A Highly Selective and Sensitive Sequential Recognition Probe Zn²⁺ and H₂PO₄⁻ Based on Chiral Thiourea Schiff Base

Abstract

A series of novel chiral thiourea fluorescent probes HL₁–HL₆ were designed and synthesized from (1R,2R)-1,2-diphenylethylenediamine, phenyl isothiocyanate, and different substituted salicylic aldehydes. All of the compounds were confirmed by ¹H NMR, ¹³C NMR, and HRMS. They exhibit high selectivity and sensitivity to Zn²⁺ in the presence of nitrate ions with the detection limit of 2.3 × 10⁻⁸ M (HL₅). Meanwhile, their zinc (II) complexes (L-ZnNO₃) showed continuous response to H₂PO₄⁻ in acetonitrile solution. The identification processes could further be verified by supramolecular chemistry data analysis, X-ray single-crystal diffraction analysis, and theoretical study. The research provides reliable evidence for an explanation of the mechanism of action of thiourea involved in coordination, which is important for the application of thiourea fluorescent probes. In short, the sensors HL₁–HL₆ based on chiral thiourea Schiff base will be promising detection devices for Zn²⁺ and H₂PO₄⁻.

1. Introduction

In recent years, widespread research has been undertaken on the detection of metal ions [1,2,3,4,5] and inorganic anions [6,7,8,9], particularly within the development of selective ion probes, which has more generally become a popular topic in scientific research. Zn²⁺ is the second most common element in the human body, after iron, and not only plays an important role in protein structure, catalysis, transcription, and regulation, but is also closely associated with nerve signal transmission, enzyme regulation, and gene expression [10]. Excessive concentrations of Zn²⁺ can cause neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease [11,12,13,14]. Therefore, the development of methods to accurately determine trace amounts of Zn²⁺ and for its removal prior to entering the body are important research goals.

Among the various anions, the detection of phosphates most commonly the subject of research. Phosphates are the key substrates of many biochemical reactions and the main components of biomolecules [15,16,17,18]. Phosphate and its derivatives are important nutrients, and the basic life activities of organisms are closely related to phosphate. Phosphate is not only a component of some important structures of plants but is also a catalyst for many key biochemical reactions in plants, which can promote the development of plant roots and the early growth of seedlings. The life activities of organisms are also closely related to phosphates [18]. Phosphates can regulate the level of 1,25-(OH) 2-vitamin D in human plasma [19] and react with hydroxyl groups to form phosphate esters, thus allowing monomers to polymerize into relatively stable long chain skeletons (DNA and RNA skeletons) [20]. In addition, phosphate is also involved in ATP energy supply, ion channel regulation, enzymatic reactions, and intercellular signal transduction [8]. Unfortunately, a large amount of phosphate deposition can lead to a lack of dissolved oxygen in water and algae eutrophication [18].

At present, there are many conventional methods to detect Zn²⁺ ions including ion chromatography, atomic absorption spectrometry, and ion exchange [21]. Among these methods, chemical sensors are widely used to detect many biological and environmental heavy metal pollutants because of their advantages such as higher sensitivity, lower cost, and faster detection [22,23]. In particular, the multi-functional sensors used for the sequential recognition of cations and anions have remarkable advantages in special applications for their low cost, quick response, and convenient operation. Most of the currently available fluorescent probes can only analyze certain types of anions or cations, and there are few fluorescent sensors with the sequential recognition of Zn²⁺ and H₂PO₄⁻ [24,25].

In this work, a series of chiral thiourea Schiff base fluorescent probes (HL₁–HL₆) were designed and synthesized starting from (1R,2R)-1,2-diphenylethylenediamine, phenyl isothiocyanate, and different substituted salicylic aldehydes (Figure 1). They were explored for the first time for fluorescent-responsive Zn²⁺ detection in the presence of nitrate ions in acetonitrile. Meanwhile, their zinc (II) complexes (HL-Zn²⁺-NO₃⁻) showed continuous response to H₂PO₄⁻ in acetonitrile solution. Up to now, the detection mechanisms of thiourea probes have been reported mostly by ¹H NMR titration or Job’s plot titration experiments to speculate the possible coordination mode, and some of the assays do not provide practical and reliable data [26,27]. Here, we successfully elucidated the mechanism of metal ion recognition by this kind of thiourea Schiff base fluorescent probe using supramolecular chemistry data analysis, X-ray single-crystal diffraction analysis, and theoretical study. The research provides reliable evidence for an explanation of the mechanism of action of thiourea involved in coordination, which is important for the application of thiourea fluorescent probes. The probes in our work have the advantages of high sensitivity and low detection limits. In short, the sensors HL₁–HL₆ based on chiral thiourea Schiff base will be promising detection devices for Zn²⁺ and H₂PO₄⁻.

