1,8-Naphthalimide derivative-based turn-off fluorescent probe for the detection of picrate in organic aqueous media

2.1 Materials and general methods

Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. ¹H NMR spectra were recorded on a Varian VR300 spectrometer and ¹³C NMR spectra were obtained with a Mercury plus 400 MHz NMR spectrometer with tetramethylsilane (TMS) as an internal standard and [D₆] dimethyl sulfoxide (DMSO) as the solvent. The corrected excitation and fluorescence spectra were performed on a Perkin Elmer LS 55 fluorescence spectrophotometer. MS spectra were recorded on a mass spectrometer micrOTOF, and elemental analyses were determined using a Carlo Erba 1106 elemental analyzer. The IR spectra were recorded in the range 4000–400 cm^–1 with a Nicolet FT-VERTEX 70 spectrometer using KBr pellets. Electronic spectra were obtained using a Lab-Tech UV Bluestar spectrophotometer. Thin-layer chromatography (TLC) was performed on silica gel, Fluka F60 254, 20 × 20, 0.2 mm, using ethyl acetate as eluant. The melting points were determined by means of a Kofler melting point microscope. The synthetic route to compound 3 is shown in Scheme 1.

Scheme 1:

Chemical structure and synthetic route to compound 3.

2.2 Synthesis of compounds

2.3 Fluorescence measurements

A 3 × 10^–3m stock solution of compound 3 was prepared by dissolving compound 3 in absolute DMF. A stock standard solution of anions (3 × 10^–3m) was prepared by dissolving an appropriate amount of alkali metal salt in water and adjusting the volume to 50 mL in a volumetric flask. The solution of 3-anion was prepared by adding 10 μL of the stock solution of compound 3, 200 μL of the stock solution of anions and 3 mL of the DMF solution in a cuvette. The fluorescence measurements were carried out at the maximum excitation wavelength of 417 nm and the maximum emission wavelength of 514 nm. Before each measurement, the solutions were allowed to stand for a moment to mix well the components.

3.1 Synthesis and characterization

The synthesis of 1,8-naphthalimide 3 was achieved in three steps as shown in Scheme 1. Intermediate 2 was tried to be synthesized by two methods. One is a two-step reaction process. First, acyl chloride was prepared by the reaction of iminodiacetic acid with thionyl chloride in dichloromethane. Second, compound 2 was obtained by the reaction of acyl chloride with butylamine in benzene solution. In the other method, compound 2 was prepared by the reaction of diethyl iminodiacetate with butylamine, and compound 2 could be obtained with high yield. The second method is one step less than the first method. Moreover, the first reaction system is complicated and the purification of the target product is difficult, which may result from the instability of the intermediate acyl chloride. Thus, we chose this method to prepare the designed compound 2.

Two new methods (i and ii) were developed for the reaction of compound 1 with compound 2. (i) The target compound 3 was obtained through solvent reactions such as refluxing 1 with compound 2 in DMF, pyridine or 2-methoxyethanol. Many byproducts are generated with this solvent method resulting in the formation of a mixture of products that were difficult to separate. Moreover, the target compound was obtained only in low yield. (ii) Compound 3 was prepared through a solvent-free reaction. Compared with other traditional solvent reactions [35, 36], this synthesis is more cost-effective and environmentally benign. The target molecule 3 was formed in high purity and in 47% yield via column chromatography. Thus, we chose this method to prepare the designed materials.

Compound 3 is stable under air. It is soluble in dipolar aprotic solvents such as DMF, DMSO, acetonitrile and pyridine; slightly soluble in chloroform, acetone and dichloromethane; and insoluble in Et₂O and water. The structure and purity of the synthesized compound 3 were confirmed by conventional techniques – melting point, TLC (R_f values) and UV/Vis spectra, and identified by ¹H and ¹³C NMR spectra, FT-IR and elemental analysis data. The data are presented in the Experimental section.

3.2 Sensitivity of sensor 3 to pH value

In order to examine the disturbance of picrate detection by proton, the fluorescence spectra of 3 in DMF–water (15/1, v/v) solutions with different pH values were determined (Fig. 1). The spectra did not show an obvious change from pH 1.81 to 11.58, which well matches the above design idea. This pH insensitivity of 3 is beneficial to the detection of picrate in different media.

Fig. 1:

The emission intensity (emission at λ = 514 nm) of 3 was measured in DMF–water (15:1, v/v) solution at different pH values. Excitation at λ = 417 nm (slit: 10.0 nm/10.0 nm).

3.3 Fluorescence response of probe 3 to different anions

In order to detect the selectivity of probe 3, adding different anions to probe buffer solutions, then observe the changes of fluorescence. The experimental steps are as follows: adding 10 L of the stock solution of compound 3, 200 L of the stock solution of anions and 3 mL of the DMF solution in a cuvette, the fluorescence measurements were carried out at the maximum excitation wavelength of 417 nm and the maximum emission wavelength of 514 nm (Fig. 2). The experimental result showed that 3 displayed substantial fluorescence quenching (93.2%) in the presence of picrate. In contrast, only a slight fluorescence decrease (<10.3%) was observed upon the addition of F^–, Cl^–, Br^–, I^–, OH^–, Ac^–, NO₃^–, ClO₄^–, SCN^–, SO₃^2–, SO₄^2–, H₂PO₄^– or HPO₄^2–. As can be seen from the photograph in Fig. 3, the distinct yellow-green fluorescence of 3 disappears in the presence of picrate anions, but the presence of other anions induces no significant change on the emission spectra. The above-mentioned results suggested that probe 3 exhibited an excellent selectivity for picrate among the various ions tested.

