MHz) using tetramethylsilane (TMS) as an internal standard. Matrix-assisted laser desorption/ionization mass spectrometry (MS-MALDI) was performed on a Bruker Daltonics Biflex III. Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. Solvents used were purified and dried by standard procedures prior to use. CH3CN in chromatographic grade was used throughout the experiments as solvent. For the CH3CN stock solutions of the various metal ions (0.01 mm), the perchlorate salts of Fe3+, Cu2+, Ba2+, Ni3+, Mg2+, Na+, and Ca2+, the nitrate salts of Pb2+ and Ag+, and the chloride salts of Co3+, Zn2+, and Hg2+ were used. The stock solution of compound RAE (0.01 m) was prepared by dissolving accurately weighed RAE in CH3CN.

General procedure: Typically, 3.0 mL of the solution of RAE (1 μm) was placed in a quartz cell (1.0 cm width), and the appropriate aliquot of Fe3+ solution was added. The resulting solution was stirred thoroughly and allowed to stand at rt for 2 min, and then the fluorescence spectrum was recorded. For fluorescence intensity measurements, the excitation and emission wavelengths were at 520 nm and 548 nm, respectively. The slit width was 5 nm/5 nm. The synthetic route is shown in Scheme 1.

2-[3’,6’-Bis(ethylamino)-2’,7’-dimethyl-9:[13] To a solution of rhodamine 6G 1 (540 mg, 1.13 mmol) in EtOH (14 mL) was added NaOH (135 mg, 3.39 mmol) in H2O (2 mL), and the reaction mixture was stirred for 2 h at reflux. After addition of distilled H2O (15 mL), the solution was cooled to rt. The resulting precipitate was isolated by filtration and dried at 70 °C for 30 min to give compound 2 (400 mg, 85 %). No further purification was conducted.

2-[3’,6’-Bis(ethylamino)-2’,7’-dimethyl-9H-xanthen-9-yl]benzoyl chloride (3):[14] To a solution of 2 (400 mg, 0.96 mmol) in CH2Cl2 (10 mL) was added POCl3 (0.26 mL, 2.88 mmol) dropwise over 2 min. The solution was heated at reflux for 3 h. The reaction mixture was cooled to rt and evaporated in vacuo to give compound 3 (380 mg, 91 %), which was used in the next step without purification.

3′,6′-Bis(ethylamino)-2-hydroxy-2′,7′-dimethyl-spiro{1: To the crude acid chloride 3 dissolved in CH2Cl2 (10 mL), Et3N (0.3 mL, 2.15 mmol) was added dropwise after addition of NH2OH⋅HCl (150 mg, 2.16 mmol). The reaction mixture was stirred for 6 h at rt, then extracted with CH2Cl2 (3×20 mL), and the combined organic layers were dried over anhydrous Na2SO4. The solution was filtered, concentrated in vacuo, and the crude product was purified by column chromatography (hexanes/EtOAc, 2:1→1:1) to give compound 4 as a pink solid (150 mg, 0.35 mmol, 36 % from rhodamine 6G). The pink colored product was recrystallized from CH2Cl2/hexanes (1:1, v/v) to give compound 4 as a white solid (117 mg, 31 %); 1H NMR (400 MHz, CDCl3): δ=7.87–7.84 (m, 1 H), 7.47–7.42 (m, 2 H), 7.07–7.04 (m, 2 H), 6.40 (s, 2 H), 6.36 (s, 2 H), 3.53 (br s, 2 H), 3.25–3.20 (m, 4 H), 2.13 (s, 6 H), 1.24 ppm (t, J=7.2 Hz, 6 H); 13C NMR (100 MHz, CDCl3): δ=163.6, 152.3, 150.9, 147.7, 132.8, 128.6, 128.3, 127.9, 123.8, 123.0, 117.9, 104.7, 97.1, 65.9, 38.5, 16.9, 14.9 ppm; IR (film):=3390, 2963, 2924, 1683, 1645, 1623, 1519, 1467, 1416, 1377, 1342, 1277, 1208, 1156, 1091, 1044, 1014 cm−1.

3′,6′-Bis(ethylamino)-2-acetoxyl-2′,7′-dimethyl-spiro[1: To the solution of 4 (1.5 g, 3.73 mmol) in anhydrous CH3CN (45 mL), NaH (0.11 g, 4.5 mmol) was added at 0–5 °C. The mixture was stirred in an ice bath for 30 min. CH3COCl (0.39 g, 4.11 mmol) dissolved in CH3CN (10 mL) was added at 0–5 °C dropwise over 20 min. The mixture was stirred in an ice bath for more than 30 min. Impurities were removed by filtration, and the filtrate was concentrated in vacuo. Purification by flash column chromatography (hexanes/EtOAc, 5:1) gave RAE as an ivory-white solid (0.62 g, 41 %); 1H NMR (500 MHz, CD3COCD3): δ=10.11 (s, 1 H), 7.87 (d, J=7.0 Hz, 1 H), 7.62–7.56 (m, 2 H), 7.05 (d, J=7.5 Hz, 1 H), 6.42 (s, 2 H), 6.32 (s, 2 H), 4.55 (s, 2 H), 3.25 (t, J=6.0 Hz, 4 H), 1.95 (s, 9 H), 1.29 ppm (q, J=5.5 Hz, 6 H); 13C NMR (125 MHz, CD3COCD3): δ=167.5, 163.4, 153.2, 152.3, 149.1, 134.4, 129.6, 129.0, 124.9, 123.7, 118.9, 105.2, 97.0, 66.6, 39.0, 18.0, 17.3, 14.9 ppm; MS-MALDI: m/z=472.1 [M+H]+.