One Single Molecule as a Multifunctional Fluorescent Probe for Ratiometric Sensing of Fe³⁺, Cr³⁺ and Colorimetric Sensing of Cu²⁺

Abstract

The reagent , incorporating a rhodamine moiety and a coumarin backbone and prepared via click chemistry, was developed as the first single molecule for detecting Cu²⁺, Fe³⁺ and Cr³⁺. Its response to Cu²⁺ in different solutions is visible to the naked eye and it exhibits a ratiometric fluorescence response to Fe³⁺ in methanol and Cr³⁺ in acetonitrile.

1. Introduction

Metal ions play very important roles in living system and have significant impacts on human health. Cu²⁺, Fe³⁺ and Cr³⁺ are indispensable nutrients for life, and both their deficiency and excesses are connected to various disorders [1–3]. For instance, during the uptake, storage and trafficking of Cu²⁺, a deficiency is proposed to be associated with severe diseases such as Menkes syndrome, Wilson's disease and Alzheimer's disease [4–6]. Fe³⁺ is one of the most essential metals in oxygen transport and electron transport [2,7,8]. Fe³⁺deficiency in our bodies will lead to low oxygen delivery, which could result in low blood pressure, anemia and decreased immunity [9,10]. On the contrary, excess Fe³⁺ can result in the formation of reactive oxygen species (ROS), which have damaging effects on lipids, nucleic acids, and proteins [11,12]. A normal person needs to absorb 25–35 mg of Cr³⁺ per day to improve the metabolism of glucose and lipids [13], while excessive intake of Cr³⁺ has an adverse effect on cellular structure [14,15]. In consideration of the necessity and toxicity of Cu²⁺, Fe³⁺ and Cr³⁺, the design of new probes for exact and convenient detection of Cu²⁺, Fe³⁺ and Cr³⁺ in environmental and biological samples is of much interest.

In recent years, because of the inexpensiveness, high sensitivity and simplicity of fluorescence analysis [16], fluorescent indicators have attracted widespread attention, and a rapidly increasing number of metal-responsive fluorescent sensors have been studied [17–19]. Although chemists have developed a great deal of fluorescent probes for Cu²⁺ [20–23], Fe³⁺ [24] and Cr³⁺ [25–30], respectively, probes which can indicate Cu²⁺, Fe³⁺ and Cr³⁺ at the same time via a single molecular species have not been reported. Herein, we intend to develop a fluorescent probe that could distinguish Cu²⁺, Fe³⁺ and Cr³⁺ under different conditions with one single molecule.

According to previous studies of other researchers, rhodamine dyes are generally used as fluorescent probes because of their excellent photostability, photophysical properties and suitable water-solubility [31,32]. On the other hand, coumarin, as another well-known strongly fluorescent compound, is easy and convenient to synthesize in general [33]. To date, some FRET probes based on rhodamine and coumarin to achieve ratiometric fluorescent responses have been reported [34–36]. Inspired by these works, we sought to design a FRET ratiometric fluorescent probe Rh-C by connecting the rhodamine B and coumarin moieties with a 1,2,3-triazole linker (Scheme 1), in the hope that the introduction of the triazole system might provide an additional coordination site [37–39]. Rh-C and the intermediates were characterized by ¹H-NMR, ¹³C-NMR, MS (see Supporting Information).

2. Results and Discussion

Firstly, the fluorescence emission profiles of Rh-C in different solvents (DMSO, DMF, THF, EtOH, MeOH, MeCN, H₂O) were studied. As shown in Figure 1, the emission peak of Rh-C at ∼470 nm which represented the characteristic peak of coumarin was almost unchanged in different organic solvents. Meanwhile, no peak was found at ∼580 nm corresponding to the emission of rhodamine B, indicating that the spirolactam of rhodamine moiety remained closed [31,32]. Surprisingly, the fluorescence of Rh-C was fairly weak and the emission peak was obviously red-shifted in water, which might be due to the TICT property of the 7-diethylamino group of the coumarin moiety [40]. Thus, the properties of Rh-C were mainly studied in organic solutions.

Then, the cation selectivity of Rh-C in different solvents was investigated through UV-Vis absorption and fluorescence emission spectroscopy. As illustrated in Figure 2, the absorption peaks of Rh-C (5 μM) were located at around 415 nm in MeOH, MeCN and THF and the addition of 20 equiv of a range of physiologically and environmentally relevant metal ions (Li⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Ba²⁺, Zn²⁺, Hg²⁺, Pb²⁺, Mn²⁺, Ni²⁺, Co²⁺ and Ag⁺) did not cause any significant changes in the absorption spectra. However, in MeOH solution, the addition of 20 equiv of Cr³⁺, Fe³⁺ and Cu²⁺ led to the appearance of a typical peak at 550 nm related to the opening of the spiro ring of rhodamine B (for Cr³⁺ 11-fold, for Fe³⁺ 17-fold, and for Cu²⁺ 114-fold), accompanied by a color change from light yellow to pink. In MeCN solution of Rh-C, the addition of Cu²⁺ and Cr³⁺ could cause a slight enhancement of the absorption at 558 nm, but only Cu²⁺ could induce a color change from light yellow to pink. In THF solution of Rh-C, only the addition of Cu²⁺ could cause a remarkable increase of absorption at ∼550 nm, implying that Rh-C exhibited an excellent selectivity towards Cu²⁺ in THF.