Fig. 2:

Fluorescence spectrum of 3 (9.3 × 10^–6m) in DMF–water (15:1, v/v) solution in the presence of anions ([anion] = 1.8 × 10^–4m). Excitation at λ = 417 nm (slit: 2.0 nm/10.0 nm).

Fig. 3:

Fluorescent distinction of anions as seen by naked eyes under a 365 nm UV light irradiation. From left to right: F^–, Cl^–, Br^–, I^–, OH^–, Pic^–, Ac^–, NO₃^–, ClO₄^–, SCN^–, SO₃^2–, SO₄^2–, H₂PO₄^–, HPO₄^2–. Excitation at λ = 417 nm (slit: 2.0 nm/10.0 nm). [Probe 3] = 9.3 × 10^–6m, [anion] = 1.8 × 10^–4m.

3.4 Fluorescence titration experiments

In order to better understand the sensing process, fluorescence spectra changes of probe 3 (20 μm) were measured with different concentrations of picrate (Fig. 4). Upon the addition of increasing amounts of picrate, the fluorescence intensity of 3 gradually decreased. The total fluorescence intensity at 514 nm of 3 was almost fully quenched when about 35 equivalents of picrate were present (inset of Fig. 4). Furthermore, the fluorescence intensity at 514 nm decreased almost linearly up to about 7 equivalents of Pic^– ions (6.82 × 10^–5m) (Fig. 5) without any significant changes in the fluorescence profile. From these concentration-dependent fluorescence changes, the detection limit of 3 for the determination of picrate in DMF was estimated to be 5.8 × 10^–7m. The detection limit (DL) of sensor 3 for picrate was calculated as 6.5 × 10^–7m by the Stern–Volmer plot [37]:

Fig. 4:

Fluorescence spectra of 3 (9.3 × 10^–6m) in DMF–water (15:1, v/v) solution in the presence of different concentrations of picrate (0–35 equiv). Inset: curve of fluorescence intensity at 514 nm of probe 3 versus increasing concentrations of picrate. Slit: 2 nm/10 nm.

Fig. 5:

Fluorescence intensity ratio (F₀–F)/F₀ of 3 (9.3 × 10^–6m) as a function of picrate concentrations (final concentration: 0.5–7.0 equiv). Each spectrum was acquired at room temperature, and picrate solution was freshly prepared before use (F and F₀ are the fluorescence intensities of 3 with and without adding the picrate in DMF–water (15:1, v/v) solution, respectively).

DL = 3σ/S=6.5 × 10−7 M

where σ is the standard deviation of the blank solution, and S is the slope of the calibration curve. It is demonstrated that probe 3 can be a sensitive fluorescent probe for the quantitative detection of picrate, which was sufficiently low for the detection of picrate in many chemical and biological systems.

3.5 Competition experiments

For an excellent fluorescent probe, high selectivity is a matter of necessity. To confirm the selectivity of probe 3 (9.3 × 10^–6m), the competition experiments were also conducted by the addition of 20 equivalents of other anions, such as F^–, Cl^–, Br^–, I^–, OH^–, Ac^–, NO₃^–, ClO₄^–, SCN^–, SO₃^2–, SO₄^2–, H₂PO₄^– or HPO₄^2–, to the DMF solution of 3 in the presence of 20 equivalents of picrate. As outlined in Fig. 6, experimental results show that the anions have no obvious interference for picrate detection, indicating that the probe has a better selectivity for picrate over other anions tested under the same conditions. Therefore, it seems feasible to use this probe for practical applications, which also strongly supports the above conclusions.

Fig. 6:

Results of the competition experiments between the Pic^– ion and other selected anions in DMF–water (15:1, v/v) solution at 298 K; the concentrations of the Pic^– ion and other competing anions were all 1.8 × 10^–4m; the excitation wavelength was 417 nm and the emission intensity was measured at 514 nm.

3.6 Investigation of sensing mechanism

From the above results, we learned that the yellow-green fluorescence of compound 3 disappears in the presence of picrate. As shown in Fig. 7, we suggest the following mechanism for fluorescence quenching. The fluorescence of compound 3 is in “off-state” because the fluorescence of naphthalimide fluorophore is quenched by the electron transfer process. Consequently, we propose that picrate recognition occurs by the initial hydrogen bonding of the anion to the N–H protons of the amide, followed by deprotonation which makes the receptor highly electron-rich and enhances the electron transfer quenching of the naphthalimide excited state. To confirm the assumption, ¹H NMR spectra were taken (Fig. 8). The N–H protons of the amide were observed at higher field than in the sample containing picrate, indicating that these signals are attributable to the initial hydrogen bonding of the anion to the N–H protons of the amide. This indicates that experimental results are consistent with our assumption.

Fig. 7:

The PET inhibition mechanism of compound 3 and picrate.

Fig. 8:

Partial ¹H NMR spectra of probe 3 + picrate and 3. Up: the reaction mixture of probe 3 and one equivalent about of picrate; below: probe 3.