2. Results and Discussion

2.1. Fluorescence Spectroscopic Studies of HL₁–HL₆ and 3 in the Presence of Metal Ions

2.1.1. Cation Selectivity Experiments

Selectivity experiments were carried out for HL₁–HL₆ and 3 by examining the emission spectra in the presence of different metal ions (Ag⁺, Na⁺, K⁺, Co²⁺, Fe³⁺, Cu²⁺, Cd²⁺, Cr³⁺, Mg²⁺, Pb²⁺, Ni²⁺, Zn²⁺, Fe²⁺, Hg²⁺, Ca²⁺) in filtered Milli-Q water. As shown in Figure 2a, HL₁ displayed a weak emission peak at 447 nm in acetonitrile solution when excited at 380 nm. Upon the addition of various metal ions in HL₁, the emission spectrum was only significantly enhanced with Zn²⁺, whereas Cd²⁺ and Pb²⁺ caused a very small enhancement in the emission intensity at wavelengths corresponding to 400 and 600 nm, respectively. This observation demonstrates the high selectivity of HL₁ towards Zn²⁺ compared to other metal ions, including Cd²⁺ and Pb²⁺. Molecular probes that show turn-on fluorescence signaling upon interaction with Zn²⁺ also often interact with Cd²⁺/Pb²⁺, resulting in turn-on signaling [28]. In contrast, blue fluorescence was observed in the probe solution upon interaction with Zn²⁺ under a 365 nm UV lamp (Figure 2a, inset). In the case of other metal cations, the fluorescence spectra almost remained unchanged (Figure 2a). Subsequently, the selectivity of the HL₁–HL₆ and 3 probes for various metal ions was investigated (Figures S1 and S2). The addition of Zn²⁺ resulted in a prominent luminescence enhancement (Figures S1 and S2), whereas the addition of a large excess of other competitive cations caused only slight luminescent changes (Figure S2), with the exception of 3 (Figures S1f and S2g). The fluorescence emission produced by the interaction between HL₅ and Zn²⁺ was the strongest, whereas that with HL₂ was the weakest. The strength of the other observed emissions was at a medium level, except for compound 3. The difference in the fluorescence intensity of other compounds mainly depends on the difference in the side substituents of salicylaldehyde. The fluorescence intensity of HL₅ is better than that of HL₃, and obviously stronger than that of HL₄, HL₆, and HL₂. When salicylaldehyde contains electron-donating groups, such as –OH, –CH₃, and –OCH₃, it can be increased the density of the electron cloud on the benzene ring, resulting in the weakening of the coordination ability of HL with zinc ions. Under the combined influence of the inductive effect and mesomeric effect, HL₅ containing –Cl has the strongest coordination ability with Zn²⁺. Compound 3 without the –OH in the benzene ring structure of the benzylidene group was not able to recognize Zn²⁺, which demonstrates that the phenolic hydroxyl group was involved in the formation of complexes. HL₁–HL₆ probes showed high selectivity in sensing Zn²⁺ and can be used as “turn-on” luminescent molecular probes toward Zn²⁺.

2.1.2. Cation-Competitive Experiments

The competitive experiments between the HL₁–HL₆ probes and coexisting metal ions were investigated on the basis of emission spectra. As shown in Figure 3a, whether in the absence or presence of competitive metal ions, the strong increase in the emission intensity of the HL₁ probe was observed upon the addition of Zn²⁺. Although Cd²⁺ and Pb²⁺, among the verified cations, triggered enhancements in emission, these were negligible compared to that of Zn²⁺. When HL₂–HL₆ were treated with 10 equiv. of other metal ions and 2 equiv. of Zn²⁺, similar luminescence changes were also observed. The effect was almost the same as that resulting from the addition of Zn²⁺ (Figure S3). In all, the coexistence of these cations had a negligible effect on the detection of Zn²⁺ in the tested media.

