A Naphthoquinoline-Dione-Based Cu²⁺ Sensing Probe with Visible Color Change and Fluorescence Quenching in an Aqueous Organic Solution

Abstract

Copper metal ions (Cu²⁺) are widely used in various industries, and their salts are used as supplementary components in agriculture and medicine. As this metal ion is associated with various health issues, it is necessary to detect and monitor it in environmental and biological samples. In the present report, we synthesized a naphthoquinoline-dione-based probe 1 containing three ester groups to investigate its ability to detect metal ions in an aqueous solution. Among various metal ions, probe 1 showed a vivid color change from yellow to colorless in the presence of Cu²⁺, as observed by the naked eye. The ratiometric method using the absorbance ratio (A₄₁₃/A₄₇₆) resulted in a limit of detection (LOD) of 1 µM for Cu²⁺. In addition, the intense yellow-green fluorescence was quenched upon the addition of Cu²⁺, resulting in a calculated LOD of 5 nM. Thus, probe 1 has the potential for dual response toward Cu²⁺ detection through color change and fluorescence quenching. ¹H-NMR investigation and density functional theory (DFT) calculations indicate 1:1 binding of the metal ion to the small cavity of the probe comprising four functional groups: the carbonyl group of the amide (O), the amino group (N), and two t-butyl ester groups (O). When adsorbed onto various solid surfaces, such as cotton, silica, and filter paper, the probe showed effective detection of Cu²⁺ via fluorescence quenching. Probe 1 was also useful for Cu²⁺ sensing in environmental samples (sea and drain water) and biological samples (live HeLa cells).

1. Introduction

Potential adaptability, easy synthesis and structural modification, and outstanding photo-physical properties of small molecular probes led to their wide applications in various fields, including medicinal chemistry, bio-imaging, drug delivery, solar cells, and OLEDs [1,2,3,4]. Recently, significant attention has been paid to molecular probes that exhibit a vibrant color change in the presence of specific analytes [5,6,7,8]. Metal ion sensing in aqueous systems is particularly demanding due to the critical roles of various metal ions in physiology. However, at their individual optimal concentrations, these metal ions perform diverse functions, including pH regulation, osmotic balance, metabolism, and signaling transduction [9,10]. Their abnormal concentrations could lead to damage of organs that can eventually show detrimental effects on the human body, such as physical disorders and acute/chronic diseases [11,12,13]. Heavy metal ions, such as Hg²⁺/CH₃Hg⁺ and Pb²⁺, are toxic to humans [14,15]. Thus, selective detection of individual metal ions is indispensable, and monitoring the quantity/concentration of these metal ions in environmental sources is required and should be regulated, especially in drinking water (e.g., tap/lake water) [16,17,18,19]. Cu²⁺ is the third most common heavy metal ion in the human body and functions to sustain bones, blood vessels, the immune system, nerves, and metabolism, and it serves a critical function in iron absorption in the human body [20]. However, excessive Cu²⁺ exposure, even for a limited time, can lead to serious health concerns, such as liver/kidney issues, gastrointestinal (GI) dysfunction, and many serious neurodegenerative diseases (e.g., Menkes disease) [21,22,23,24,25]. It is also widely used in construction, transport, and electronics, and many of its salts are being used as supplementary components in agriculture and medicine [26,27]. This metal ion seeps into groundwater and deteriorates the quality of drinking water, posing a threat to human health. The U.S. Environmental Protection Agency (EPA) [28] currently sets the limit of Cu²⁺ concentration in drinking water at 1.3 ppm (~20 µM).

