A Highly Selective and Sensitive Fluorescent Turn-Off Probe for Cu2+ Based on a Guanidine Derivative

A new highly selective and sensitive fluorescent probe for Cu2+, N-n-butyl-4-(1′-cyclooctene-1′,3′,6′-triazole)-1,8-naphthalimide (L), was synthesized and evaluated. The structure of compound L was characterized via IR, 1H-NMR, 13C-NMR and HRMS. The fluorescent probe was quenched by Cu2+ with a 1:1 binding ratio and behaved as a “turn-off” sensor. An efficient and sensitive spectrofluorometric method was developed for detecting and estimating trace levels of Cu2+ in EtOH/H2O. The ligand exhibited excitation and emission maxima at 447 and 518 nm, respectively. The equilibrium binding constant of the ligand with Cu2+ was 1.57 × 104 M−1, as calculated using the Stern-Volmer equation. Ligand L is stable and can be used to detect Cu2+ in the range of pH from 7 to 12. The sensor responded to Cu2+ rapidly and a large number of coexisting ions showed almost no obvious interference with the detection.


Introduction
Fluorescent sensors for chemical species used in biological and environmental detection are currently an attractive field of research [1][2][3]. In particular, pollution by heavy metal ions has attracted much attention because these metal ions not only play important roles in various physiological processes but also have distinctly toxic impact on human health [4]. It is highly desired to detect these in relevant systems. The importance of metal ion sensing can never be overestimated because of their widespread distribution in environmental systems and biological processes [5]. Copper is a significant metal pollutant because of its widespread application and is also an essential trace element in biological systems. Cu 2+ is present in various biological processes, as the third most abundant transition metal ion in the human body. In addition, its homeostasis is critical for the metabolism and development of living organisms [6][7][8]. For example, Cu 2+ plays a critical role as a catalytic cofactor for a variety of metalloenzymes, including superoxide dismutase, cytochrome oxidase and tyrosinase [9,10]. The misregulation or excess accumulation of Cu 2+ could cause serious diseases [11]. Its concentration in the neuronal cytoplasm may contribute to the etiology of Alzheimer's or ParkinsoN s disease [12][13][14]. However, exposure to high levels of Cu 2+ will cause gastrointestinal disturbance and even liver or kidney damage. The detection of Cu 2+ is meaningful for both disease diagnosis and environmental monitoring [15]. The normal average concentration of Cu 2+ in blood is in the range of 15.7-23.6 µM [16]. The U.S. Environmental Protection Agency (EPA) set a limit for Cu 2+ in drinking water of 20 µM [17]. The detection of Cu 2+ levels in water requires an efficient, selective and highly sensitive chemosensor [18,19]. Numerous methods to detect metal ions have been developed, such as atomic absorption spectrometry [20], inductively coupled plasma atomic emission spectroscopy [21], visual detection [22,23], chemiluminescence detection [24] and electrochemical techniques [25,26]. However, these methods have disadvantages, such as expensive instrumentation, complicated sample preparation and tedious operating procedures, resulting in the need for other sensitive, simple detection methods for copper ions. Fluorescent molecules offer many advantages over other systems in this area as a result of their emissive behavior, easy signal detection, sensitivity, selectivity and low cost [27,28]. The development of the fluorescence method has been demonstrated to be promising because of its advantages of wide applicability, ease of manipulation, high selectivity, sensitivity, rapidity, nondestructive methodology and direct visual perception [29,30]. Thus, a number of high-performance sensors have been developed to detect metal ions [31]. Cu 2+ is a typical ion with a pronounced quenching effect on fluorophores via mechanisms that are inherent to the paramagnetic species [32][33][34][35].