To sum up, Rh-C exhibited selective colorimetric response towards Cu²⁺ in different organic solution, particularly in THF. The fluorescence responses of Rh-C (5 μM) towards various metal ions are shown in Figure 3.

As envisioned, in MeOH, MeCN and THF, there were no significant changes in the fluorescence spectra after the addition of 20 equiv of Li⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Ba²⁺, Zn²⁺, Hg²⁺, Pb²⁺, Mn²⁺, Ni²⁺ and Co²⁺, but surprisingly, the addition of Cu²⁺ did not result in the appearance of the typical emission peak of rhodamine B at ∼580 nm but rather led to more or less fluorescence quenching at ∼470 nm in all solvents, while the spiro ring opening of rhodamine B indeed occurred in the presence of Cu²⁺. We assumed that although the coordination between Cu²⁺ with Rh-C caused the spiro ring opening of rhodamine B, the fluorescence of the rhodamine B moiety as well as the coumarin was simultaneously quenched by Cu²⁺ due to the heavy metal ion effect [41]. In MeOH, upon addition of 20 eq. Cr³⁺ and Fe³⁺, an obvious fluorescence increment at 579 nm and a moderate fluorescence reduction at 470 nm was observed, indicating the generation of a FRET process between coumarin and rhodamine B. The emission intensity change ratios induced by Fe³⁺ and Cr³⁺ were 59-fold and 22-fold, respectively. In MeCN, only Cr³⁺ could induce the fluorescence increment of Rh-C at 581 nm, suggesting that Rh-C could selectively detect Cr³⁺ in MeCN via fluorimetry. In THF, the fluorescence of Rh-C showed almost no changes upon addition of the tested metal ions. In short, Rh-C exhibited a moderate selectivity toward Fe³⁺ in MeOH and an outstanding selectivity toward Cr³⁺ in MeCN through a FRET pathway.

Subsequently, the fluorescence titrations of Rh-C (5 μM) toward Fe³⁺ in MeOH and Cr³⁺ in MeCN were exploited, respectively. As depicted in Figure 4, with the addition of more of Fe³⁺ into the MeOH solution of Rh-C, the emission of Rh-C at 470 nm gradually decreased, and a new peak at 579 nm corresponding to the spiro ring opening of rhodamine B appeared (Figure 4A). A FRET process was speculated to emerge between the coumarin moiety and the rhodamine B fluorophore. The fluorescence intensity ratio (I₅₇₉/I₄₇₀) reached a plateau when 10 equiv Fe³⁺ was added, accompanied by a remarkable enhancement of the emission intensity ratio from 0.02 to 0.94. A similar phenomenon was observed upon addition of Cr³⁺ into the MeCN solution of Rh-C. The emission of Rh-C at 463 nm decreased gradually as the amount of Cr³⁺ in MeCN increased, accompanied with a fluorescence increase at 581 nm (Figure 4B). The fluorescence intensity ratio (I₅₈₁/I₄₆₃) became stable after 10 equiv of Cr³⁺ was added, with an enhancement of the emission intensity ratio from 0.02 to 0.68.

We believe that the different selectivity of Rh-C towards metals in different solvents (THF, MeOH, and MeCN) is closely related to the complexing abilities of the metals. All the ions (Cu²⁺, Fe³⁺, and Cr³⁺) complexed with Rh-C could induce ring-opening of the rhodamine moiety. In addition, the coumarin moiety, which shows an absorption band that peaks at∼415 nm, behaves as an energy donor for the FRET system, and the open form of the rhodamine moiety, which shows an absorption band that peaks at ∼550 nm, acts as an energy acceptor.

3. Experimental Section

3.1. General Information

¹H-NMR and ¹³C-NMR spectra were measured on a Bruker AM400 NMR spectrometer (Fällanden, Switzerland). Proton chemical shifts of NMR spectra are given in ppm relative to the internal reference TMS (1H, 0.00 ppm). ESI-MS and HRMS spectral data were recorded on a Thermo Finnigan LCQ^DECA (San Jose, CA, USA) and a BrukerDaltonics Bio TOF mass spectrometer (Billerica, MA, USA), respectively. Fluorescence emission spectra were obtained using FluoroMax-4 Spectrofluorophotometer (HORIBA JobinYvon, Paris, France) at 298 K. Unless otherwise noted, materials were obtained from commercial suppliers and were used without further purification. All of the solvents were either HPLC or spectroscopic grade in the optical spectroscopic studies and they were dried according to the standard methods prior to use.

3.2. Synthesis of Various Compounds

Rh-1, Rh-2, C-1 and C-2 were synthesized according to the literature [42–44].

