Synthesis of an Antipyrine-Based Fluorescent Probe with Synergistic Effects for the Selective Recognition of Zinc Ion

Abstract

A novel fluorescent probe containing an imine structure was synthesized through a condensation reaction based on the skeleton of antipyrine. Due to the synergistic effect of photoinduced electron transfer (PET), excited-state intramolecular proton transfer (ESIPT), and E/Z isomerization, the probe itself has weak fluorescence. When zinc ions are added to the ethanol solution of the probe, the formed complex inhibits PET, ESIPT, and E/Z isomerization while activating chelation-enhanced fluorescence (CHEF), resulting in fluorescent “turn-on” at 462 nm. Under optimal detection conditions, the probe can rapidly respond to zinc ions within 3 min, with a linear range of 60–220 μM and a lower limit of detection (LOD) of 0.63 μM. It can also specifically identify zinc ions in the presence of 13 common metal ions.

1. Introduction

Metal ions play a pivotal role in human health, yet an overabundance of essential metal ions such as iron and zinc can induce various maladies, including Parkinson’s disease, Alzheimer’s disease, muscular dystrophy, ischemic stroke, etc. [1,2,3,4]. Consequently, the rapid and efficient selective detection of metal ions has remained a focal point of research endeavors. Up to now, a myriad of assays, including traditional titrimetry [5], atomic absorption spectroscopy (AAS) [6], ultraviolet–visible spectrophotometry (UV–vis) [7], inductively coupled plasma (ICP) [8], ion selective electrode (ISE) [9], biosensors [10], and fluorescence analysis (FA) have been developed for metal ion detection. Among them, fluorescent probes have attracted considerable attention due to their exceptional sensitivity, rapid response rate, uncomplicated equipment requirements, and user-friendly operation, enabling them to facilitate “naked eye” detection [11,12,13,14,15]. Additionally, by meticulously modifying the probe molecules chemically, one can achieve targeted and specific recognition of the desired analytes.

The presence of a ketone group and two nitrogen atoms within the molecular structure of antipyrine derivatives endows them with exceptional biological activity and coordination capabilities. These attributes make them valuable as ligands in synthetic applications, where they demonstrate distinct characteristics in the optical, electrical, and biological domains [16,17,18]. Particularly, 4-aminoantipyrine derivatives are frequently employed as chromogenic agents for the detection of heavy metal ions. For example, Gupta’s group reported that the condensation products of 4-aminoantipyrine with α-diones and β-diones can serve in the colorimetric detection of aluminum and chromium ions [19]. Das’s group synthesized a 4-aminoantipyrine Schiff base derived from 1-formylpyrene, which exhibits a selective blue shift in the UV–vis spectrum specifically for copper ions [20]. Furthermore, Kim et al. developed a 4-aminoantipyrine Schiff base obtained from 4-diethylaminosalicylaldehyde, enabling the dual colorimetric detection of copper and fluoride ions [21]. Additionally, Kumar’s team crafted an antipyrine-based hydrazone fluorescent probe that allows for the rapid detection of copper ions [22]. It is clear that the identification of metal ions using antipyrine compounds often relies on discernible colorimetric responses.

Given that the sensitivity of fluorescence detection is two orders of magnitude superior to that of colorimetry, the enhancement in fluorescent probe functionalities would be significant for practical applications. Luxami’s group introduced a sulfonamide probe created from 4-aminoantipyrine and dansyl chloride. When this probe complexes with copper ions, it inhibits the photoinduced electron transfer (PET) from the dimethylamino group to the antipyrine moiety, thereby releasing fluorescence and enabling the selective fluorometric detection of copper ions [23]. John’s team designed and synthesized a perilyl antipyrine fluorescent probe that activates fluorescence by suppressing the PET process through the coordination of Th(IV) with imines [24]. In 2020, Maity et al. synthesized an aggregation-induced emission enhancement (AIEE) probe based on the condensation reaction between ortho-vanillin and 4-aminoantipyrine for the dual sensing detection of Al³⁺ and Zn²⁺ [25]. Chattopadhyay et al. utilized a benzotriazole framework in conjunction with antipyrine to construct a zinc ion fluorescent probe exhibiting an aggregation-induced enhancement (AIE) effect [26]. In addition to the aforementioned work, several articles and patents have also reported fluorescence probes based on the antipyrine motif [27,28,29].

