Coumarin-Based Dual Chemosensor for Colorimetric and Fluorescent Detection of Cu²⁺ in Water Media

Abstract

A novel coumarin derivative (5) was synthesized and used as a colorimetric and fluorescent probe for selective detection of Cu²⁺ ions in the presence of other metal ions, with the detection limits of 5.7 and 4.0 ppb, respectively. Cu²⁺ ion reacts with probe 5 to form a 1:1 stoichiometry complex, resulting in a remarkable redshift of absorption maximum from 460 to 510 nm, as well as almost completely quenching fluorescence intensity of probe 5 at the wavelength of 536 nm. These changes can be distinctly observed by naked eyes. In addition, the working pH range of probe 5 is wide and suitable for physiological conditions, thus probe 5 may be used for detection of Cu²⁺ ions in living cells. The stable structures of probe 5 and its 1:1 complex with Cu²⁺ ion were optimized at the PBE0/6-31+G(d) level of theory. The presence and characteristics of bonds in compounds were studied through atoms in a molecule and natural bond orbital analysis. The formation of the complex led to a strong transfer of electron density from probe 5 as a ligand to Cu²⁺ ion, resulting in breaking the π-electron conjugated system, which is the cause of fluorescence quenching and color change of 5-Cu²⁺ complex.

1. Introduction

Copper is both an essential trace element and a toxic substance to plants, animals, and humans.^1,2 Copper toxicosis is very rare compared to its deficiency in plants.³ This element exhibits high toxicity to microorganisms such as algae, bacteria, viruses, and aquatic animals.⁴ Copper is present in almost all tissues of the human body and plays an important role in physiological processes, immunity, and resistance to oxidative stress.⁵⁻⁷ The copper deficiency or copper overload is believed to be related to diseases like Alzheimer’s, Parkinsons, Menkes, Wilsons, and Prion.⁸⁻¹¹

There are different methods that can determine Cu²⁺ ions at ppb levels such as absorption atomic spectrometry (AAS),^12,13 inductively coupled plasma mass spectrometry (ICP-MS),¹⁴ inductively coupled plasma optical emission spectrometry (ICP-OES),¹⁵ and voltammetry.¹⁶ However, these methods require expensive equipment, with complicated sample preparation and measurement techniques. Meanwhile, a fluorescence-based analytical method can also determine Cu²⁺ ions at ppb levels, which is simpler and less expensive than the above methods.^17,18 Furthermore, a fluorescence-based analytical method can be used to monitor substances in living cells to detect abnormal cases and causes of diseases.¹⁹⁻²¹ Therefore, the development of new fluorescent probes for Cu²⁺ ions has attracted special attention of scientists.²²⁻²⁴ So far, many fluorescent probes for Cu²⁺ ions have been reported. The development of new fluorescent probes is, however, still being concerned due to the limitations of reported probes such as low sensitivity and selectivity,^25,26 a narrow range of pH,^20,27−29 working in solutions with a high proportion of organic solvents,^26,30 and excitation wavelength in the ultraviolet region.^26,28,31,32 These limitations reduce the application of probes to monitor Cu²⁺ ions in living cells.

In this study, a novel derivative of coumarin, (E)-3-((2-(benzo[d]thiazol-2-yl)hydrazono)methyl)-7-(diethylamino) coumarin (5) that was successfully synthesized could be used as a colorimetric and fluorescent probe for detection of Cu²⁺ ions in the preeminent operating conditions such as high sensitivity and selectivity, a wide range of pH, and excitation wavelength in the visible region. In addition, quantum chemical calculations have also been used to determine the optimized geometry of 5-Cu²⁺ complex and shed light on the cause of the changes in fluorescence properties.

