A Novel Series Colorimetric and Off–On Fluorescent Chemosensors for Fe³⁺ Based on Rhodamine B Derivative

Abstract

A novel series colorimetric and off–on fluorescent chemosensors (2a, 2b, 2c) were designed and synthesized, which showed reversible and highly selective and sensitive recognition toward Fe³⁺ over other examined metal ions. Upon addition of Fe³⁺, sensors (2a, 2b) exhibit remarkably and 2c exhibits moderate enhanced absorbance intensity and color change from colorless to pink in CH₃OH–H₂O(1:1, v/v). The three compounds (2a, 2b, 2c) may therefore be applicable as rhodamine-based turn-on type fluorescent chemosensors.

Introduction

The design and development of sensors for the detection of heavy and transition metals are significant due to their vital role in biological and environmental application [1–3]. Iron is one of the most essential metals in the biological systems and plays crucial roles in cellular metabolisms. Especially, ferric iron (Fe³⁺) is widely retained in many proteins and enzymes either for structural purposes or as part of a catalytic site. Detection of trace amounts of Fe³⁺ is of great importance as iron is the most abundant essential trace element in the human body, and performs an important role in many fundamental physiological processes in organisms. However, only a few sensors for Fe³⁺ have been reported despite its importance in many biochemical processes at the cellular level [4–10]. Furthermore, Fe³⁺ is a well-known fluorescence quencher due to its paramagnetic nature, which makes it difficult to develop a sensitive turn-on fluorescent sensor [10]. There have been reported several methods for detecting iron such as atomic absorption, spectrophotometry, colorimetry and voltammetry techniques, but they generally require exorbitant equipment, intricate sample preparation procedures, and trained operators.

The rhodamine moiety has been used widely in the field of chemosensors, especially as a chemodosimeter, given its fluorescence OFF-ON behavior that results from its particular structural properties [11]. A lot of successful attempts have been made to develop selective fluorescent sensors based on rhodamine B, such as Cu²⁺ [12], Pb²⁺ [13], Hg²⁺ [14], Fe³⁺ [15] and Cr³⁺ [16]. As reported, the OFF/ON fluorescence switching of these chemosensors is based on structure change of the rhodamine moiety between spirocyclic and open-ring forms [17], the mechanism involves the formation of a ring-opened form of the spirolactam upon cation binding, resulting in fluorescence enhancement (550–600 nm).

Herein, we report three new rhodamine-based fluorescent chemosensors (2a, 2b, 2c) containing a 1,2,4-triazoles moiety, which is synthesized by two-step facile condensation (Scheme 1). 1,2,4-triazoles are important molecules with significant properties that have found widespread applications in foremost sectors of chemical sciences [18]. They are also largely used as ligands in coordination chemistry finding applications as molecular magnetic materials and dye-molecules in regenerative solar cells [19].

Scheme 1

Synthetic route of 2a, 2b, 2c

Experimental Section

Apparatus

Fluorescence spectra measurements were performed on a HITACHI F-4500 fluorescence spectrophotometer, and the excitation and emission wavelength band passes were both set at 4.0 nm. Absorption spectra were measured on a Lambda 35 UV/VIS spectrometer, Perkin Elmer precisely. The melting points were determined by a X-4 microscopic melting point apparatus with a digital thermometer (Shanghai, China). ¹H and ¹³C NMR spectra were recorded using a Bruker DTX-400 spectrometer. Samples were dissolved in CDCl₃ and placed in 5 mm NMR tubes. TMS was used as internal reference. Electrospray ionization(ESI) mass spectra was conducted in positive ionmode using a Bruker Esquire 3000 instrument(CH₃OH was used as solvent).

Materials

All chemicals and reagents were used as received from commercial sources without further purification. Solvents for chemical synthesis were purified according to standard procedures. Chloride salts of metal ions (K⁺, Na⁺, Ca²⁺, Mg²⁺, Ba²⁺, Zn²⁺, Fe³⁺, Fe²⁺, Mn²⁺, Pb²⁺, Cu²⁺, Co²⁺, Ni²⁺, Cr³⁺, Hg²⁺) and the nitrate salt of Ag⁺ ions were used to evaluate the metal ion binding properties by synthesized compounds. The metal ions were prepared as 10.00 mmol/L in water solution.