Herein, we designed and synthesized a novel antipyrine derivative P2 that employs the phenolic hydroxyl group within the molecule as a proton donor and the Schiff base structure (-CH=N-) as a proton acceptor. Based on the excited-state intramolecular proton transfer (ESIPT) effect, it achieves precise recognition of zinc ions; meanwhile, the integration of the methyl butynol structure within the molecule extends the conjugated region of the compound, serving as a zinc ion probe with heightened sensitivity.

2. Materials and Methods

2.1. Instruments and Reagents

In this study, we used FA1004 Electronic Analytical Balance (Shanghai Liangping Instrument & Meter Co., Ltd., Shanghai, China), DF-101Z Heat Collection Constant Temperature Heating Magnetic Stirrer (Jintan Fuhua Instrument Co., Ltd., Changzhou, China), RE-201 Rotary Evaporator (Shanghai Hongguan Instrument Equipment Co., Ltd., Shanghai, China), DZG-6020 Vacuum Drying Oven (Shanghai Senxin Laboratory Instrument Co., Ltd., Shanghai, China), and F-4600 Fluorescence Spectrophotometer (Hitachi Technology Corporation, Tokyo, Japan). Nuclear magnetic resonance spectra were obtained using a Bruker AV 400 NMR instrument, and FT-IR spectra were recorded on a Bruker Tensor 27 spectrophotometer (Bruker Corporation, Billerica, MA, USA).

We used 5-bromosalicylaldehyde, 2-methyl-3-butyn-2-ol, 4-aminoantipyrine, CuI, PPh₃, Pd(PPh₃)₂Cl₂, tetrahydrofuran, triethylamine, anhydrous ethanol, petroleum ether (PE), ethyl acetate (EA), and metal salts (Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China); all reagents used were of analytical purity.

2.2. Synthesis and Characterization of Probe P2

Synthesis of P1: Under a nitrogen atmosphere, 5-bromosalicylaldehyde (2.01 g, 10 mmol), CuI (0.038 g, 0.2 mmol), PPh₃ (0.052 g, 0.2 mmol), Pd(PPh₃)₂Cl₂ (0.07 g, 0.1 mmol), 15 mL THF, and 20 mL Et₃N were added into a 50 mL three-necked flask. After stirring for 15 min, 2-methyl-3-butyn-2-ol (1.26 g, 15 mmol) was injected into the reaction mixture with a syringe. The reaction was conducted under reflux for 11 h and monitored using thin-layer chromatography until the starting material was completely consumed. Then, the reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated to obtain a black oily substance and purified by silica gel column chromatography (V_PE:V_EA = 10:1) to give 2-hydroxy-5-(3-hydroxy-3-methylbut-1-yn-1-yl)benzaldehyde (intermediate P1, 1.43 g) as a pale yellow solid, with a yield of 70.3%. ¹H NMR (400 MHz, CDCl₃) δ 11.08 (s, 1H), 9.85 (s, 1H), 7.65–7.54 (m, 2H), 6.94 (d, J = 8 Hz, 1H), 1.62 (s, 6H). IR(KBr), ν, cm⁻¹ 3132, 2250, 1666, 689.

Synthesis of P2: P1 (0.25 g, 1.2 mmol) and 4-aminoantipyrine (0.25 g, 1.2 mmol) were added to a 50 mL three-necked flask, followed by the addition of 25 mL of anhydrous ethanol. The reaction was carried out at 75 °C for 2.5 h. After cooling to room temperature, the reaction mixture was filtered, and the filter residue was washed with anhydrous ethanol and then recrystallized from anhydrous ethanol to obtain (E)-4-((2-hydroxy-5-(3-hydroxy-3-methylbut-1-yn-1-yl)benzylidene)amino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (probe P2, 0.32 g) as an orange-red crystal, with a yield of 68.4%. ¹H NMR (400 MHz, DMSO-d6) δ 13.03 (s, 1H), 9.66 (s, 1H), 7.62–7.49 (m, 3H), 7.45–7.34 (m, 3H), 7.31 (dd, J = 8.5, 2.1 Hz, 1H), 6.90 (d, J = 8.5 Hz, 1H), 5.43 (s, 1H), 3.22 (s, 3H), 2.41 (s, 3H), 1.46 (s, 6H). ¹³C NMR (101 MHz, DMSO-d6) δ 159.63, 159.47, 156.33, 150.82, 134.63, 133.71, 129.71, 127.79, 125.52, 121.16, 117.45, 114.46, 113.79, 94.86, 80.40, 64.07, 35.56, 32.17, 10.33. IR(KBr), ν, cm⁻¹ 3473, 2250, 1658, 762.