Synthesis and Characterization
Compound L was synthesized through the route outlined in Scheme 2. The structure modification was focused on the C-4 position. A dihydroxyethylamino moiety was introduced to replace the Br atom, and the subsequent reaction modified the -OH group to N3; compound 4 was obtained in a 67% yield from the amination. Thiourea is easily transformed into guanidine; however, the conversion from thiourea to guanidine has attracted little attention as a potential fluorogenic reaction because most thiourea derivatives have the same color or fluorescence as their guanidine analogues [39]. Initially, we envisioned that a similar molecular recognition/reactivity motif might be incorporated into fluorophores to serve as a ratiometric fluorescent chemodosimeter for the selective detection of Hg 2+ , as reported in the literature [40], because compound 4 has two free amino groups. The expected thiourea derivative was not achieved; however, the product transformed into the inner guanidine ring because compound 4 contained a primary amine subunit. Compound L was obtained through intramolecular cyclization with a recognition/reactivity motif that could be incorporated into fluorophores so that metal ion addition led to a variation of the fluorescence.

Synthesis and Characterization
Compound L was synthesized through the route outlined in Scheme 2. The structure modification was focused on the C-4 position. A dihydroxyethylamino moiety was introduced to replace the Br atom, and the subsequent reaction modified the -OH group to N 3 ; compound 4 was obtained in a 67% yield from the amination. Thiourea is easily transformed into guanidine; however, the conversion from thiourea to guanidine has attracted little attention as a potential fluorogenic reaction because most thiourea derivatives have the same color or fluorescence as their guanidine analogues [39]. Initially, we envisioned that a similar molecular recognition/reactivity motif might be incorporated into fluorophores to serve as a ratiometric fluorescent chemodosimeter for the selective detection of Hg 2+ , as reported in the literature [40], because compound 4 has two free amino groups. The expected thiourea derivative was not achieved; however, the product transformed into the inner guanidine ring because compound 4 contained a primary amine subunit. Compound L was obtained through intramolecular cyclization with a recognition/reactivity motif that could be incorporated into fluorophores so that metal ion addition led to a variation of the fluorescence.
Compound L was obtained with a yield of 57% in the final reaction, and its structure was characterized by 1 H-NMR, 13 C-NMR and HRMS. As evidenced by the signals at 6.57-8.42 ppm in the 1 H-NMR spectrum, the number of Ar-H is 10, which indicated that benzoyl isothiocyanate had reacted with compound 4. The signals at δ = 9.02 ppm indicated the existence of benzamide, and the signals at δ = 1.26 ppm showed that -NH 2 was substituted. Furthermore, the molecular ion peak in the HRMS-ESI ([M + H] + , 484.2332 m/z) spectrum was consistent with the theoretically calculated molecular mass of compound L.

Spectral Characteristics
The photophysical characteristics of compound L in EtOH/H2O (4:1 v/v) were investigated. Figure 1 shows the excitation (λ = 447 nm) and emission (λ = 518 nm) spectra of compound L. The Stokes shift (υA-υF), an important parameter indicating the difference in properties and structure between the ground state (S0) and first excited state (S1), was calculated by Equation (1) to be 3062 cm −1 .
The ability of the molecules to emit absorbed light energy is characterized quantitatively by the fluorescence quantum yield (ΦF). The quantum yields of fluorescence were calculated using coumarin 6G (Φsample = 0.94) in EtOH as the standard according to Equation (2), where Aref, Sref, and nref and Asample, Ssample, and nsample represent the absorbance at the exited wavelength, the integrated emission band area and the solvent refractive index of the standard and the sample, respectively [41,42]. Thus, the fluorescence quantum yield of probe L in EtOH/H2O (4:1 v/v) solution was 0.06.
The photophysical properties of compound L as a ligand in the presence of different metal cations were investigated in EtOH/H2O (4:1 v/v) to determine its potential application as a sensor for detecting these metal ions. The binding behavior of compound L toward different metal ions such as their corresponding chlorides, acetates, sulfides or nitrates was studied using fluorescence spectroscopy. The anions did not affect the spectroscopic results.