2. Results and Discussion

2.1. Synthesis of (E)-3-((2-(Benzo[d]thiazol-2-yl)hydrazono)methyl)-7-(diethylamino)coumarin (5)

Probe 5 was synthesized through four steps as shown in Figure 1. First, 7-diethylamino-3-ethylacetate-coumarin (2) was synthesized from a condensation reaction between 4-diethylaminosalicylaldehyde (1) and diethylmalonate in the presence of triethylamine. Second, 7-diethylamino-coumarin (3) was obtained from the decarboxylation reaction of 2 using concentrated HCl and glacial acetic acid. Third, 7-diethylaminocoumarin-3-aldehyde (4) was obtained from the aldehyde reaction of 3 in the presence of POCl₃ and N,N-dimethylformamide (DMF). Finally, probe 5 was obtained from the condensation reaction of 7-diethylaminocoumarin-3-aldehyde (4) with 2-hydrazinobenzothiazole in about 30% overall yield (Supporting Information). The structures of the products 3, 4, 5, and intermediates were confirmed by ¹H NMR, ¹³C NMR, and electrospray ionization mass spectrometry (ESI-MS) analysis (Figures S1–S4, Supporting Information).

Figure 1.

Synthetic pathway for the probe 5.

2.2. Experimental Characterization and Application of Probe 5

The absorption and fluorescence titration spectra of probe 5 with Cu²⁺ ions in aqueous solution are presented in Figure 2. It shows that free probe 5 has a characteristic absorption band peaked at the wavelength of 460 nm in aqueous solution. The absorption maximum is progressively redshifted with gradual addition of Cu²⁺ ions. The absorption maximum at 460 nm is gradually decreased, whereas a new absorption maximum at 510 nm appears. In companion with this, there is a change in the color of the solution from orange to pink. Furthermore, three distinct isosbestic points are observed at 340, 400, and 475 nm. These results indicate that the concentration of the light-absorbing compounds has been converted back and forth in the solution. This is seen as a piece of evidence for the formation of a new compound between probe 5 and Cu²⁺ ions.³³⁻³⁵

Figure 2.

(a) Absorbance titration spectra of probe 5 (5 μM) in ethanol/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH 7.4, 1/1, v/v) at 25 °C with gradual addition of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, and 2.0 equiv of Cu²⁺ ions. (b) Fluorescence titration spectra of probe 5 (5 μM) in ethanol/HEPES (pH 7.4, 1/1, v/v) at 25 °C with gradual addition of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, and 2.0 equiv of Cu²⁺ ions with an excitation of 460 nm.

As shown in Figure 2, free probe 5 has a strong emission band peaked at the wavelength of 536 nm with an excitation wavelength of 460 nm. The fluorescence quantum yield (Φ) of free probe 5 in aqueous solution is determined to be 10.5%, using the fluorescein in 0.1 N NaOH solution as a reference.³⁶ Addition of Cu²⁺ ions greatly quenches the fluorescence intensity of probe 5, along with a slight redshift in the emission maximum. Upon addition of 1 equiv of Cu²⁺ ions to the solution of probe 5, the fluorescence intensity is quenched over 95% of its original intensity, along with a shift in the emission maximum from 536 to 545 nm. The fluorescence quantum yield (Φ) of the product between probe 5 and Cu²⁺ ions in aqueous solution is found to be 0.15% based on the obtained experimental data.

The Job plot in Figure 3a shows a complexation between probe 5 and Cu²⁺ ions in a molar ratio of 1:1. The stoichiometry is also confirmed by the plot of the fluorescence intensity of probe 5 with different concentrations of Cu²⁺ ions. Figure 3b shows that the fluorescence intensity is gradually quenched upon an increasing addition of Cu²⁺ ions. Upon addition of 1 equiv of Cu²⁺ ions, the fluorescence intensity is quenched to more than 95% and then becomes almost unchanged as the concentration of Cu²⁺ ions is further increased.

Figure 3.

(a) Job plot of probe 5 and Cu²⁺ ions. (b) Plot and linear fitting of the fluorescence intensity of probe 5 (5 μM) with different concentrations of Cu²⁺ ions (excitation wavelength: 460 nm, emission wavelength: 536 nm).