Synthesis

Synthesis of Compound 2a

N,N-Dimethylformamide Azine Dihydrochloride (3) and compound 1a was syn- thesized by reported methods [18, 20].

To a stirred solution of compound 1a (0.97 g, 2 mmol) in toluene (30 mL), N,N- Dimethylformamide Azine Dihydrochloride (3) (0.284 g, 2 mmol)was added. The solution was refluxed for about 15 h and the mixture was filtrated, the filtrate was concentrated by evaporation. Purification by column chromatography on silica gel (CH₂Cl₂/CH₃OH = 25/1) gave 0.407 g of white solid in a yield of 38 %. The proposed molecular structure and its purity were confirmed by various spectroscopic analyses. ¹H NMR (400 MHz, CDCl₃, ppm): 1.17–1.21 (t, J = 8.0 Hz, 12H, H¹), 3.33–3.38 (m, 8H, H²), 3.45–3.48(t, J = 6.0 Hz, 2H, H¹⁷), 3.76–3.80(t, J = 8.0 Hz, 2H, H¹⁸), 6.26–6.36 (m, 4H, H^8,4), 6.42(s, 2H, H⁷), 7.13–7.15(d, J = 8.0 Hz, 1H, H¹¹), 7.50–7.51(t, J = 2.0 Hz, 2H, H^12,13), 7.91–7.94(t, J = 6.0 Hz, 3H, H^14,19) . ¹³C NMR(100 MHz, CDCl₃ ppm): δ 168.1(C¹⁶), 153.4(C⁵), 152.7(C³), 149.0(C¹⁹), 142.7(C¹⁰), 133.0(C¹⁵), 130.8(C⁷), 128.6(C¹²), 128.4(C¹³), 124.0(C¹¹), 123.0(C¹⁴), 108.4(C⁶), 104.8(C⁸), 97.5(C⁴), 65.0(C⁹), 44.4(C²), 42.6(C¹⁷), 40.1(C¹⁸), 12.6(C¹). ESI-MS: Calcd for [C₃₂H₃₆N₆O₂]: 536.3. Found: 537.4[M+H⁺]⁺, 559.3[M+Na⁺]⁺. (Supporting Information, Figs. S1, S2, S3). M.p.:122–124 °C.

Synthesis of Compound 2b

The following compound was prepared using a general procedure which is essentially similar to that used for 2a. Compound 1b was synthesized by reported methods [20]. Yield of 2b: 38.2 %. ¹H NMR (400 MHz,CDCl₃, ppm): δ1.13–1.25 (m, 12H, H¹), 2.37–2.40(t, J = 6.0 Hz, 2H, H¹⁸), 2.73–2.76(t, J = 6.0 Hz, 2H, H¹⁹), 3.23–3.26(t, J = 6.0 Hz, 2H, H¹⁷), 3.30–3.35(m, 8H, H²), 3.85–3.88(t, J = 6.0 Hz, 2H, H²⁰), 6.23–6.26 (t, J = 6.0 Hz, 2H, H⁸), 6.35–6.39(m, 4H, H^4,7), 7.08–7.10(m, 1H, H¹¹), 7.44–7.48(m, 2H, H^12,13), 7.90–7.92(m, 1H, H¹⁴), 8.14(s, 2H, H²¹); ¹³C NMR(100 MHz, CDCl₃ ppm): δ168.6(C¹⁶), 153.3(C⁵), 149.0(C³), 143.0(C²¹), 142.7(C¹⁰), 132.9(C¹⁵), 131.1(C⁷), 128.9(C¹²), 128.5(C¹³), 124.0(C¹¹), 122.7(C¹⁴), 108.3(C⁶), 105.1(C⁸), 97.6(C⁴), 65.1(C⁹), 48.7(C¹⁹), 47.4(C¹⁸), 44.4(C²), 41.9(C¹⁷), 39.8(C²⁰), 12.6(C¹). ESI-MS: Calcd for [C₃₄H₄₁N₇O₂]: 579.3. Found: 580.3[M+H⁺]⁺, 602.5[M+Na⁺]⁺. (Supporting Information, Figs. S4, S5, S6). M.p.: 110–112 °C.