2.3. Spectral Analysis

For fluorescent spectra measurement, the excitation wavelength was set to 352 nm, and the emission spectrum was scanned from 360 nm to 650 nm, with both excitation and emission slit widths set to 10 nm.

For the preparation of stock solutions, the stock solutions of P2 (1 mM) were prepared in ethanol (EtOH), dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF), separately. The stock solutions of metal ions (10 mM) were prepared in deionized water.

For the solvent screening experiment, stock solutions (EtOH, DMSO, DMF) of probe P2 and Zn²⁺ were added into a 1 mL cuvette and diluted with the corresponding organic solvent/water mixture to form the test solution with P2 (100 μM) and Zn²⁺ (100 μM). The fluorescence spectra were measured 3 min later,.

For the water content test, various amounts of Zn²⁺ and P2 stock solutions were added into dilutes with H₂O/EtOH ratios ranging from (10:0) to (0:10), diluting to the testing solution consisting of P2 (100 μM) and Zn²⁺ (100 μM). The fluorescence spectra were measured 3 min later to determine the optimal water content of the system.

For the response time test, the Zn²⁺ stock solution was mixed with the probe solution and diluted with 30% water content in EtOH to be the test solution, in which the concentration of zinc ions and P2 were both 100 μM. Fluorescence spectra measurements were taken every 30 s to determine the optimal incubation time. At the same time, a blank group without zinc ions was set up to eliminate the fluorescence interference of the detection system itself.

For fluorescence titration, we mixed 10 μL P2 stock solution and various amounts of Zn²⁺ stock solution in a 1 mL cuvette. After diluting with 30% water content in EtOH, a series of test solutions with different concentrations were prepared, where the concentration of P2 was 100 μM, and the concentration of Zn²⁺ ranged from 0 to 320 μM with a concentration gradient of 10 μM. The fluorescence spectra of the test solutions were analyzed 3 min later. At the same time, a fluorescence titration curve was plotted, and the limit of detection (LOD) was calculated.

For the anti-interference test, CuSO₄·5H₂O, MgSO₄, ZnSO₄, CoCl₂·6H₂O, Al₂(SO₄)₃, Fe₂(SO₄)₃·9H₂O, Ce(NO₃)₃·6H₂O, NiSO₄·6H₂O, Pb(NO₃)₂, AgNO₃, Sr(NO₃)₂, Eu(NO₃)₃·6H₂O, Pr(NO₃)₃·6H₂O, Gd(NO₃)₃·6H₂O were dissolved in deionized water to prepare separate 100 μM metal ion solutions. In a cuvette, 1 mL of interfering metal ion solution was mixed with 1 mL P2 solution, and then the fluorescence spectrum of each mixed solution as the control group was measured. Subsequently, 0.5 mL of interfering metal ion solution was mixed with 1 mL of P2 solution in a cuvette, followed by adding 0.5 mL of zinc ion solution and measuring the fluorescence spectrum.

3. Results and Discussion

3.1. Synthesis of Probe P2

The synthesis procedure of P2 is shown in Scheme 1. Similar to a previous report [30], P1 was synthesized via Sonogashira coupling, and then P1 was reacted with 4-aminoantipyrine via condensation to generate probe P2. The structural identification of intermediate P1 and probe P2 was confirmed by NMR and FT-IR spectroscopies (Figures S1–S5).