The selectivity of probe 5 toward Cu²⁺ ions compared to the other metal ions was investigated. Figure 4 gives the variations of absorption and fluorescence spectra of probe 5 caused by miscellaneous ions including Na⁺, K⁺, Ca²⁺, Mg²⁺, Fe³⁺, Co²⁺, Ni²⁺, Zn²⁺, Pb²⁺, Cd²⁺, Hg²⁺, and Cu²⁺ ions. The results show that Cu²⁺ ions cause a remarkable redshift in absorption maximum from 460 to 510 nm with increased intensity; Hg²⁺ ions induce a medium change from 460 to 500 nm, whereas other metal ions only produce slight changes in their absorption spectra (Figure 4a). On the other hand, probe 5 shows a strong emissive band peaked at 536 nm. Cu²⁺ ions can quench the fluorescence of probe 5 to more than 95%, whereas Hg²⁺ ions can quench fluorescence to about 40%. Other miscellaneous competitive ions do not lead to any significant changes in fluorescence (Figure 4b). The above changes in color and fluorescence can be distinctly observed by naked eyes, and their photos are presented in Figure 5. All these show a remarkably high selectivity of probe 5 for Cu²⁺ ions over other competitive ions except for Hg²⁺ ions.

Figure 4.

Absorption spectra (a) and fluorescence spectra (b) of probe 5 (5 μM) in ethanol/HEPES (pH 7.4, 1/1, v/v) at 25 °C upon addition of 1 equiv of Cu²⁺ ions and other metal ions including Na⁺, K⁺, Ca²⁺, Mg²⁺, Fe³⁺, Co²⁺, Ni²⁺, Zn²⁺, Pb²⁺, Cd²⁺, and Hg²⁺ ions (excitation wavelength: 460 nm, emission wavelength: 536 nm).

Figure 5.

Color changes (a) and fluorescence changes (b) of probe 5 with Na⁺, K⁺, Ca²⁺, Mg²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Pb²⁺, Cd²⁺, Cu²⁺, and Hg²⁺.

The possibility of using probe 5 to quantify Cu²⁺ ions in the presence of Hg²⁺ ions is also investigated. As shown in Figure 6a, the influence of Hg²⁺ ions on the quantification of Cu²⁺ ions can be prevented by adding 8–10 equiv of NaCl to the solution containing Hg²⁺ ions. Furthermore, when 10 equiv of Na₄ EDTA is added to the solution of probe 5 containing 1 equiv of Cu²⁺ ions, the solution returns to strong fluorescence with the same intensity as the solution of free probe 5 (Figure 6b). This result shows that the reaction between Cu²⁺ ions and probe 5 is reversible. Therefore, probe 5 acts as a chemosensor for Cu²⁺ ions.

Figure 6.

Fluorescence spectra of probe 5 (5 μM) in ethanol/HEPES (pH 7.4, 1/1, v/v) at 25 °C (a) upon addition of 1 equiv of Hg²⁺ ions and NaCl (0–50 μM) and (b) upon addition of 1 equiv of Cu²⁺ ions and EDTA (50 μM) (excitation wavelength: 460 nm).

For practical applicability, a proper range of pH values for probe 5 was also evaluated. Figure 7a shows that the absorbance of free probe 5 and of probe 5 in the presence of Cu²⁺ ions reaches a plateau in the pH range from 5 and 9. Under acidic conditions (pH < 5) or alkaline conditions (pH > 9), the absorbance of these solutions is not stable upon pH changes, it decreases as pH increases. A similar result also comes from the fluorescence spectra with the change of pH conditions, as presented in Figure 7b. These results show that probe 5 can detect Cu²⁺ ions with a wide pH span, from 5 to 9.

Figure 7.

(a) Absorption intensity at 510 nm and (b) fluorescence intensity at 536 nm of probe 5 (5 μM) in the absence and presence of 1 equiv of Cu²⁺ ions in ethanol/HEPES (pH 7.4, 1/1, v/v) solution.

The changes of absorbance and fluorescence intensity of probe 5 (5 μM, in ethanol/HEPES, pH 7.4, 1/1, v/v) after adding 1 equiv of Cu²⁺ ions with different times are investigated and presented in Figure 8. The results show that the complexation process between probe 5 with Cu²⁺ ions is very fast. It can reach the equilibrium within several seconds, indicating that probe 5 can be used for real-time detection of Cu²⁺ ions.