Synthesis of Compound 2c

The following compound was prepared using a general procedure which is essentially similar to that used for 2a. Compound 1c was synthesized by reported methods [20]. Yield of 2c: 35.2 %. ¹H NMR (400 MHz, CDCl₃, ppm): δ1.12–1.15 (t, J = 6.0 Hz, 18H, H^1,18,19,20), 1.62(s, 2H, H²¹), 3.09(s, 2H, H¹⁷), 3.30–3.32(d, J = 8.0 Hz, 8H, H²), 3.86–3.90(t, J = 8.0 Hz, 2H, H²²), 6.23–6.25(d, J = 8.0 Hz, 2H, H⁸), 6.36–6.41 (t, J = 10.0 Hz, 4H, H^4,7), 7.05–7.06(d, J = 4.0 Hz, 1H, H¹¹), 7.40–7.42(t, J = 4.0 Hz, 2H, H^12,13), 7.87–7.88(d, J = 4.0 Hz, 1H, H¹⁴), 8.10(s, 2H, H²³). ¹³C NMR(100 MHz, CDCl₃ ppm): δ168.1(C¹⁶), 153.3(C⁵), 148.7(C³), 142.6(C²³), 132.3(C¹⁵), 131.4(C⁷), 128.9 (C¹²), 128.0(C¹³), 123.8(C¹¹), 122.6(C¹⁴), 107.9(C⁶), 105.8(C⁸), 97.6(C⁴), 64.8(C⁹), 45.1(C²²), 44.4(C²), 39.8(C¹⁷), 30.3(C¹⁸), 27.7(C²¹), 26.1(C¹⁹), 25.7(C²⁰), 12.6(C¹). ESI-MS: Calcd for [C₃₆H₄₄N₆O₂]:592.3. Found: 593.4[M+H⁺]⁺, 615.5[M+Na⁺]⁺. (Supporting Information, Figs. S7, S8, S9). M.p.: 98–100 °C.

Results and Analysis

Fluorescence and UV absorption studies were performed using a 10 μM solution of 2a, 2b and 2c in a CH₃OH–H₂O(1:1, v/v) solution with appropriate amounts of metal ions. Solutions were shaken for 30 s before measuring the absorption and fluorescence. All compounds 2a, 2b and 2c were colorless and found to be very stable in the above-mentioned solution system for more than 1 week. The absorption spectra of compounds 2a, 2b and 2c in solutions did not show any peaks above 400 nm indicating the ring-closed spirolactone is predominant. In addition, a very weak fluorescence signal was observed at 580 nm upon excitation at 510 nm, confirming the presence of ring-closed spirolactone [21].

Steady-State Optical Properties

As shown in Fig. 1, UV–vis spectrum of 2a (10 μM) exhibited only very weak bands over 450 nm. Addition of Fe³⁺ (500 μM) for both sensor molecules resulted in the appearance of the characteristic rhodamine B absorption at 560 nm. As shown in Fig. 1, in the presence of Fe³⁺, 2a and 2b show better absorption spectra than 2c. We select 2b as the representation when expatiating the characters of the three compounds in the following discussion.

Fig. 1

Absorption spectra of 2a, 2b and 2c (10 μM) in CH₃OH–H₂O(1:1, v/v) with the presence of Fe³⁺(50 eq.)

UV–vis Spectral Responses of 2b

As shown in Fig. 2, UV–vis spectrum of 2b (10 μM) exhibited only very weak bands over 500 nm. Addition of 50 equiv Fe³⁺ into solution immediately resulted in a significant enhancement of absorbance at about 560 nm simultaneously the color change into red. Under the identical condition, no obvious response could be observed upon the addition of other ions including Zn²⁺, Mg²⁺, Ca²⁺, Cd²⁺, Pb²⁺, Cu²⁺, Hg²⁺, Ba²⁺, Ni²⁺, Fe²⁺, K⁺, Ag⁺, Co²⁺, Cr³⁺ and Na⁺. The results demonstrated that 2b was characteristic of high selectivity toward Fe³⁺ over other competitive metal ions.