3.2. Influence of Solvents on Fluorescence Detection

Given the propensity for the luminescence of most fluorophores to be impacted by the surrounding environment, an initial assessment was conducted to evaluate the solvent’s impact on the detection system. The data presented in Figure 1 elucidate that different aqueous solvents—EtOH, DMSO, and DMF—when employed as media for the probe, result in varying levels of fluorescence response. The effectiveness of these aqueous solvents in enhancing fluorescence followed the hierarchy EtOH > DMSO > DMF. Consequently, 70% EtOH emerged as the solvent of choice due to its superior ability to enhance fluorescence, thereby optimizing the performance of the detection probe.

3.3. Influence of Water Content on Fluorescence Detection

The tolerance of the detection system to water content is crucial for assessing its suitability for in situ detection within biological systems. To evaluate this, Zn²⁺ ions were added to the detection system with varying water contents and allowed to stand for 3 min before measuring the fluorescence changes. A plot was created with the water content as the x-axis and the relative fluorescence intensity I/I₀ at 462 nm as the y-axis. Figure 2 reveals that the relative fluorescence intensity increased with the water content up to 30%, reaching a maximum at this level. However, a further increase in water content led to a decrease in relative fluorescence intensity, which could be attributed to the reduced probe solubility in the mixed solvents with excessively high water proportions. Consequently, for subsequent detections, a 30% water content in ethanol was determined to be the optimal solvent system for the detection setup.

3.4. Influence of Incubation Time on Fluorescence Detection

Considering the importance of incubation time in sensing systems, we investigated the detection efficiency of the system toward zinc ions under different incubation durations. The concentrations of the probe and zinc ions were both 100 μM. A graph was plotted with reaction time on the x-axis and the fluorescence intensity at 462 nm on the y-axis. As seen in Figure 3, in the absence of Zn²⁺, the fluorescence intensity of the probe remained stable over extended reaction times, indicating that the probe was stable in a 30% water content ethanol solvent. Upon addition of Zn²⁺ to the system and commencement of the timing test, it was observed that the fluorescence intensity gradually increased with longer reaction times. The fluorescence intensity peaked at 3 min, with a 5-fold enhancement. Afterward, the fluorescence intensity stabilized. Based on these results, we established an optimal incubation time of 3 min for the system, which was then used as the optimal response time in subsequent experiments.

3.5. Fluorescence Titration

To establish a fluorescence titration curve, we prepared test solutions with varying concentrations of Zn²⁺ and conducted fluorescence spectroscopy measurements. As shown in Figure 4a, under an excitation wavelength of 352 nm, the probe alone exhibited a weak characteristic emission peak. With the addition of zinc ions, the fluorescence intensity of the probe at 462 nm gradually increased. When the concentration of zinc ions reached 240 μM, the fluorescence intensity peaked, enhancing by approximately seven times. After the zinc ion concentration exceeded 240 µM, the fluorescence intensity at 462 nm slightly decreased instead (Figure S5). Based on the fluorescence spectroscopy data, a working curve was fitted with zinc ion concentration on the x-axis and the fluorescence intensity at 462 nm on the y-axis, as shown in Figure 4b. Within the range of zinc ion concentrations from 60 to 220 μM, there is a good linear relationship (R² = 0.9776), indicating that this detection system can be used for the quantitative detection of zinc ions.

Furthermore, the sensitivity of fluorescence detection determines the application prospects of this detection system, so we needed to test the system’s detection sensitivity toward zinc ions. Using the detection limit formula reported in the literature, LOD = 3σ/k, where σ is the standard deviation of the blank signal, and k is the slope of the calibration curve, we determined that at a signal-to-noise ratio (S/N) of three, the detection limit for zinc ions using the probe was 0.63 μM. Additionally, the performance of typical Zn²⁺ sensors are summarized in

Table 1. Comparison of the previously reported Zn²⁺ probes with P2.

Entry	Probe	Solvent System	LOD (μM)	Linear Range (μM)	Ref.
1		CH3OH	0.84	0–40	[31]
2		CH3CN/H2O (7/3)	0.65	-	[32]
3		DMSO/H2O (9/1)	3.65	-	[33]
4		CH3CN/HEPES (1/1)	1.79	0–40	[34]
5		DMSO/H2O (1/1)	1.24	-	[35]
This work		EtOH/H2O (7/3)	0.63	60–220

in terms of the solvent system, limit of detection (LOD), and linear range. In comparison to reports in the literature, our method demonstrates superior sensitivity, a lower detection limit, and an expanded linear range. Notably, our analytical approach offers significant benefits, including straightforward operation, convenience, and cost-effectiveness.