Figure 8.

(a) Changes of absorbance at 510 nm and (b) fluorescence intensity at 536 nm of probe 5 (5 μM) after adding 1 equiv of Cu²⁺ ions in ethanol/HEPES (pH 7.4, 1/1, v/v) with different times.

The possibility to use 5 as a chemosensor for quantification of Cu²⁺ ions by colorimetric and fluorescent methods was investigated. The results obtained from the absorption spectra show that the optimal absorption wavelength for quantification of Cu²⁺ ions is 525 nm, where the difference between absorbances is the largest, and the absorbance of Cu²⁺ ions is approximately equal to zero (Figure 9a). Figure 9b,c show that, in the concentration range of Cu²⁺ ions from 0 to 300 ppb, the linear relationships between the concentration of Cu²⁺ ions and the absorbance or fluorescence intensity of 5 solution (5 μM) are very good and expressed by the equations that were found from calibration curves: A_525 nm = (0.013 ± 0.002) + (0.001 ± 0.000) × [Cu²⁺] (ppb) or FI_536 nm = (967.793 ± 4.262) – (3.004 ± 0.029) × [Cu²⁺] (ppb). The calculated linear correlation coefficients are 0.9996 and 0.9996, respectively. These results indicate that 5 can be used as a colorimetric or fluorescent chemosensor for quantification of Cu²⁺ ions. The limit of detection is 5.7 ppb for the colorimetric method and 4.0 ppb for the fluorescent method (Figure S5), much lower than that for the recently published colorimetric and fluorescent sensors.³⁷⁻⁴⁰ The method of using probe 5 to quantify Cu²⁺ ions in the presence or absence of Hg²⁺ ions has been assessed for accuracy through the repeatability (relative standard deviation, RSD) and recovery rate. The obtained results were reliable (see more details in Supporting Information).

Figure 9.

(a) Absorption spectra of probe 5 (5 μM) and probe 5 (5 μM) + 1 equiv of Cu²⁺ ions in ethanol/HEPES (pH 7.4, 1/1, v/v). (b) Variation of absorbance at 525 nm of 5 solution (5 μM) in ethanol/HEPES (pH 7.4, 1/1, v/v) versus the concentration of Cu²⁺ ions (0–300 ppb). (c) Variation of fluorescence intensity at 536 nm of 5 solution (5 μM) in ethanol/HEPES (pH 7.4, 1/1, v/v) versus the concentration of Cu²⁺ ions (0–300 ppb).

2.3. Investigations on the Structural Properties of Probe 5 and Its Complex with Cu²⁺ ions

For the optimized geometries of probe 5, the 1:1 complexes between probe 5 and Cu²⁺ ions were optimized at the PBE0/6-31+G(d) level of theory. The stable structures corresponding to minima on the potential energy surface are shown in Figure 10. All their Cartesian coordinates are presented in Tables S1 and S2 of Supporting Information. The calculated bond lengths, bond angles, and dihedral angles of probe 5 and its 1:1 complex with Cu²⁺ ions are presented in Table S3 of Supporting Information.

Figure 10.

Stable structures of probe 5 (a) and its 1:1 complex with Cu²⁺ ions (b) at the PBE0/6-31+G(d) level of theory.

The calculated results show that in free probe 5, most of the atoms are coplanar except for the ones of ethylamino groups. Two chains C(16)–C(15)–C(17)–N(36) and N(36)–N(35)–C(41)–N(34) in free probe 5 are in trans-configurations (Figure 10a). Meanwhile, the 1:1 complexation leads to the changes in configurations of these two chains, being cis-configuration (Figure 10b).