2.1.3. Study of the Reversibility and Dependence of Fluorescence on Solvent, pH, Different Zinc Salts

To examine whether the complexation of HL and Zn²⁺ is reversible, we added an aqueous solution of Na₂EDTA during detection. Upon the addition of Na₂EDTA (1.0 equiv.) to the solution of HL₁ containing Zn²⁺, the emission intensity of the mixture solution decreased significantly with the luminescence quenching.t It seems logical that the fluorescent intensity observed in Figure 4a is inversely proportional to the stability of the complex formed by L-Zn with the anions as the fluorescence of the complex can be quenched by Na₂EDTA [29]. Changes in the solvent and pH had non-negligible effects on the detection effect of the fluorescent probe. Therefore, we investigated the dependence of the fluorescence emission intensity on solvent and pH during the recognition of Zn²⁺ by HL₁. Interestingly, the intensity of the fluorescence emission spectrum in the acetonitrile solvent was significantly enhanced, and CH₃CH₂OH was also a good choice under some conditions (Figure 4b). Further investigation of the effect of pH on the detection of Zn²⁺ was carried out by adjusting the required pH using 0.01 M HCl or 0.01 M NaOH. The fluorescence emission intensity was stable within the pH range of 5.5–9.0, which includes physiological range basically (Figure 4c). Under strong acid or base conditions, the stability of the C=N bond and the presence of -OH can have a significant impact, resulting in fluorescence quenching. To judge whether the probe can selectively recognize Zn²⁺ in different zinc salts, an examination of the sensitivity of the L₁ probe to several different zinc salts (zinc chloride, zinc gluconate, and zinc nitrate) was undertaken using fluorescence spectroscopy. However, the fluorescence spectrum enhancement of the L₁ probe was not obvious when it was complexed with zinc chloride and zinc gluconate, and the fluorescence enhancement phenomenon only appeared when it interacted with zinc nitrate (Figure 4d). Therefore, we preliminarily speculate that the HL probe recognizes Zn²⁺ in the presence of NO₃⁻, which is involved in the coordination process.

2.1.4. Zn²⁺ Titration Analysis

Fluorescence titration spectra were examined to obtain additional information regarding the binding form of the HL₁ probe with Zn²⁺ (Figure 3b). After adding Zn²⁺ to the solution, HL₁ exhibited a broad emission profile peaking at 447 nm in acetonitrile solution and displayed a linear enhancement in the emission intensity. The increase in the intensity of the emission spectra stopped after the addition of 1 equivalent of metal ions, which suggested 1:1 binding stoichiometry between HL₁ and Zn²⁺ (Figure 3b, inset). In addition, the photoluminescence titration experiments of individual complexes of HL₂–HL₆ with Zn²⁺ in varying concentrations were carried out by monitoring the emission intensity changes. Similarly, the emission intensity of HL₂–HL₆ increased continuously until the addition of 1 equiv. of Zn²⁺; the further addition of Zn²⁺ induced only minor changes in the luminescence spectra (Figure S4).

2.2. Binding Mechanism

According to literature reports [30,31,32], the supramolecular chemical data analysis method was adopted to analyze the combination of host and guest, and the binding constants of host and guest 1:1, 1:2, and 2:1 were simulated, respectively. The results are shown in

Table 1. Job plot shapes of HL₁ at various concentrations and K^1:1, K^1:2 to K^2:1 ratios. K^1:1 = 1000, K^1:2 = 100, K^2:1 = 100 were assumed.

Parameter (Bounds)	Optimized	Error	Initial
K (0 → ∞)	8607.30 M−1	±9.1006%	100.00 M−1
K11 (0 → ∞)	3376.76 M−1	±5.2092%	1000.00 M−1
K12 (0 → ∞)	4790.08 M−1	±26.9494%	100.00 M−1
K11 (0 → ∞)	3576.53 M−1	±9.1090%	1000.00 M−1
K21 (0 → ∞)	−277,004.73 M−1	±−4.6365%	100.00 M−1

. The detection limit of HL₁ for Zn²⁺ was calculated as 3σ/k, where σ is the standard deviation of the blank measurement and k is the slope of the plot of the emission intensity ratio versus Zn²⁺ concentration [33]. The limit of detection (LOD) of HL₂–HL₆ for Zn²⁺ are 2.8 × 10⁻⁸ M, 2.2 × 10⁻⁶ M, 3.3 × 10⁻⁸ M, 1.4 × 10⁻⁷ M, 2.3 × 10⁻⁸ M, and 1.74 × 10⁻⁷ M, respectively. The fluorescence intensity of the HL probe had a good linear relationship with the concentration of Zn(NO₃)₂ and had a lower detection limit, which could be used for the quantitative detection of Zn(NO₃)₂.