Spectral Characteristics
The photophysical characteristics of compound L in EtOH/H 2 O (4:1 v/v) were investigated. Figure 1 shows the excitation (λ = 447 nm) and emission (λ = 518 nm) spectra of compound L. The Stokes shift (υ A -υ F ), an important parameter indicating the difference in properties and structure between the ground state (S 0 ) and first excited state (S 1 ), was calculated by Equation (1) to be 3062 cm −1 .
The ability of the molecules to emit absorbed light energy is characterized quantitatively by the fluorescence quantum yield (Φ F ). The quantum yields of fluorescence were calculated using coumarin 6G (Φ sample = 0.94) in EtOH as the standard according to Equation (2), where A ref , S ref , and n ref and A sample , S sample , and n sample represent the absorbance at the exited wavelength, the integrated emission band area and the solvent refractive index of the standard and the sample, respectively [41,42]. Thus, the fluorescence quantum yield of probe L in EtOH/H 2 O (4:1 v/v) solution was 0.06.
The photophysical properties of compound L as a ligand in the presence of different metal cations were investigated in EtOH/H 2 O (4:1 v/v) to determine its potential application as a sensor for detecting these metal ions. The binding behavior of compound L toward different metal ions such as their corresponding chlorides, acetates, sulfides or nitrates was studied using fluorescence spectroscopy. The anions did not affect the spectroscopic results. To evaluate the selectively of compound L for Cu 2+ , changes in the fluorescence intensity upon the addition of various metal ions under the same conditions were investigated. The fluorescence spectrum of compound L exhibited strong fluorescence emission when excited at 447 nm in EtOH/H2O (4:1 v/v; Figure 2). Under the same conditions as those used for adding the metal ions, significant fluorescence changes were observed in the presence of Cu 2+ . Fluorescence quenching was observed immediately after Cu 2+ was added. When other metal ions were added, the fluorescence exhibit few changes. As shown in Figure 2, remarkable color changes ranging from yellow to colorless and yellow floccules floating in the solution could be distinguished by the naked eye. In addition, the introduction of Cu 2+ to the mixture solution of compound L led to evident fluorescence quenching, while the rest of the metal cations exerted a negligible or very small influence under identical conditions, which was further evidenced by the illumination taken under a 365 nm portable UV lamp. On the basis of the literature, the fluorescence quench of probe L might have been due to the coordination to paramagnetic Cu 2+ [43].  To evaluate the selectively of compound L for Cu 2+ , changes in the fluorescence intensity upon the addition of various metal ions under the same conditions were investigated. The fluorescence spectrum of compound L exhibited strong fluorescence emission when excited at 447 nm in EtOH/H 2 O (4:1 v/v; Figure 2). Under the same conditions as those used for adding the metal ions, significant fluorescence changes were observed in the presence of Cu 2+ . Fluorescence quenching was observed immediately after Cu 2+ was added. When other metal ions were added, the fluorescence exhibit few changes. As shown in Figure 2, remarkable color changes ranging from yellow to colorless and yellow floccules floating in the solution could be distinguished by the naked eye. In addition, the introduction of Cu 2+ to the mixture solution of compound L led to evident fluorescence quenching, while the rest of the metal cations exerted a negligible or very small influence under identical conditions, which was further evidenced by the illumination taken under a 365 nm portable UV lamp. On the basis of the literature, the fluorescence quench of probe L might have been due to the coordination to paramagnetic Cu 2+ [43]. To evaluate the selectively of compound L for Cu 2+ , changes in the fluorescence intensity upon the addition of various metal ions under the same conditions were investigated. The fluorescence spectrum of compound L exhibited strong fluorescence emission when excited at 447 nm in EtOH/H2O (4:1 v/v; Figure 2). Under the same conditions as those used for adding the metal ions, significant fluorescence changes were observed in the presence of Cu 2+ . Fluorescence quenching was observed immediately after Cu 2+ was added. When other metal ions were added, the fluorescence exhibit few changes. As shown in Figure 2, remarkable color changes ranging from yellow to colorless and yellow floccules floating in the solution could be distinguished by the naked eye. In addition, the introduction of Cu 2+ to the mixture solution of compound L led to evident fluorescence quenching, while the rest of the metal cations exerted a negligible or very small influence under identical conditions, which was further evidenced by the illumination taken under a 365 nm portable UV lamp. On the basis of the literature, the fluorescence quench of probe L might have been due to the coordination to paramagnetic Cu 2+ [43].