The calculated values of ρ(r) and ∇²(ρ(r)) and the topological properties of 5-Cu²⁺ complex at the critical points obtained from AIM analysis are presented in Table 1 and Figure 11, respectively. The calculated results confirmed the presence of chemical bonds in each of the O(2)···Cu(49), N(36)···Cu(49), and N(34)···Cu(49) contacts. All values of ∇²(ρ(r)) for these contacts are negative. In addition, the contact distances of atom pairs O(2)···Cu(49), N(36)···Cu(49), and N(34)···Cu(49) are 1.845, 1.952, and 1.898 Å, respectively. They are significantly smaller than the sum of the van der Waals atomic radii of corresponding atom pairs (2.920, 2.950, and 2.950 Å). As a result, these contacts are thought to form covalent bonds. RCPs are found in the center of O(2)···C(16)···C(15)···C(17)···N(36)···Cu(49) and N(36)···N(35)···C(41)···N(34)···Cu(49) atom groups, indicating a cyclic structure present in each group of atoms.

Table 1. Electron density (ρ(r), in au) and the Laplacian (∇²(ρ(r)), in au) at the contact points of atom pairs in 5-Cu²⁺ complex at the PBE0/6-31+G(d) level of theory.

bond	ρ(r)	λ1	λ2	λ3	∇2(ρ(r))	critical point
O(2)···Cu(49)	0.1127	–0.1589	–0.1573	0.9036	–0.1469	BCP
N(36)···Cu(49)	0.1032	–0.1393	–0.1343	0.6041	–0.0826	BCP
N(34)···Cu(49)	0.1148	–0.1525	–0.1488	0.6977	–0.0991	BCP
O(2)···C(16)···C(15)···C(17)···N(36)···Cu(49)	0.0146	–0.0101	0.0367	0.0526	–0.0198	RCP
N(36)···N(35)···C(41)···N(34)···Cu(49)	0.0275	–0.0229	0.0642	0.1283	–0.0424	RCP

Figure 11.

Topological properties of 5-Cu²⁺ complex at the BCPs (yellow points) and RCPs (red points).

The intermolecular orbital interaction and bond properties are investigated by natural bond orbital (NBO) analysis to explain the fluorescent properties of probe 5 and 5-Cu²⁺ complex. The calculated results in Table 2 and Figure 11 show that in free probe 5, the presence of π bonds between atom pairs with their significantly large interactive energies (E⁽²⁾), including C4–C10, C11–C8, C14–C15, C17–C36, and O2–C16, has led to the formation of a π-electron conjugated system in the first moiety of probe 5 containing the diethylaminocoumarin. This π-electron conjugated system also enhances electron density from the electron pair of the N3 atom by the donor–acceptor interaction, with the interactive energy (E⁽²⁾) of 52.50 kcal·mol^–1. These intermolecular orbital interaction and bond properties are typical in fluorescent compounds.⁴¹⁻⁴³ These findings are considered as evidence for the fluorescence of probe 5.

Table 2. Significant second-order interaction energies at the PBE0/6-31+G(d) level of theory (kcal·mol^–1) between donor and acceptor orbitals in 5 and 5-Cu²⁺ complex.