Fig. 2

UV–vis spectrum of 2b (10 μM) in CH₃OH–H₂O (1:1, v/v) with different metal ions (50 eq.)

To determine the stoichiometry of the ferric-ligand complex, Job’s method for absorbance measurement was applied [22]. Keeping the sum of the initial concentration of Fe³⁺ and 2b at 100 μM, the molar ratio of Fe³⁺ was varied from 0 to 1. A plot of $\left[ {{\text{F}}{{\text{e}}^{{{3} + }}}} \right]/\left\{ {\left[ {{\text{F}}{{\text{e}}^{{{3} + }}}} \right] + \left[ {{{\bf 2b}}} \right]} \right\}$ versus the molar fraction of Fe³⁺ was provided in Fig. 3. It showed that the $\left[ {{\text{F}}{{\text{e}}^{{{3} + }}}} \right]/\left\{ {\left[ {{\text{F}}{{\text{e}}^{{{3} + }}}} \right] + \left[ {{{\bf 2b}}} \right]} \right\}$ value went through a maximum at a molar fraction of 0.5, indicating a 1:1 stoichiometry of the Fe³⁺ to 2b in the complex. Another more direct evidence was obtained by comparing the ESI mass spectra of 2b and 2b-FeCl₃. As shown in Fig. 4, the cluster peak at m/z = 705.2 (calcd = 705.2) corresponding to [2b+Fe³⁺+2Cl^-]⁺ and m/z = 741.3(calcd = 741.2) corresponding to [2b+Fe³⁺+3Cl^-+H⁺]⁺ was clearly observed when 5 equiv of FeCl₃ was added to 2b, whereas 2b without FeCl₃ exhibited peaks only at m/z = 580.3 and 602.5, which corresponded to [2b+H⁺]⁺ and [2b+Na⁺]⁺ (Fig. S6), respectively. This indicating the formation of a 1:1 metal-ligand complex.

Fig. 3

The titration probe evaluated from the absorption at 560 nm. Job’s plot for determining the stoichiometry of 2b and ${\text{F}}{{\text{e}}^{{{3} + }}}\left( {\left[ {{{\bf 2b}}} \right] + \left[ {{\text{F}}{{\text{e}}^{{{3} + }}}} \right] = {1}00\mu {\text{M}}} \right)$

Fig. 4

ESI mass spectra (positive) of 2b in the presence of FeCl₃ (5 equiv), indicating the formation of a 1:1 metal-ligand complex

We also do the same measurement with compounds 2a and 2c, they also show the same absorbance and peaks with the addition of FeCl₃. (Figs. S10, S11, S12, S13)

Fluorescence Spectral Responses of 2b

As shown in Fig. 5, 2b (10 μM) shows a very weak fluorescence in the absence of metal ions. When 10 equiv. metal ions of Zn²⁺, Mg²⁺, Ca²⁺, Cd²⁺, Pb²⁺, Cu²⁺, Hg²⁺, Ba²⁺, Ni²⁺, Fe²⁺, K⁺, Ag⁺, Co²⁺, Cr³⁺ and Na⁺ were added, no obvious changes on fluorescence intensity and color could be observed. However, under the same conditions, the addition of Fe³⁺(10 μM) resulted in a remarkably enhancement of fluorescence at 580 nm. The color of the solution also changed from colorless to pink (Fig. 5, top). This strongly suggested that 2b can serve as a “naked eye” probe and a high sensitivity for Fe³⁺. Moreover, the competitive experiments also confirmed that the background metal ions showed very low interference with the detection of Fe³⁺ in CH₃OH–H₂O(1:1, v/v) (Fig. 6).

Fig. 5

Fluorescence spectra (λ_ex = 565 nm) of 2b (10 μM) in CH₃OH –H₂O(1:1, v/v) with the presence of 10 equivalents of various species. Top shows the photo of sensor 2b with different metal ions

Fig. 6

Fluorescence intensity (at 580 nm) of 2b (10 μM) upon the addition of 10 μM Fe³⁺ in the presence of 10 μM background metal ions in CH₃OH–H₂O (1:1, v/v). (λ_ex = 565 nm)

To further investigate the binding stoichiometry of 2b and Fe³⁺ ion, a fluorescence titration experiment was carried out. An increase of fluorescence intensity of 2b could be observed with gradual addition of Fe³⁺ ion(Fig. 7). Under optimal conditions, the detection limit for Fe³⁺ was as low as 10 μM(Fig. 7, inset).