Calculation of Binding Constants and Different Concentrations of Cu 2+
A Job plot was generated by continuously varying the mole fraction of Cu 2+ from 0 to 1 in a solution of [L-Cu 2+ ] with a total concentration of 50 µM to determine the stoichiometry of the [L-Cu 2+ ] complex. The Job plot analysis revealed an approximate maximum at a 0.5 mole fraction, which indicated a 1:1 stoichiometry of the ligand L and Cu 2+ complex ( Figure 3).

Calculation of Binding Constants and Different Concentrations of Cu 2+
A Job plot was generated by continuously varying the mole fraction of Cu 2+ from 0 to 1 in a solution of [L-Cu 2+ ] with a total concentration of 50 μM to determine the stoichiometry of the [L-Cu 2+ ] complex. The Job plot analysis revealed an approximate maximum at a 0.5 mole fraction, which indicated a 1:1 stoichiometry of the ligand L and Cu 2+ complex ( Figure 3).

Calculation of Binding Constants and Different Concentrations of Cu 2+
A Job plot was generated by continuously varying the mole fraction of Cu 2+ from 0 to 1 in a solution of [L-Cu 2+ ] with a total concentration of 50 μM to determine the stoichiometry of the [L-Cu 2+ ] complex. The Job plot analysis revealed an approximate maximum at a 0.5 mole fraction, which indicated a 1:1 stoichiometry of the ligand L and Cu 2+ complex (Figure 3).   Upon the addition of Cu 2+ , the fluorescence was quenched. The changes in the fluorescence emission intensities of compound L (1 × 10 −5 mol L −1 ) with different concentrations of Cu 2+ (0-3 × 10 −4 mol L −1 ) are presented in Figure 5. A progressive decrease in the fluorescence intensity was observed. The plot of the fluorescence intensity versus externally added Cu 2+ (Figure 6) reaches saturation after a certain amount of externally added Cu 2+ . Curvilinearity was obtained for up to a value of 6 times (6 × 10 −5 mol L −1 ) the amount of externally added Cu 2+ . The results indicated that compound L could detect Cu 2+ at a micromole level. Upon the addition of Cu 2+ , the fluorescence was quenched. The changes in the fluorescence emission intensities of compound L (1 × 10 −5 mol L −1 ) with different concentrations of Cu 2+ (0-3 × 10 −4 mol L −1 ) are presented in Figure 5. A progressive decrease in the fluorescence intensity was observed. The plot of the fluorescence intensity versus externally added Cu 2+ (Figure 6) reaches saturation after a certain amount of externally added Cu 2+ . Curvilinearity was obtained for up to a value of 6 times (6 × 10 −5 mol L −1 ) the amount of externally added Cu 2+ . The results indicated that compound L could detect Cu 2+ at a micromole level.

Response Time
The response time is a significant practical parameter [45]. Time-dependence of probe L for sensing Cu 2+ was examined and the results are illustrated in Figure 7. Upon the addition of Cu 2+ , the fluorescence intensity decreased to the minimum immediately and tended to be stable during the Upon the addition of Cu 2+ , the fluorescence was quenched. The changes in the fluorescence emission intensities of compound L (1 × 10 −5 mol L −1 ) with different concentrations of Cu 2+ (0-3 × 10 −4 mol L −1 ) are presented in Figure 5. A progressive decrease in the fluorescence intensity was observed. The plot of the fluorescence intensity versus externally added Cu 2+ (Figure 6) reaches saturation after a certain amount of externally added Cu 2+ . Curvilinearity was obtained for up to a value of 6 times (6 × 10 −5 mol L −1 ) the amount of externally added Cu 2+ . The results indicated that compound L could detect Cu 2+ at a micromole level.