Among various analytical techniques to detect metal ions in biological and environmental samples [29], fluorescence spectroscopy possesses many advantages over others in terms of affordability, quick response, convenience, easy operation, on-site detection/monitoring, and naked eye recognition [30,31,32,33,34,35]. Thus, many researchers have made efforts to create selective, reliable, convenient, and efficient sensors with a variety of fluorescent platforms, such as fluorescein, rhodamine B, triphenylamine, quinolone, pyrene, anthraquinone, chalcone, benzothiazole/benzimidazole amine, coumarin-conjugated di-(2-picolyl)amine, peptide, and Schiff base compounds [36,37,38,39,40,41,42,43]. Many of these probes utilized ethylene diamine-like structures for strong and selective Cu²⁺ binding among different metal ions, leading to high selectivity toward Cu²⁺ detection. For instance, S. Anbu et al. reported a benzimidazole-based bis-Schiff base probe as a fluorogenic chemosensor. The Schiff base-benzimidazole conjugate mimics ethylene diamine as a metal ion-binding group. This probe detected Cu²⁺ and Zn²⁺ in an aqueous solution via fluorescence ‘turn-on’ and ‘turn-off’ mechanisms, respectively, with limits of detection (LODs) of 24 and 2 nM [36]. K. Guo and J. Li used an imine-linked triphenylamine–benzothiazole conjugate as a metal ion-sensing probe. The probe containing an ethylenediamine-like binding site for metal ions was able to detect two metal ions (Cu²⁺ and Fe³⁺) in CH₃CN, with LODs of 0.3 and 3 μM, respectively [37]. The probe showed a color change from colorless to red in the presence of Cu²⁺, while the interaction of the probe with Fe³⁺ resulted in the ‘switch on’ fluorescence emission. In an aqueous CH₃CN solution, this benzothiazole-based probe was selective for Zn²⁺, with an LOD of 3 μM. D. Aydin et al. reported a phenolphthalein-based Schiff base probe for selective Cu²⁺ detection. The metal binding site of the probe is structurally similar to ethylenediamine but comprises a different spacer (propylene) and atom (oxygen) from the latter. Upon the addition of the metal ion, the probe displayed a color change from yellow to colorless in an HEPES:CH₃CN (1:1, pH = 7) solution. In addition, the fluorescence emission of the probe turned off in the presence of Cu²⁺, giving an LOD of 84 nM [38]. G. Singh et al. reported a pyrene-bearing organosilane-based nanoparticle for the selective detection of Cu²⁺. This pyrene-decorated nanoparticle also contains an ethylenediamine-like binding site and showed a sensitive Cu²⁺ detection (LOD = 6 µM) in methanol [43]. Inspired by the structural feature of such ethylenediamine-like binding sites present in these previously reported probes, we prepared a new probe (probe 1) containing three ester groups based on the naphthoquinoline-dione platform in the current study. The resulting three ester group probe selectively detected Cu²⁺ by luminescence/fluorescence and absorption/color change. The probe 1 solution changed from bright yellow to colorless upon Cu²⁺ binding, demonstrating a low calculated LOD of 5 nM. Cu²⁺ sensing was also realized using this probe adsorbed onto solid surfaces (cotton, filter paper, silica gel, and TLC plate). In addition, Cu²⁺ added in environmental samples (sea/drain water) or bio-samples (HeLa cells) was successfully detected by the probe.

2. Results and Discussion

2.1. Synthesis and Characterization of Probe 1

Probe 1 was synthesized from compound 3 via a two-step reaction (Scheme 1). The amino group bearing the naphthoquinoline-dione derivative (compound 3) was reacted with t-butyl bromoacetate in DMSO using K₂CO₃ (base) and TBAHSO₄ (catalyst) to produce compound 2 (50%). The formation of compound 2 was verified by its ¹H-NMR spectrum, where the amidic NH of compound 3 was lost at 12.45 ppm, and two new singlets of NCH₂ and t-butyl groups appeared in the aliphatic region at δ 5.13 and 1.49 ppm, respectively (Figure 1A). A further reaction of compound 2 with t-butyl bromoacetate gave probe 1 a moderate yield (50%). The ¹H NMR spectrum of the probe showed a loss of the NH₂ signal at δ 5.89 ppm with the simultaneous appearance of four doublets at δ 3.95, 4.93, 5.16, and 5.63 ppm, corresponding to two CH₂ groups attached to the amino group. These spectral features match the chemical structure of probe 1 containing three t-butyl acetate groups (Figure 1A). The 2D NMR (¹H-¹H NOESY and COSY) spectra verify the chemical structure of probe 1 (Figure 1B–E and Figures S1–S4). The 2D NMR (¹H-¹H NOESY) spectrum displayed a strong correlation between the three t-butyl protons, their neighboring protons (N^αCH₂), and the nearest aromatic protons (Figures S1 and S2). The CH₂ attached to the amidic N correlates with the aromatic proton (H_g), while two rigid CH₂ groups attached to the amino N strongly correlate with the nearby aromatic proton (H_a) (Figure 1B,C). Further correlations were observed between H_g (doublet) and H_f (triplet), as well as between H_f (triplet) and H_e (doublet) (Figure 1B,C). The 2D NMR (¹H-¹H COSY) spectrum of the probe revealed correlations between two neighboring protons (Figure 1D,E and Figures S3–S5). The ¹³C NMR and HRMS spectra further confirmed the identity of the probe (see electronic Supplementary Materials).