To further confirm the coordination mechanism of ligand HL with zinc nitrate, the crystals were obtained and analyzed. Single crystals of HL₄ (CCDC: 2077389) and L₄-ZnNO₃ (CCDC: 2077390) suitable for X-ray diffraction study were obtained by the slow evaporation of mixed solutions of acetonitrile/methanol at room temperature. L₄-ZnNO₃ was cultured in proportions of equal amounts and an excessive amount of Zn(NO₃)₂, and the complex single crystal with a coordination mode of 1:1 was obtained. The ellipsoid diagrams of HL₄ and L₄-ZnNO₃ shown in Figure 5 were obtained by X-ray single crystal diffraction analysis. The corresponding crystallographic data are summarized in Table S1, and selected bond lengths and angles are listed in Table S2. The single crystal of the complex L₄-ZnNO₃ clearly showed the formation of a six-membered ring and a seven-membered ring. The central Zn atom adopted a four-coordination method, which was, respectively, connected to the S atom (Zn-S = 2.314(16) Å), the N atom of C=N (Zn-N3 = 2.020(5) Å), the O atom of the salicylaldehyde phenolic hydroxyl group (Zn-O1 = 1.946(5) Å), and the O atom of NO₃ (Zn-O3 = 2.043(5) Å). This coordination mode is consistent with the coordination mode of monodentate ligands reported in the literature [34,35,36]. The crystal structure indicates that the N atom on the thiourea group was not coordinated, and that only the S atom is involved.

Coordination mechanism studies confirm that HL and Zn(NO₃)₂ form strongly fluorescent complexes, which suggests 1:1 binding stoichiometry between HL and Zn(NO₃)₂. Through the single-crystal structure of the complex, it can be visually seen that the atoms coordinated with Zn²⁺ include O atoms provided by phenolic hydroxyl groups, N atoms provided by Schiff bases, and S atoms provided by thiourea structural units. Zn²⁺ with HL forms a six-membered ring and a seven-membered ring with coordination atoms.

2.3. Theoretical Study

Density Functional Theory (DFT) calculations and time-dependent DFT (TD-DFT) using B3LYP were carried out with Gaussian 09 software package ADDIN EN.CITE [37]. The 6-31G(d,p) basis set was employed for C, H, N, O, and S atoms, and the LANL2DZ basis set was employed for the Zn atom [38,39]. Full geometry optimizations of the HL₄ and L₄-ZnNO₃ in the singlet ground-state were carried out using the DFT method (Figure 6). The assignment of the type of each MO is made on the basis of its composition. The frontier molecular orbital results from the extension geometries show that the spatial distributions of the HOMO (HOMO = highest occupied molecular orbital) and LUMO (LUMO = lowest unoccupied molecular orbital) are both localized with the phenol moiety with a bandgap of 4.243 eV. However, after the complexation of HL₄ with Zn²⁺, the HOMOs of L₄-ZnNO₃ were spread over the phenol moiety, and the LUMOs extended to the Zn²⁺ and thiourea moiety with the bandgap of 3.732 eV, which indicates the possible electron transfer from ligand to metal (LMCT). The theoretical bond length between S and O was 5.12262 Å before adding Zn²⁺, and was shortened to 4.05971 Å after forming a complex with HL₄, which was consistent with the change in the crystal data.