Response Time
The response time is a significant practical parameter [45]. Time-dependence of probe L for sensing Cu 2+ was examined and the results are illustrated in Figure 7. Upon the addition of Cu 2+ , the fluorescence intensity decreased to the minimum immediately and tended to be stable during the Figure 6. Fluorescence intensity vs Cu 2+ ; inset shows that up to 6-fold Cu 2+ curvilinearity was sustained; λ ex : 437 nm; λ em : 518 nm; slit width: 10 nm.

Response Time
The response time is a significant practical parameter [45]. Time-dependence of probe L for sensing Cu 2+ was examined and the results are illustrated in Figure 7. Upon the addition of Cu 2+ , the fluorescence intensity decreased to the minimum immediately and tended to be stable during the test time. No changes in the fluorescence of probe L in the absence of Cu 2+ were detected with the increased time. This showed that probe L exhibited a reliable rapid response to Cu 2+ .

Effect of pH
In order to investigate the application of probe L within complex media, the fluorescence spectra response of probe L was measured in the absence or presence of 5 × 10 −5 mol L −1 Cu 2+ under different pH conditions. As shown in Figure 8, the fluorescence intensity of probe L did not reveal any clear differences over a pH range of 2-12, which was consistent with the goal of our experimental design. The fluorescence of the [L-Cu 2+ ] solution could not remarkably decrease when the pH values were lower than 5, possibly because the imino groups of probe L were protonated at acid conditions, resulting in weak coordination with Cu 2+ . In addition, the fluorescence intensity of the [L-Cu 2+ ] solution showed a 5-fold change from pH 4 to pH 7, which suggested that it is sensitive to pH changes. These results indicated that probe L could be used to detect biological as well environmental Cu 2+ in the range of pH from 7 to 12.

Effect of pH
In order to investigate the application of probe L within complex media, the fluorescence spectra response of probe L was measured in the absence or presence of 5 × 10 −5 mol L −1 Cu 2+ under different pH conditions. As shown in Figure 8, the fluorescence intensity of probe L did not reveal any clear differences over a pH range of 2-12, which was consistent with the goal of our experimental design. The fluorescence of the [L-Cu 2+ ] solution could not remarkably decrease when the pH values were lower than 5, possibly because the imino groups of probe L were protonated at acid conditions, resulting in weak coordination with Cu 2+ . In addition, the fluorescence intensity of the [L-Cu 2+ ] solution showed a 5-fold change from pH 4 to pH 7, which suggested that it is sensitive to pH changes. These results indicated that probe L could be used to detect biological as well environmental Cu 2+ in the range of pH from 7 to 12.

Effect of pH
In order to investigate the application of probe L within complex media, the fluorescence spectra response of probe L was measured in the absence or presence of 5 × 10 −5 mol L −1 Cu 2+ under different pH conditions. As shown in Figure 8, the fluorescence intensity of probe L did not reveal any clear differences over a pH range of 2-12, which was consistent with the goal of our experimental design. The fluorescence of the [L-Cu 2+ ] solution could not remarkably decrease when the pH values were lower than 5, possibly because the imino groups of probe L were protonated at acid conditions, resulting in weak coordination with Cu 2+ . In addition, the fluorescence intensity of the [L-Cu 2+ ] solution showed a 5-fold change from pH 4 to pH 7, which suggested that it is sensitive to pH changes. These results indicated that probe L could be used to detect biological as well environmental Cu 2+ in the range of pH from 7 to 12.

Selectivity
The selectivity is clearly one of the most important characteristics of a cation-selective chemosensor. Thus, the influence of other metal ions on the fluorescence intensity of the proposed Cu 2+ chemosensor was investigated (Figure 9). In this study, Ag + , Na + , K + , Ba 2+ , Ca 2+ , Co 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Pb 2+ , Sn 2+ , Zn 2+ , Al 3+ , Cu + , Hg 2+ and Fe 3+ (100 µM) were added to the compound L (10 µM)/Cu 2+ (50 µM) solution, and the fluorescence was observed. The fluorescence emission intensity at 518 nm significantly decreased as a result of binding with Cu 2+ . The coexisting ions showed little influence on the detection of Cu 2+ . Common anions, including oxalate, sulfate, nitrate and chloride, did not interfere with the fluorescence intensity of the ligand or [L-Cu 2+ ] complex. Compared to the other common cations, compound L exhibited good selectivity for Cu 2+ , indicating that L has a good anti-interference ability in detecting Cu 2+ .