2.2. Spectroscopic Characterization and Sensing Ability of Probe 1

To explore the probe potential in metal ion sensing, we performed fluorescence studies of probe 1 in acetonitrile (ACN)). In the range of excitation wavelengths from 390 to 500 nm, the probe exhibited maximum emission or the highest quantum yield (ø = 0.45) at 470 nm (Figure S6). Thus, this excitation wavelength was chosen for further fluorescence studies of the probe. Different trivalent (Cr³⁺, Fe³⁺, Ga³⁺, Al³⁺, and Ru³⁺), divalent (Mg²⁺, Ca²⁺, Ba²⁺, Fe²⁺, Co²⁺, Zn²⁺, Cd²⁺, Pb²⁺, Cu²⁺, Ni²⁺, and Hg²⁺), and monovalent metal ions (Ag⁺, Cs⁺, Na⁺, and K⁺) were added individually to the ACN solution containing probe 1 at 5 µM. Among these metal ions, only Cu²⁺ was able to selectively quench the green luminescence of probe 1 (Figure S7A,B and Figure 2A). Of note, compound 3, a starting material for the preparation of probe 1, was previously found to be Hg²⁺ selective [44]. Interestingly enough, the structural modification from compound 3 to probe 1 changed the probe selectivity toward Cu²⁺ from Hg²⁺. In the titration, upon the sequential addition of Cu²⁺ in ACN, the fluorescence intensity of probe 1 (5 µM, ACN) was reduced regularly and became saturated at about 60 µM [Cu²⁺] (Figure 2B and Figure S7C). This titration data indicate 1:1 stoichiometry between probe 1 and the metal ion with a high association constant (K_a = 3.98 × 10⁴ M⁻¹) (Figure S7C). The fluorescence emission intensity at 528 nm (I₅₂₈) linearly decreased with increasing [Cu²⁺], demonstrating an LOD of 0.5 µM for this sensing system (inset of Figure 2B). In addition, no significant interference was observed from other metal ions (Figure S7D).

The UV–Vis spectrum of probe 1 (50 µM, ACN) exhibited a strong band at 476 nm, likely originating from the n → π* transition of the naphthoquinoline-dione system. Among the various metal ions studied, the addition of only Cu²⁺ resulted in a significant change in the UV–Vis absorption spectrum. The absorbance maximum was blue shifted from 476 to 413 nm, along with the appearance of a shoulder at 431 nm (Figure 2C). Upon the addition of Cu²⁺, the probe 1 solution changed from yellow-green to pale green in daylight, as detected by the naked eye and reflected in the absorption spectra obtained under the same conditions (Figure 2C,D). Over the course of UV–Vis titration carried out in ACN, the absorption band of probe 1 at 476 nm decreased with an increasing amount of Cu²⁺. At the same time, a new band of the probe at 413 nm appeared, with an isosbestic point observed at 445 nm Figure 2E,F). The absorption spectrum of the probe failed to result in further changes when more than 10 equiv. of Cu²⁺ was added to the probe solution. Non-linear regression analysis of this titration data suggests the formation of a 1:1 complex between the probe and the metal ion (K_a = 3.41 × 10³ M⁻¹) (Figure 2F). The linear relationship between A₄₇₆ and [Cu²⁺] observed at the initial stage of the titration was used to obtain an LOD of 1 µM for the sensing system (Figure S8A). A ratiometric approach using the ratio of two absorption values (A₄₁₃/A₄₇₆) allowed us to detect Cu²⁺ over a wide range of concentrations (1 µM to 1 mM), with an observed LOD of 1 µM (Figure S8B). The probe 1 solutions containing different metal ions showed no noticeable alteration in absorption spectra upon the addition of Cu²⁺, indicating that these metal ions did not interfere with the Cu²⁺ detection of the probe (Figure S9). In addition, the reversibility of probe 1 in Cu²⁺ binding was demonstrated by the use of EDTA (disodium ethylene-diamine tetra-acetic acid) as a metal ion chelator. The green luminescence observed for the free probe was fully recovered after adding EDTA to the [probe 1-Cu²⁺] solution (Figure S10).