Fig. 7

The fluorescence emission spectra of 2b (10 μM) in the presence of different concentrations of Fe³⁺(0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 eq.) in CH₃OH –H₂O(1:1, v/v). (λ_ex = 565 nm). Inset: Changes in the emission intensity at 580 nm

Effect of pH

In order to investigate the influence of the different acid concentration on the spectra of sensor 2b and find a suitable pH span in which sensor 2b can selectively detect Fe³⁺ efficiently, the acid titration experiments were performed. As shown in Fig. 8, the fluorescence titration curve of free sensor did not show obvious enhancement of fluorescence between pH 3.0 and 10.0, suggesting that spirolactam tautomer of sensor 2b was insensitive to the pH changes in this range. However, the addition of Fe³⁺ led to the enhancement of fluorescence over a comparatively wide pH range (3.0–7.0), which is attributed to opening of the rhodamine ring. Consequently, sensor 2b may be used to detect Fe³⁺ in approximate physiological conditions.

Fig. 8

Fluorescence intensity (580 nm) of free sensor 2b (10 μM) and in the presence of 10 equiv. Fe³⁺ in CH₃OH/Tris–HCl buffer (1:1, v/v) solutions with different pH conditions

Further, it was of great interest to investigate the reversible binding nature of the sensor. To demonstrate the reversibility of 2b, EDTA (10 eq.), as a strong affinity for Fe³⁺, was introduced into the solution containing 2b (10 μM) and Fe³⁺ (100 μM). Upon addition of CH₃OH–H₂O (1:1, v/v) solution of EDTA (up to 10 eq.) to a solution mixture of 2b (10 μM) and Fe³⁺(100 μM), the fluorescence intensity at 580 nm was decreased (blue line) due to decomplexation of Fe³⁺ from 2b by EDTA, and further addition of 10 eq. Fe³⁺ could recover the strong fluorescence again (green line) (shown in Fig. 9). This observation is assumed to be due to decomplexation of Fe³⁺ by EDTA followed by a spirolactam ring closure reaction. Thus, 2b can be classified as a reversible chemosensor for Fe³⁺.

Fig. 9

Fluorescence intensity of 2b (10 μM) to Fe³⁺ in CH₃OH –H₂O(1:1, v/v), (λ_ex = 565 nm)

Fluorescence spectral responses of 2a, 2c is similar with 2b, and the result should be seen in the supporting information (Fig. S14, S15, S16, S17, S18, S19, S20, S21).

Fluorescence Spectral Responses Contrast of 2a, 2b and 2c

We also find that there have some differences among the ability of 2a, 2b and 2c interact with Fe³⁺ ion. As shown in Fig. 10, 2a, 2b and 2c exhibit 63-fold, 98-fold, 33-fold enhancement of fluorescence intensity at peak wavelength 580 nm in the presence of 10 equiv. Fe³⁺, respectively. 2a and 2b may therefore be applicable as rhodamine-based turn-on type fluorescent chemosensors.

Fig. 10

Fluorescence intensity (at 580 nm) of 2a, 2b, 2c (10 μM) upon the addition of 10 μM Fe³⁺ in CH₃OH–H₂O (1:1, v/v). (λ_ex = 565 nm)

The fluorescence enhancement of 2c is not as good as that of 2a and 2b, but it also displays moderate selectivity for Fe³⁺. It is maybe due to the long carbon linker between the triazole with the rhodamine group and that result in amide and triazole group having a bad affinity toward Fe³⁺.

Conclusions

In conclusion, we synthesized three easily available fluorescent chemosensors (2a, 2b and 2c) for Fe³⁺. 2a and 2b exhibited a strong fluorescence enhancement upon addition of Fe³⁺ while showing almost no response to other cations. The colorimetric and fluorescent response to Fe³⁺ can be conveniently detected even by the naked eye, which provides a facile method for visual detection of Fe³⁺. 2a, 2b and 2c may therefore be applicable as rhodamine-based turn-on type fluorescent chemosensors.