Selectivity
The selectivity is clearly one of the most important characteristics of a cation-selective chemosensor. Thus, the influence of other metal ions on the fluorescence intensity of the proposed Cu 2+ chemosensor was investigated (Figure 9). In this study, Ag + , Na + , K + , Ba 2+ , Ca 2+ , Co 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Pb 2+ , Sn 2+ , Zn 2+ , Al 3+ , Cu + , Hg 2+ and Fe 3+ (100 μM) were added to the compound L (10 μM)/Cu 2+ (50 μM) solution, and the fluorescence was observed. The fluorescence emission intensity at 518 nm significantly decreased as a result of binding with Cu 2+ . The coexisting ions showed little influence on the detection of Cu 2+ . Common anions, including oxalate, sulfate, nitrate and chloride, did not interfere with the fluorescence intensity of the ligand or [L-Cu 2+ ] complex. Compared to the other common cations, compound L exhibited good selectivity for Cu 2+ , indicating that L has a good anti-interference ability in detecting Cu 2+ .

Thermal Studies
The stability of probe L and its Cu 2+ complex was studied by thermogravimetry to prove the binding of ligand L with Cu 2+ ions, and the results are presented in Figures 10 and 11, respectively. The thermal stability of the Cu 2+ complex (up to 324.76 °C) was better than that of the free ligand (up to 288.57 °C).

Thermal Studies
The stability of probe L and its Cu 2+ complex was studied by thermogravimetry to prove the binding of ligand L with Cu 2+ ions, and the results are presented in Figures 10 and 11, respectively. The thermal stability of the Cu 2+ complex (up to 324.76 • C) was better than that of the free ligand (up to 288.57 • C).

Selectivity
The selectivity is clearly one of the most important characteristics of a cation-selective chemosensor. Thus, the influence of other metal ions on the fluorescence intensity of the proposed Cu 2+ chemosensor was investigated (Figure 9). In this study, Ag + , Na + , K + , Ba 2+ , Ca 2+ , Co 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Pb 2+ , Sn 2+ , Zn 2+ , Al 3+ , Cu + , Hg 2+ and Fe 3+ (100 μM) were added to the compound L (10 μM)/Cu 2+ (50 μM) solution, and the fluorescence was observed. The fluorescence emission intensity at 518 nm significantly decreased as a result of binding with Cu 2+ . The coexisting ions showed little influence on the detection of Cu 2+ . Common anions, including oxalate, sulfate, nitrate and chloride, did not interfere with the fluorescence intensity of the ligand or [L-Cu 2+ ] complex. Compared to the other common cations, compound L exhibited good selectivity for Cu 2+ , indicating that L has a good anti-interference ability in detecting Cu 2+ .

Thermal Studies
The stability of probe L and its Cu 2+ complex was studied by thermogravimetry to prove the binding of ligand L with Cu 2+ ions, and the results are presented in Figures 10 and 11, respectively. The thermal stability of the Cu 2+ complex (up to 324.76 °C) was better than that of the free ligand (up to 288.57 °C).