To evaluate the solvent effects on the probe’s ability to sense Cu²⁺, we prepared probe 1 solutions at 5 µM using various solvents and added 20 equiv. of Cu²⁺ to each (Figure 3 and Figure S11). The yellow-greenish luminescence was significantly quenched (60 to 98%) for the probe dissolved in ACN, THF, acetone, DCM, or CHCl₃. Upon the addition of Cu²⁺, the probe dissolved in EtOH, MeOH, ^tBuOH, or AW11 (ACN/water (1:1)) resulted in moderate luminescence quenching, whereas little change in luminescence intensity was observed for the probe in pure water, dioxane, or toluene (Figure 3C). To explore the potential utility in real sample applications, we investigated the Cu²⁺-induced changes in photoluminescence and fluorescence of the probe using a binary solvent system of ACN (A) and water (W) (Figure 4 and Figure S12). With water percentage increasing up to 60% in ACN, the fluorescence emission peak of probe 1 gradually red shifted from 528 nm (green) to 567 nm (yellow), with slight decreases in emission intensity (Figure 4A and Figure S12A). When 80% water in ACN was used as a medium, we found a 60% decrease in the fluorescence intensity of probe 1 compared to that in pure ACN. This could be due to the occurrence of substantial probe aggregation, as expected from the presence of three bulky t-butyl groups in the probe. Upon the addition of Cu²⁺, significant fluorescence quenching was observed for probe 1 dissolving in the different binary solvents (Figure 4B and Figure S12B). This quenching effect on probe fluorescence became less obvious when the water content was greater than 80% in ACN (Figure 4C). Thus, Cu²⁺ can be effectively detected via fluorescence quenching when the water content is less than 80% in ACN. We chose 20% aqueous ACN as a binary solvent system for further studies of the probe.

Upon excitation at 470 nm, the probe 1 solution (5 µM, HEPES (pH 7.4): ACN = (1:4)) exhibited an intense yellow fluorescence emission at 550 nm, along with a good quantum yield (Φ = 0.40) (Figure S13). Among the various metal ions, the probe responded only to Cu²⁺; the yellow luminescence and fluorescence emission of probe 1 were quenched (Figure 5A). Titration with Cu²⁺ showed a gradual decrease in fluorescence emission intensity of the probe at 550 nm (I₅₅₀) with increasing [Cu²⁺] (Figure 5B and Figure S14). The titration data (I₅₅₀ vs. [Cu²⁺]) are consistent with a 1:1 complex formation between probe 1 and Cu²⁺ with an association constant (K_a = 1.91 × 10⁴ M⁻¹). This association constant corresponds to nearly half of that observed for the [probe 1-Cu²⁺] complex in ACN (Figure S14), indicating weaker binding of the metal ion to the probe in aqueous ACN solutions compared to pure ACN. The probe detected Cu²⁺ at concentrations as low as 0.5 µM, while the calculated LOD was 5 nM (inset of Figure 5B and Figure S15). This LOD is comparable to or better than those of many recently reported Cu²⁺-sensing probes (Table S1). We further applied the probe for metal ion sensing in seawater and drain water from a small lake, which showed the detection of Cu²⁺ as low as 0.5 µM. A slight decrease was found in probe sensitivity for the application (Figure S16).

2.3. Mechanism of Cu²⁺ Interaction with Probe 1

To explore the molecular interaction between probe 1 and Cu²⁺, we conducted ¹H NMR titration of the probe with Cu²⁺ in DMSO-d₆. With a gradual increase in Cu²⁺ concentration from 0 to 1 equiv., most of the probe signals (e.g., N(CH₂) signals) were up-field shifted, along with notable broadening of the aromatic proton peaks (H_a, H_f, and H_e). These changes are likely due to the paramagnetic character of Cu²⁺ combined with probe aggregation upon binding to the metal ion (Figure 6 and Figure S17). This result indicates that the tertiary N of naphthoquinoline-dione and the carbonyl (-CO) oxygen of the ester group coordinate in Cu²⁺ binding. Further addition of Cu²⁺ up to 2 equiv. failed to produce a further change in the ¹H NMR signals, ensuring the 1:1 complex formation between the probe and Cu²⁺.