donor NBO (i)	acceptor NBO (j)	E(2)	donor NBO (i)	acceptor NBO (j)	E(2)
5			5-Cu2+
π(C4–C10)	π*(C7–C9)	14.80	π(C7–C9)	π*(N3–C4)	14.05
π(C4–C10)	π*(C8–C11)	32.07	π(C8–C14)	π*(C7–C9)	12.19
π(C7–C9)	π*(C4–C10)	24.47	π(C10–C11)	π*(N3–C4)	13.15
π(C7–C9)	π*(C8–C11)	13.91	π*(N3–C4)	π*(C7–C9)	48.82
π(C8–C11)	π*(C4–C10)	17.70	π*(N3–C4)	π*(C10–C11)	24.57
π(C8–C11)	π*(C7–C9)	27.97	π*(C8–C14)	π*(C10–C11)	23.42
π(C8–C11)	π*(C14–C15)	24.83	LP(O1)	LP*(C16)	43.31
π(C14–C15)	π*(O2–C16)	27.96	LP(O1)	π*(C7–C9)	14.11
π(C14–C15)	π*(C8–C11)	13.99	LP(O2)	LP*(C16)	64.86
π(C14–C15)	π*(C17–N36)	19.81	LP(C15)	π*(C8–C14)	30.79
LP (O1)	π*(O2–C16)	40.59	LP(C15)	π*(C17–N36)	57.01
LP (O1)	π*(C7–C9)	33.17	LP(N35)	π*(C17–N36)	12.97
LP (O2)	δ*(O1–C16)	39.11	LP(O2)	LP*(Cu49)	13.89
LP (O2)	δ*(C15–C16)	18.09	LP(O2)	LP*(Cu49)	20.19
LP (N3)	π*(C4–C10)	52.50	LP(N34)	LP*(Cu49)	19.52
δ*(O1–C16)	δ*(O1–C9)	22.77	LP(N34)	LP*(Cu49)	23.49
π*(O2–C16)	π*(C14–C15)	125.95	LP(N34)	LP*(Cu49)	11.14
π*(C4–C10)	π*(C8–C11)	431.68	LP(N36)	LP*(Cu49)	14.70
π*(C8–C11)	π*(C14–C15)	218.22	LP(N36)	LP*(Cu49)	29.53
π*(C17–N36)	π*(C14–C15)	76.60	π(C37–C39)	LP*(C42)	20.29
π*(C4–C10)	π*(C7–C9)	352.98	π(C37–C39)	π*(N34–C38)	13.32
π(N34–C41)	π*(C37–C38)	18.64	π(C40–C43)	LP*(C42)	22.65
π(C37–C38)	π*(N34–C41)	10.85	π(C40–C43)	π*(N34–C38)	19.41
π(C37–C38)	π*(C39–C42)	21.99	π*(N34–C38)	π*(C37–C39)	42.65
π(C37–C38)	π*(C40–C43)	17.54	π*(N34–C38)	π*(C40–C43)	29.85
π(C39–C42)	π*(C37–C38)	19.24	LP(C41)	π*(N34–C38)	24.08
π(C39–C42)	π*(C40–C43)	20.75	LP*(C42)	π*(C37–C39)	51.77
π(C40–C43)	π*(C37–C38)	22.80	LP*(C42)	π*(C40–C43)	34.21
π(C40–C43)	π*(C39–C42)	20.58	π(C34–C38)	LP*(C41)	45.19
LP (S33)	π*(N34–C41)	29.69	LP(N35)	π*(C17–N36)	12.97
LP (S33)	π*(C37–C38)	17.92	LP(S33)	LP(C41)	66.00
LP (N34)	δ*(S33–C41)	19.21	LP(N35)	LP(C41)	47.19
LP (N35)	π*(C17–N36)	33.15
LP (N35)	π*(N34–C41)	53.86
π*(N34–C41)	π*(C37–C38)	119.73
π*(C37–C38)	π*(C39–C42)	197.32
π*(C37–C38)	π*(C40–C43)	158.69

In 5-Cu²⁺ complex, the binding between 5 ligand and Cu²⁺ ion is stabilized by the electron donor–acceptor interactions between electron lone pairs (LP) on the O and N atoms of the ligand and on the Cu atom. The hyperconjugation energies of the LP(2)O2 → LP*(5)Cu49, LP(2)O2 → LP*(6)Cu49, LP(1)N34 → LP*(5)Cu49, LP(1)N34 → LP*(6)Cu49, LP(1)N34 → LP*(7)Cu49, LP(1)N36 → LP*(5)Cu49, and LP(1)N36 → LP*(6)Cu49 in the complex are 13.89, 20.19, 19.52, 23.49, 11.14, 14.70, and 29.53 kcal·mol^–1, respectively. These interactions have caused a strong transfer of electron density from 5 ligand to Cu²⁺ ion. The calculated charge of Cu atom in 5-Cu²⁺ complex is +1.00e, indicating that 5 ligand becomes deficient in electron density. The charge of the first moiety of 5 in the complex is +0.62e, while the charge of the second moiety of 5 in the complex is +0.38e. The charge values of C17 and N3 in the complex are significantly more positive than those in free 5 (The NBO charges of atoms are presented in Table S4). The deficiency in electron density at C17 and N3 has resulted in breaking the π-electron conjugated system in the first moiety of 5 at the C15 atom. The π bonds between C15–C14 or C15–C17 are not found, while the LP(1)C15 → π*(C8–C14) and LP(1)C15 → π*(C17–N36) interactions are found with the hyperconjugation energies of 30.79 and 57.01 kcal·mol^–1, respectively. On the other hand, the calculated results have confirmed the presence of the π(N3–C4) bond and the absence of interactions from the electron lone pairs of the N3 atom in the form of donor–acceptor interaction. These findings shed light on the cause of fluorescence quenching in 5-Cu²⁺ complex.