IR Spectra
The structural matching played an important role in selective recognition. According to the examples in the literature, a similar mode of probes containing a tridentate structure binding with Cu 2+ has been reported [46,47]. To further study the possible binding mode of compound L and Cu 2+ , IR spectroscopy was employed to elucidate the possible coordination mode. The IR spectra of compound L and the [L-Cu 2+ ] complex are compared in Figure 12. The IR spectra of compound L (Figure 12a) has two slender peaks appearing between 3350 and 3290 cm −1 . One of the extremely sharp peaks in this range vanished when the complex was formed (Figure 12b) and only appeared at 3325.72 cm −1 . This result suggested that the aromatic amide was not involved in the coordination of [L-Cu 2+ ]. However, the extremely sharp peak in the IR spectra corresponding to the imine disappeared. For fluorescence quenching, the N atom directly connected to naphthalene must be present in the coordination of the complex. On the basis of the aforementioned fact, a plausible binding mode of the complex was proposed, as shown in Scheme 3.

IR Spectra
The structural matching played an important role in selective recognition. According to the examples in the literature, a similar mode of probes containing a tridentate structure binding with Cu 2+ has been reported [46,47]. To further study the possible binding mode of compound L and Cu 2+ , IR spectroscopy was employed to elucidate the possible coordination mode. The IR spectra of compound L and the [L-Cu 2+ ] complex are compared in Figure 12. The IR spectra of compound L (Figure 12a) has two slender peaks appearing between 3350 and 3290 cm −1 . One of the extremely sharp peaks in this range vanished when the complex was formed (Figure 12b) and only appeared at 3325.72 cm −1 . This result suggested that the aromatic amide was not involved in the coordination of [L-Cu 2+ ]. However, the extremely sharp peak in the IR spectra corresponding to the imine disappeared. For fluorescence quenching, the N atom directly connected to naphthalene must be present in the coordination of the complex. On the basis of the aforementioned fact, a plausible binding mode of the complex was proposed, as shown in Scheme 3.

IR Spectra
The structural matching played an important role in selective recognition. According to the examples in the literature, a similar mode of probes containing a tridentate structure binding with Cu 2+ has been reported [46,47]. To further study the possible binding mode of compound L and Cu 2+ , IR spectroscopy was employed to elucidate the possible coordination mode. The IR spectra of compound L and the [L-Cu 2+ ] complex are compared in Figure 12. The IR spectra of compound L (Figure 12a) has two slender peaks appearing between 3350 and 3290 cm −1 . One of the extremely sharp peaks in this range vanished when the complex was formed (Figure 12b) and only appeared at 3325.72 cm −1 . This result suggested that the aromatic amide was not involved in the coordination of [L-Cu 2+ ]. However, the extremely sharp peak in the IR spectra corresponding to the imine disappeared. For fluorescence quenching, the N atom directly connected to naphthalene must be present in the coordination of the complex. On the basis of the aforementioned fact, a plausible binding mode of the complex was proposed, as shown in Scheme 3.

Chemicals and Instruments
All of the solvents and reactants were commercially available and used without purification. The IR spectra were recorded on a Bruker ALPHA-T spectrometer (KBr, BRUKER Inc., Beijing, China). The melting points were determined using a Beijing Taike melting point apparatus (X-4) and were uncorrected, and 1 H-NMR and 13 C-NMR spectra were recorded on a Bruker AVANVE 300 or 400 MHz nuclear magnetic resonance spectrometer (Energy Chemical, Shanghai, China) using CDCl3 or DMSO-d6 as the solvent and TMS as the internal standard. The high-resolution mass spectrometry was recorded on a FT-ICR MS spectrometer (BRUKER Inc., Beijing, China). Fluorescence emission spectra were obtained using a PerkinElmer LS55 fluorescence spectrometer (PerkinElmer, Liantrisant, UK). The pH values were measured using a PHS-3C pH meter (Inesa, Shanghai, China). Thermal analysis was performed using a 449F3 thermal analyzer (Netzsch, Bavaria, Germany). The spectrogram data of the synthesized compounds could be found in the Supplementary Materials.