2.4. Fluorescence Studies of L-ZnNO₃ in the Presence of Anions

2.4.1. Anion Selectivity Experiments

Different zinc salt selectivity experiments revealed that different kinds of anions could affect the Zn²⁺ recognition performance. To investigate the effect of different kinds of anions on zinc complexes, anion selectivity experiments were performed. When excited at 377 nm, L₁-ZnNO₃ displayed a strong emission peak at 449 nm. Among a set of different anions (Cl⁻, Br⁻, F⁻, CH₃COO⁻, NO₃⁻, C₆H₅COO⁻, C₆H₅SO₂⁻, PO₄²⁻, H₂PO₄⁻, HPO₄²⁻, SO₃²⁻, SO₄²⁻, H₂PO₂ ⁻, NO₂⁻, NO₃⁻, and SCN⁻), a prominent change in the emission spectra of L₁-ZnNO₃ manifested upon the addition of H₂PO₄⁻ alone (Figure 7). When L₁-ZnNO₃ was combined with H₂PO₄⁻, the bright blue color of the solution disappeared, resulting in a colorless transparent solution (Figure 7, insert and Figure S5). A similar phenomenon also occurred when adding H₂PO₄⁻ to L₂-ZnNO_3, L₃-ZnNO₃, L₄-ZnNO₃, L₅-ZnNO₃, and L₆-ZnNO₃ (Figure S6). The above experimental results proved that this type of fluorescent probe could realize the “turn-on–turn-off” behavior, thus realizing the recognition function of cation and anion.

2.4.2. Anion-Competitive Experiments

To verify the influence of other anions on the probe recognition of H₂PO₄⁻, the emission intensity of the probe and H₂PO₄⁻ at 450 nm (λ_ex = 370 nm) was monitored (Figure 8a). The result showed that the L₁-ZnNO₃ could specifically detect H₂PO₄⁻ in the presence of other related anions. The anion-competitive experiments of L₂-ZnNO_3, L₃-ZnNO₃, L₄-ZnNO₃, L₅-ZnNO₃, and L₆-ZnNO₃ are shown in the Supplementary Materials (Figure S7), which were generally consistent with L₁-ZnNO₃.

The titration of L₁-ZnNO₃ with H₂PO₄⁻ triggered a distinct decrease in the emission intensity at 450 nm (Figure 8b). L₂-ZnNO₃~L₆-ZnNO₃ and H₂PO₄⁻ are exhibited in the Supplementary Materials (Figure S8).

In this work, a series of novel chiral thiourea fluorescent probes HL₁–HL₆ were prepared for the first time for fluorescence-responsive Zn²⁺, while their complex L-Zn-NO₃ was continuously identified and detected the phosphate anion (H₂PO₄⁻). Compared with other working probes for the detection of analytes (Table S4) [7,40,41,42,43,44,45,46], the probes synthesized in this work have the advantages of high sensitivity and low detection limits.

3. Materials and Methods

3.1. General Procedures

3.1.1. Reagents and Instruments

All of the materials used for synthesis were purchased from commercial suppliers and used as received. Nitrate salts of Ag⁺, Na⁺, K⁺, Co²⁺, Fe³⁺, Cu²⁺, Cd²⁺, Cr³⁺, Mg²⁺, Pb²⁺, Ni²⁺, and Zn²⁺, sulfate salts of Fe²⁺ and Hg²⁺, and chlorine salt of Ca²⁺ were used for the spectroscopic studies. The different sodium salts of F⁻, Cl⁻, Br⁻, CH₃COO⁻, C₆H₅COO⁻, C₆H₅SO₂⁻, PO₄³⁻, H₂PO₄⁻, HPO₄²⁻, SO₃²⁻, SO₄²⁻, H₂PO₂⁻, NO₂⁻, NO₃⁻, and SCN⁻ were used for investigating anion effects. All salts were dissolved in filtered Milli-Q water to prepare aqueous ion solutions.

The melting points of the products were determined on an X-4 binocular microscope. ¹H and ¹³C NMR spectra were recorded on a Bruker 400 MHz instrument at room temperature. Chemical shifts were measured relative to residual solvent peaks of CDCl₃ (¹H: δ = 7.26 ppm; ¹³C: δ = 77.0 ppm) or DMSO-d₆ (¹H: δ = 2.50 ppm; ¹³C: δ = 39.5 ppm) with tetramethylsilane (TMS) as internal standard. The following abbreviations are used to describe spin multiplicities in ¹H NMR spectra: s = singlet; d = doublet; t = triplet; m = multiplet. Specific rotations were measured on a PerkinElmer 341 MC polarimeter. HRMS data were obtained using a 7.0T FT-ICR MS spectrometer. X-ray crystal diffraction measurements of L were acquired using a Bruker SMART APEX II CCD diffractometer. Fluorescence spectra were examined on a Hitachi FL-2700 spectrophotometer using 10 mm path length quartz cuvettes at 298 K. The pH was calculated by STARTER 3100/F.