3.6. Anti-Interference Experiments

The impact of coexisting ions on the performance of P2 is illustrated in Figure 5. Although several ions, such as Ag⁺, Sr²⁺ Al³⁺, and Ce⁴⁺, caused a slight increase in the fluorescence intensity at 462 nm, the addition of Zn²⁺ to the system resulted in a significant enhancement in fluorescence intensity. For other ions, aside from the test solutions containing Pb²⁺ and Gd³⁺, which significantly reduced the fluorescence intensity at 462 nm, noticeable fluorescence enhancements were observed with the rest of the test solutions. This indicates that the probe has a strong anti-interference capability for the recognition of Zn²⁺ in the presence of common metal ions.

3.7. Job’s Plot Curve and Recognition Mechanism

To further investigate the coordination mode between P2 and Zn²⁺, test solutions were prepared with a total concentration of 200 μM and a [Zn²⁺]/([Zn²⁺] + [P2]) ratio ranging from 0 to 0.9. Fluorescence spectroscopy measurements were conducted, and the fluorescence intensity at 462 nm was plotted to create a Job’s plot, as shown in Figure 6. The plot reveals that the fluorescence intensity was maximized when the coordination ratio of Zn²⁺ to the probe was 1:1. Based on this observation, it can be inferred that the complexation mechanism involves Zn²⁺ coordinating with the oxygen of the phenolic hydroxyl group, the nitrogen of the imine group, and the carbonyl group in the antipyrine structure within the probe molecule, leading to the formation of a complex with both six-membered and five-membered ring structures. In this process, on the one hand, the PET effect and ESIPT effect of P2 are simultaneously inhibited; on the other hand, the coordination of the probe with metal ions results in the Schiff base double bond being unable to rotate freely, thus inhibiting E/Z isomerization. Based on the above-mentioned synergistic effect, after the probe is excited, energy is released through fluorescence emission, which results in an enhancement in fluorescence (Figure 7).

3.8. Practical Application

The probe P2 was utilized for the determination of Zn²⁺ in real water samples. Tap water, Ganjiang river water, and rainwater were gathered and pretreated with the methods described in the literature [36,37]. The introduction of exogenous Zn²⁺ (100 μM) into these samples led to a substantial increase in fluorescent intensity.

Table 2. Study of the detection of Zn²⁺ in real water samples.

Samples	pH	Added Amount (μM)	Found Amount (μM)	Std Dev (μM)	RSD (%, n = 3)	Recovery (%, n = 3)
Tap water	6.47	100.00	95.72	0.35	0.91	95.72
Ganjiang river water	7.22	100.00	87.77	0.54	1.02	87.77
Rainwater	5.84	100.00	96.30	0.36	0.88	96.30

Experimental conditions: Zn²⁺, 100 μM; concentration of P2, 100 μM; incubation time: 3 min. The pH values of water samples were determined by pH meter.

illustrates that the recovery rates for tap water and rainwater were 95.72% and 96.30%, respectively, with corresponding relative standard deviations of 0.88% and 0.72%. These findings indicate that the fluorescence probe P2 holds promise for the monitoring of Zn²⁺ levels in real water samples.

4. Conclusions

In summary, this work developed an imine-based fluorescent sensor built on the antipyrine scaffold for the selective detection of Zn²⁺ in ethanol/water mixtures. In the presence of Zn²⁺, the probe P2 achieves a fluorescence turn-on through the inhibition of photoinduced electron transfer (PET), excited-state intramolecular proton transfer (ESIPT), and E/Z isomerization, along with the activation of chelation-enhanced fluorescence emission (CHEF). The probe can specifically identify Zn²⁺ in the presence of 13 common interfering cations, offering a broad linear detection range and a low detection limit. It was successfully applied to the quantitative determination of Zn²⁺ in real water samples.