Testing Methods
Compound L was dissolved in EtOH to form a 10 −3 M stock solution. Cu 2+ , Ag + , Na + , K + , Ba 2+ , Ca 2+ , Co 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Pb 2+ , Sn 2+ , Zn 2+ , Al 3+ , Hg 2+ and Fe 3+ sources were dissolved in deionized water (H2O) to obtain 10 −2 M stock solutions. For the fluorescence measurement of Cu 2+ , the mixed stock solutions were diluted to 10 mL with EtOH and H2O to form EtOH/H2O (4:1 v/v) solutions. For the titration experiments, compound L stock solution (100 μL) was mixed with a certain amount of Cu 2+ stock solution and diluted to 10 mL with EtOH and H2O to form EtOH/H2O (4:1 v/v) solutions. The wide pH range solutions were prepared by adjustment of 0.05 mol L −1 Tris-HCl solution with HCl or NaOH solution. Thermal studies of compound L and its [L-Cu 2+ ] complex were also performed to prove the binding of the ligand with Cu 2+ . All fluorescence spectra were recorded at 25 °C with the excitation wavelength set at 437 nm and the slit width set at 10 nm.

Chemicals and Instruments
All of the solvents and reactants were commercially available and used without purification. The IR spectra were recorded on a Bruker ALPHA-T spectrometer (KBr, BRUKER Inc., Beijing, China). The melting points were determined using a Beijing Taike melting point apparatus (X-4) and were uncorrected, and 1 H-NMR and 13 C-NMR spectra were recorded on a Bruker AVANVE 300 or 400 MHz nuclear magnetic resonance spectrometer (Energy Chemical, Shanghai, China) using CDCl 3 or DMSO-d 6 as the solvent and TMS as the internal standard. The high-resolution mass spectrometry was recorded on a FT-ICR MS spectrometer (BRUKER Inc., Beijing, China). Fluorescence emission spectra were obtained using a PerkinElmer LS55 fluorescence spectrometer (PerkinElmer, Liantrisant, UK). The pH values were measured using a PHS-3C pH meter (Inesa, Shanghai, China). Thermal analysis was performed using a 449F3 thermal analyzer (Netzsch, Bavaria, Germany). The spectrogram data of the synthesized compounds could be found in the Supplementary Materials.

Testing Methods
Compound L was dissolved in EtOH to form a 10 −3 M stock solution. Cu 2+ , Ag + , Na + , K + , Ba 2+ , Ca 2+ , Co 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Pb 2+ , Sn 2+ , Zn 2+ , Al 3+ , Hg 2+ and Fe 3+ sources were dissolved in deionized water (H 2 O) to obtain 10 −2 M stock solutions. For the fluorescence measurement of Cu 2+ , the mixed stock solutions were diluted to 10 mL with EtOH and H 2 O to form EtOH/H 2 O (4:1 v/v) solutions. For the titration experiments, compound L stock solution (100 µL) was mixed with a certain amount of Cu 2+ stock solution and diluted to 10 mL with EtOH and H 2 O to form EtOH/H 2 O (4:1 v/v) solutions. The wide pH range solutions were prepared by adjustment of 0.05 mol L −1 Tris-HCl solution with HCl or NaOH solution. Thermal studies of compound L and its [L-Cu 2+ ] complex were also performed to prove the binding of the ligand with Cu 2+ . All fluorescence spectra were recorded at 25 • C with the excitation wavelength set at 437 nm and the slit width set at 10 nm.  N-n-butyl-4-(N ,N -dihydroxyethyl)amino-1,8-naphthalimide was obtained using a procedure similar to that reported by Guo et al. [49]. N-n-butyl-4-bromine-1,8-naphthalimide (15 g, 45.2 mmol) and diethanolamine (75 mL) were mixed in ethylene glycol monomethyl ether (100 mL). The mixture was refluxed for 6 h. The crude product was purified by column chromatography using silica gel, with EtOAc and light petroleum (1:4 v/v) as the eluent. Compound 2 was obtained as a yellow solid coexisting metal ions [15,51]. In addition, Probe L showed a 1:1 stoichiometry (sensor/Cu 2+ ) with a high binding constant. This sensor should be valuable for copper analysis in environmental samples and biological systems.
Supplementary Materials: Supplementary data associated with this article can be found in the online.