3.1.2. Synthesis of HL₁–HL₆ and Compound 3

The synthetic route for HL₁–HL₆ and compound 3 is shown in Figure 1. Under ice-cold conditions, (1R,2R)-1,2-diphenylethylenediamine (1, 4.71 mmol) was dissolved in dichloromethane under ice-cold conditions with constant stirring. Isothiocyanate (4.71 mmol) was dissolved in dichloromethane and then added dropwise to the above amine system via a constant pressure dropping funnel. The reaction mixture was stirred for 12 h and then evaporated under low pressure. The crude product was purified by silica gel column chromatography using dichloromethane as an eluent to give the chiral primary amine thiourea 2 in the form of a white powder. Compound 2 and the substituted aldehydes were dissolved in an ethanol solvent and heated to reflux under nitrogen protection for 24 h. The mixture was cooled and washed with ether (3 × 50 mL) to give the solid products HL₁–HL₆ and compound 3.

1-((1R,2R)-2-(((E)-2-Hydroxybenzylidene)amino)-1,2-diphenylethyl)-3-phenylthiourea (HL₁). White solid, 70% yield, m.p. 104–108 °C; ${\left[\alpha \right]}_{\mathrm{D}}^{23}$ = +33.44 (c = 10, CH₂Cl₂); ¹H NMR (CDCl₃, 400 MHz) δ 12.54–12.23 (m, 1H), 8.06 (s, 1H), 7.69 (s, 1H), 7.48–7.28 (m, 7H), 7.24–6.74 (m, 13H), 5.99 (s, 1H), 4.81 (d, J = 4.1 Hz, 1H); ¹³C NMR (CDCl₃, 101 MHz) δ 180.5, 167.2, 160.6, 138.6, 138.2, 137.4, 135.7, 133.0, 132.0, 130.3, 129.3, 128.5, 128.3, 127.9, 127.6, 127.3, 127.2, 126.3, 125.4, 124.0, 118.9, 118.4, 117.0, 64.2, 37.1, 34.4, 32.7, 31.9, 30.1, 29.7, 29.4, 26.9, 22.7, 14.1, 11.4; HRMS (ESI) m/z calc’d for C₂₈H₂₅N₃OS [M + H]⁺: 452.1797, found 452.1790.

1-((1R,2R)-2-(((E)-2,3-Dihydroxybenzylidene)amino)-1,2-diphenylethyl-3-phenylthiourea (HL₂). Yellow solid, 48% yield, m.p. 146–149 °C; ${\left[\alpha \right]}_{\mathrm{D}}^{23}$ = +36.61 (c = 10, CH₂Cl₂); ¹H NMR (DMSO-d_6, 400 MHz) δ 13.20 (d, J = 155.7 Hz, 1H), 9.88 (s, 1H), 9.12 (s, 1H), 8.75–8.36 (m, 2H), 7.59–6.54 (m, 18H), 6.11 (t, J = 8.3 Hz, 1H), 5.15–4.87 (m, 1H); ¹³C NMR (DMSO-d_6, 101 MHz) δ 180.4, 166.9, 149.1, 145.5, 140.2, 139.8, 139.4, 128.7, 128.4 (d, J = 10.9 Hz), 128.3, 127.8, 127.6, 127.3, 126.9, 126.7, 123.9, 122.6, 121.9, 118.8, 118.4, 118.3, 76.5, 62.8; HRMS (ESI) m/z calc’d for C₂₈H₂₅N₃O₂S [M + H]⁺: 468.1746, found 468.1749.

1-((1R,2R)-2-(((E)-2-Hydroxy-5-methylbenzylidene)amino)-1,2-diphenylethyl)-3-phe-nylthiourea (HL₃). White solid, 81% yield, m.p. 139–142 °C; ${\left[\alpha \right]}_{\mathrm{D}}^{23}$ = +42.78 (c = 10, CH₂Cl₂); ¹H NMR (DMSO-d₆, 400 MHz) δ 12.45 (s, 1H), 9.75 (s, 1H), 8.48–8.16 (m, 2H), 7.39–7.03 (m, 17H), 6.82 (d, J = 8.3 Hz, 1H), 6.14 (t, J = 7.6 Hz, 1H), 4.93 (d, J = 6.5 Hz, 1H), 2.22 (s, 3H); ¹³C NMR (DMSO-d₆, 101 MHz) δ 178.1, 164.0, 155.3, 138.1, 137.7, 136.8, 131.1, 129.1, 126.2, 126.0, 125.6, 125.3, 125.2, 125.2, 125.0, 124.6, 121.9, 120.6, 116.2, 114.0, 74.3, 60.5, 17.6; HRMS (ESI) m/z calc’d for C₂₉H₂₇N₃OS [M + H]⁺: 466.1953, found 466.1958.