3. Conclusions

A novel coumarin derivative was synthesized and could be used as a colorimetric and fluorescent probe for detection of Cu²⁺ ions with the detection limits of 5.7 and 4.0 ppb, respectively. This probe could operate over a wide range of pH from 5 to 9 and was not affected by the presence of other metal ions, including Na⁺, K⁺, Ca²⁺, Mg²⁺, Fe³⁺, Co²⁺, Ni²⁺, Zn²⁺, Pb²⁺, Cd²⁺, and Hg²⁺. The stable structures of probe 5 and its 1:1 complex with Cu²⁺ ions have been identified at the PBE0/6-31+G(d) level of theory and is confirmed by AIM analysis. The results from NBO analysis showed that the formation of the complex led to a strong transfer of electron density from 5 ligand to Cu²⁺ ion. As a result, the π-electron conjugated system was broken, resulting in fluorescence quenching and color change in the complex.

4. Materials and Methods

4.1. Instruments

A Shimadzu UV-1800 UV–Vis spectrophotometer was used for the UV–Vis absorption spectra. A Shimadzu RF-5301PC series fluorescence spectrometer was used for the fluorescence spectra. A Varian instrument was used for the ¹H NMR and ¹³C NMR spectra. A Finnigan 4021C instrument and Daltonics flex analysis software were used for the mass spectra.

4.2. Reagents

4-Diethylaminosalicylaldehyde, diethylmalonate, 2-hydrazinobenzothiazole, triethylamine, acetic acid, HEPES, POCl₃, HCl, NaOH, and all ions of Na⁺, K⁺, Ca²⁺, Mg²⁺, Fe³⁺, Co²⁺, Ni²⁺, Hg²⁺, Zn²⁺, Pb²⁺, Cd²⁺, and Cu²⁺ as their chloride or perchlorate were purchased from Aldrich Chemical Corporation and used as received. DMF was an HPLC reagent and redistilled prior to use. Ethanol was of HPLC grade without fluorescent impurity. All solvents were purchased from Merck and used as received. Deionized water was used for all experiments.

4.3. Computational Methodology

The optimized geometries of compounds were determined at the PBE0/6-31+G(d) level of theory,⁴⁴⁻⁴⁶ using the Gaussian 09 program package.^47,48 The structures and the nature of the bonds in these compounds have also been investigated through the atoms in molecules (AIM) topological analysis,⁴⁹ using the AIM2000 software.⁵⁰ The calculated results, including the electron density (ρ(r)), Laplacian ∇²(ρ(r)), the bond critical points (BCPs), and the ring critical points (RCPs), were used to evaluate the stability of bonds, the characteristics of bonds, the presence of bonds, and ring structure.^41,51−53 The electronic properties of compounds were investigated by the natural bond orbital (NBO) analysis, using the NBO 3.1 program available in Gaussian 09 package, at the PBE0/6-31+G(d) level of theory. The calculated results, including NBO charge of atoms, the second-order stabilization energy (E⁽²⁾) between the donor NBO(i) and the acceptor NBO(j), were used to evaluate interactions and shed light on the fluorescent characteristics of compounds.^41,54−57

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c03097.

• Synthetic methods, NMR spectra of synthetic products, and Cartesian coordinates of transition states in all of the studied environments (PDF)

Author Contributions

^§ These authors contributed equally to the work

The authors declare no competing financial interest.