Synthesis of 2-Azido-N-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)acetamide (Rh-3). Under nitrogen, Rh-2 (650 mg, 1.2 mmol) was dissolved in DMF (15 mL), then sodium azide (234 mg, 3.6 mmol) was added to the solution very carefully. The mixture was stirred at 50 °C for 12 h. After the reaction, the solution extracted with CH₂Cl₂ (3 × 20 mL) and the combined organic layer was washed with brine (3 × 20 mL). After the solution was dried over anhydrous sodium sulfate, the solvent was removed under the reduced pressure and the residue was further purified by column chromatography to afford Rh-3 (620 mg, 95.6%) as a white solid. ¹H-NMR (400 MHz, DMSO) δ 9.91 (s, 1H), 7.84 (d, J = 6.9 Hz, 1H), 7.63–7.48 (m, 2H), 7.03 (d, J = 7.2 Hz, 1H), 6.51 (d, J = 9.3 Hz, 2H), 6.34 (d, J = 6.6 Hz, 4H), 3.99 (s, 2H), 3.34 (s, 8H), 1.09 (t, J = 6.8 Hz, 13H). MS: m/z [M+H]⁺ calcd. 540.2719 found, 540.2723.

Synthesis of 7-(Diethylamino)-2-oxo-N-(prop-2-yn-1-yl)-2H-chromene-3-carboxamide (C-3). Under nitrogen, triethylamine (1.4 mL, 10 mmol) was added to a solution of propargylamine (0.68 mL, 10 mmol) in anhydrous DCM and then a solution of C-2(1.4 g, 5 mmol) in anhydrous DCM was added dropwise to the solution with stirring. The organic layer was washed with water after stirred for 24 h. After the solution was dried over anhydrous sodium sulfate, the solvent was removed under the reduced pressure and the residue was further purified by column chromatography to afford C-3 (1.3 g, 87.2%) as a yellow solid. ¹H-NMR (400 MHz, CDCl₃) δ 9.01 (s, 1H), 8.72 (s, 1H), 7.45 (d, J = 9.0 Hz, 1H), 6.67 (dd, J = 9.0, 2.5 Hz, 1H), 6.52 (d, J = 2.4 Hz, 1H), 4.25 (dd, J = 5.4, 2.5 Hz, 2H), 3.48 (q, J = 7.1 Hz, 4H), 2.26 (t, J = 2.5 Hz, 1H), 1.26 (t, J = 7.1 Hz, 6H).

Synthesis of N-((1-(2-((3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)amino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methyl)-7-(diethylamino)-2-oxo-2H-chromene-3-carboxamide (Rh-C). Rh-3 (369 mg, 0.68 mmol), C-3 (210 mg, 0.7 mmol) and CuI (285 mg, 1.5 mmol) were added in 20 mL THF-water (v:v, 1:1). The mixture was stirred at 50 °C for 24 h, and monitored by TLC. Then the solution was cooled down to room temperature and the solvent was removed under reduced pressure. After that, the solution was poured into distilled water and extracted with CH₂Cl₂ (3 × 30 mL). The combined extracts were dried over anhydrous sodium sulfate. The solvent was removed under the reduced pressure and the residue was further purified by column chromatography to afford Rh-C (204 mg, 35.8%) as an orange solid. ¹H-NMR (400 MHz, DMSO) δ 10.12 (s, 1H), 9.02 (t, J = 5.5 Hz, 1H), 8.70 (s, 1H), 7.82 (d, J = 6.8 Hz, 1H), 7.73–7.67 (m, 2H), 7.61–7.48 (m, 2H), 7.01 (d, J = 7.3 Hz, 1H), 6.82 (dd, J = 9.1, 2.3 Hz, 1H), 6.63 (d, J = 2.0 Hz, 1H), 6.52 (d, J = 8.5 Hz, 2H), 6.34 (d, J = 9.2 Hz, 4H), 5.07 (s, 2H), 4.54 (d, J = 5.5 Hz, 2H), 3.49 (q, J = 6.9 Hz, 4H), 3.32 (d, J = 7.7 Hz, 8H), 1.15 (t, J = 7.0 Hz, 6H), 1.08 (t, J = 6.9 Hz, 12H). MS: m/z [M+H]⁺, calcd. 838.4041, found 838.4021.

4. Conclusions

In conclusion, we have developed a fluorescent chemosensor Rh-C based on a rhodamine B and coumarin backbone. This probe allowed the selective detection Cu²⁺ in different solvents (THF, MeOH and MeCN) by the naked eye, especially in THF. Additionally, Rh-C could also sense Fe³⁺ in MeOH and Cr³⁺ in MeCN by ratiometric fluorescence change via a FRET process. Hence, we realize the optical detection of three kinds of common heavy metal ions with a single probe molecule, which might prove beneficial for the strategic design of multifunctional optical sensors.

Supplementary Materials

Supplementary materials can be accessed at: https://www.mdpi.com/1424-8220/15/1/49/s1.