1-((1R,2R)-2-(((E)-2-Hydroxy-3-methoxybenzylidene)amino)-1,2-diphenylethyl)-3-p-henylthiourea (HL₄). Brown solid, 84% yield, m.p. 150–152 °C; ${\left[\alpha \right]}_{\mathrm{D}}^{23}$ = +48.81 (c = 10, CH₂Cl₂); ¹H NMR (DMSO-d₆, 400 MHz) δ 12.55 (s, 1H), 9.35 (s, 1H), 8.27–7.34 (m, 2H), 7.23–6.53 (m, 17H), 6.44 (d, J = 7.9 Hz, 1H), 5.74 (s, 1H), 4.55 (d, J = 6.6 Hz, 1H), 3.39 (s, 3H); ¹³C NMR (DMSO-d₆, 101 MHz) δ 180.4, 166.6, 150.1, 147.8, 140.2, 139.9, 139.1, 128.5, 128.3, 127.9, 127.6, 127.5, 127.4, 126.9, 124.1, 123.0, 122.8, 118.6, 118.4, 115.0, 76.3, 62.7, 55.8; HRMS (ESI) m/z calc’d for C₂₉H₂₇N₃O₂S [M + H]⁺: 482.1902, found 482.1907.

1-((1R,2R)-2-(((E)-5-Chloro-2-hydroxybenzylidene)amino)-1,2-diphenylethyl)-3-phe-nylthiourea (HL₅). Yellow solid, 80% yield, m.p. 158–160 °C; ${\left[\alpha \right]}_{\mathrm{D}}^{23}$ = +37.11 (c = 10, CH₂Cl₂); ¹H NMR (DMSO-d₆, 400 MHz) δ 12.58 (s, 1H), 9.73 (s, 1H), 8.30 (d, J = 8.0 Hz, 2H), 7.54 (s, 1H), 7.40–7.14 (m, 16H), 7.09 (d, J = 7.2 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 6.14 (s, 1H), 4.93 (d, J = 6.3 Hz, 1H); ¹³C NMR (DMSO-d₆, 101 MHz) δ 180.5, 164.3, 158.4, 140.2, 132.3, 129.9, 128.5, 128.3, 127.9, 127.6, 127.5, 127.4, 127.0, 123.0, 122.4, 120.3, 118.5, 76.5, 62.7; HRMS (ESI) m/z calc’d for C₂₈H₂₄ClN₃OS [M + H]⁺: 482.1407, found 486.1412.

1-((1R,2R)-2-(((E)-(2-Hydroxynaphthalen-1-yl)methylene)amino)-1,2-diphenylethyl)-3-phenylthiourea (HL₆). Yellow solid, 74% yield, m.p. 154–156 °C; ${\left[\alpha \right]}_{\mathrm{D}}^{23}$ = +46.22 (c = 10, CH₂Cl₂); ¹H NMR (DMSO-d₆, 400 MHz) δ 14.92 (s, 1H), 9.65 (s, 1H), 9.25 (s, 1H), 8.42 (s, 1H), 8.04 (s, 1H), 7.93–7.71 (m, 2H), 7.49 (s, 1H), 7.36–7.06 (m, 15H), 6.96 (d, J = 9.2 Hz, 2H), 6.22 (s, 1H), 5.24 (s, 1H); ¹³C NMR (DMSO-d₆, 101 MHz) δ 180. 4, 161.3, 139.3, 138.9, 135.9, 133.1, 129.0, 128. 5, 128.0, 127. 9, 127.8, 127.6, 127.5, 127.2, 126.3, 122.9, 123.0, 122.2, 119.2, 107.2, 72.7; HRMS (ESI) m/z calc’d for C₃₂H₂₇N₃OS [M + H]⁺: 502.1953, found 502.1959.

1-((1R,2R)-2-(((E)-Benzylidene)amino)-1,2-diphenylethyl)-3-phenylthiourea (3). White solid, 76% yield, m.p. 175–179 °C; ${\left[\alpha \right]}_{\mathrm{D}}^{23}$ = +27.33 (c = 10, CH₂Cl₂); ¹H NMR (CDCl₃, 400 MHz) δ 7.99–7.76 (m, 2H), 7.73–7.27 (m, 16H), 7.26–7.02 (m, 5H), 5.92 (s, 1H), 4.60 (s, 1H); ¹³C NMR (CDCl₃, 101 MHz) δ 180.4, 162.9, 141.2, 140.5 (d, J = 65.6 Hz), 135.3, 131.2, 130.2, 128.5, 128.4, 128.3, 127.6, 127.4, 127.2, 126.7, 126.1, 77.3, 64.9; HRMS (ESI) m/z calc’d for C₂₈H₂₅N₃S [M + H]⁺: 436.1847, found 436.1841.

3.2. Spectroscopic Measurements

Stock solutions of various ions (0.50 mol/L) were prepared in filtered Milli-Q water. A stock solution of HL probe (1.0 × 10⁻² mol/L) and compound 3 in acetonitrile was freshly prepared for fluorescence measurement. Fluorescence spectroscopic measurements were recorded for samples at 1 min after the addition of various analytes. All spectroscopic measurements were performed at 25 °C. For fluorescence measurements, the excitation wavelength was fixed at 380 nm (λ_ex = 380 nm) and 357 nm for HL₁ and HL₂, and at 390 nm (λ_ex = 390 nm) for HL₃–HL₆ and compound 3, respectively. The emission wavelength was recorded from 220 to 600 nm for HL₁–HL₆ and compound 3. In the cation selectivity experiments, the test samples were prepared by interacting 20 μL of the cation stock (0.50 mol/L) with 1 mL of HL solution (1.0 × 10⁻⁴ mol/L) and compound 3. In the presence of other metal ions, the competition between Zn²⁺ and other metal ions was systematically studied by fluorescence emission spectroscopy. In the titration experiments, the solution of HL (1.0 × 10⁻⁵ mol/L) was placed in a quartz cuvette, and a certain volume of the metal ion stock solution (5.0 × 10⁻⁴ mol/L) was added gradually to achieve a concentration of 1.0 × 10⁻⁵ mol/L. Similarly, anion experiments were conducted based on L-ZnNO₃ to study the fluorescence spectra after combination with different anions.

3.3. Synthesis of the Sensor Zn(II) Complex and X-ray Crystallography

A methanol solution (2 mL) of Zn(NO₃)₂·6H₂O (0.0297 g, 1 equiv.) was added into the round bottom flask containing acetonitrile (3 mL) and compound HL₄ (0.0482 g, 0.1 mmol). Then, it was sealed with unsintered polytetrafluoroethylene (PTFE) tapes and holes were pricked in the tape with a needle. The crystals of L₄-ZnNO₃ were obtained after the solvent slowly evaporated at room temperature.

The crystal structures were determined using a Bruker SMART APEX II CCD diffractometer with a monochromator and Cu Kα radiation (λ = 0.71073 Å) at 114(2) or 146.4(3) K with an increasing ω (width of 0.3° per frame) at a scan speed of 5 s per frame. Using Olex2 [47], the structure was solved with the ShelXT [48] structure solution program using Direct Methods and refined with the ShelXL [49] refinement package using least squares minimization. All non-hydrogen atoms were refined anisotropically and the hydrogen atoms attached to all carbon atoms were geometrically fixed. The positional and temperature factors were refined isotropically.

4. Conclusions

Chiral thiourea Schiff base compounds were synthesized and their binding properties with cations were studied. The spectral data showed that the HL₁–HL₆ probes have high selectivity and sensitivity to Zn²⁺ and are not affected by the presence of other coexisting metal ions. Combined with the crystal data, it was determined that the HL and Zn(NO₃)₂ form a L-ZnNO₃ complex in a 1:1 ratio. The formed L-ZnNO₃ complex can selectively recognize H₂PO₄⁻ without interference from other anions. The probe has good prospects for application and provides a new approach for the detection of Zn(NO₃)₂ and H₂PO₄⁻ in actual samples.