To further understand the molecular interactions of Cu²⁺ with probe 1, density functional theory (DFT) calculations were performed at the B3LYP/6-31G* level (Figure 7 and Figures S18–S20). Such calculations provide information about the energy levels and electronic distributions of the frontier molecular orbitals (FMOs) (i.e., HOMO [highest occupied molecular orbital] and LUMO [lowest unoccupied molecular orbital]). As the 1:1 complex between probe 1 and the metal ion was demonstrated by various experiments (fluorescence, UV–Vis, and NMR studies), we used this stoichiometry as a model for the calculations. The calculations indicate that four atoms (i.e., amine N, two O’s of two different ester groups, and carbonyl O of the amide in probe 1) coordinate with Cu²⁺ to form the tetrahedral complex (Figure 7A and Figures S18–S20). Upon complexation, the ΔE_H/L (HOMO and LUMO energy gap) was increased from 3.16 to 3.22 eV (Figure 7B), qualitatively explaining the blue shifts of the absorption and fluorescence spectra observed for probe 1 upon interaction with Cu²⁺. A minor deviation from the experimental result may be due to the fact that we carried out the calculations under the vacuum condition. The HOMO electron density of probe 1 mainly was distributed around the tertiary amino N and the amide group-containing ring of naphthoquinoline-dione, while the LUMO electron density was localized on the naphthoquinoline-dione ring. The electron densities calculated for the aHOMO and bHOMO of probe 1 complexed with Cu²⁺ were both mainly located around the carbonyl functional group of the naphthoquinoline-dione ring. However, the LUMO electron density of the probe moved to the metal binding site upon complexation with the metal ion (Figure 7B). This electron transfer is most likely responsible for the effective quenching of the green/yellow fluorescence emission of probe 1 upon Cu²⁺ binding (Scheme 2).

2.4. Practical Application

To examine the practical utility, we first investigated the ability of the probe to detect Cu²⁺ in a solid state. For this purpose, various solid surfaces, such as a TLC plate, cotton, and Whatman filter paper, were used for probe adsorption. The probe adsorbed on these solid supports yielded bright yellow luminescence under 365 nm illumination (Figure 8). The bright yellow ‘Cu²⁺’ written on the TLC plate using the probe 1 solution (5 µM, ACN) was quenched following the application of Cu²⁺ spray (20 µM) (Figure 8A). Similar results were obtained for probe 1 on cotton buds and silica gels (Figure 8B,C). Yellow luminescence of the spots on the filter paper and TLC plate gradually weakened with increasing amounts of Cu²⁺. The filter paper and TLC plate-supported probe 1 showed the ability to detect 0.5 µM and 1 µM Cu²⁺, respectively (Figure 8D,E).

Probe 1 was further applied to detect Cu²⁺ in live HeLa cells. For this experiment, HeLa cells were incubated with probe 1 over a range of concentrations (0–20 µM) for 30 min. More than 80% of the cells treated with the probe survived under the conditions, indicating that the probe is only slightly toxic to the cells (Figure S21). Under irradiation at 488 nm, the cells without the probe displayed negligible emission, but incubation of the cells with probe 1 (5 µM) produced an intense green emission in the cytoplasm (Figure 9). This optical change confirms that the probe penetrated the plasma membranes but not the double-layered nuclear membranes. When the HeLa cells containing probe 1 were treated with 20 µM Cu²⁺, the green emission of the cells was completely quenched. This quenching likely originates from the complexation of the probe with metal ions in the cytoplasm. The bright-field images indicate that these cells were viable throughout the experiment. Hence, intracellular Cu²⁺ can be detected in a non-toxic manner using probe 1, which is crucial for bio-applications.

3. Materials and Methods

3.1. General Chemicals and Materials

All reagents and solvents used in the experiments, including t-butyl bromoacetate, metal perchlorates, tetrabutylammonium bisulfate (TBAHSO₄), dimethyl sulfoxide (DMSO), THF, acetonitrile (ACN), and potassium carbonate (K₂CO₃), were purchased from Aldrich (St. Louis, MO, USA) (analytical grade) and used without further purification. The compound 1-amino-3H-naphtho[1,2,3-de]quinoline-2,7-dione (compound 3) was synthesized according to a procedure reported previously [44]. All the tested metal ions were used in the perchlorate forms (M(ClO₄)_x), except Ru³⁺ and Au³⁺, which were used in the form of chloride salts. Stock solutions of the metal ions were prepared in ACN (1 mM) or water (10 mM) depending on the sensing medium (ACN or HEPES (pH 7.4: ACN (1:4)) used for metal ion detection. All the fluorescence spectra were recorded on a ChronosBH fluorescence lifetime spectrometer. UV–Vis (ultraviolet–visible) spectra were recorded on a Shimadzu UV-2600PC spectrophotometers with a quartz cuvette (Shimadzu, Kyoto, Japan). The cell holder was maintained at 25 °C. The ¹H and ¹³C nuclear magnetic resonance (NMR) spectra of all new compounds were recorded on a Bruker 400 MHz spectrophotometer using CDCl₃ or DMSO-d₆ as a solvent and tetramethylsilane (TMS) as an internal standard. The NMR data are reported as chemical shifts in ppm (parts per million) (δ), multiplicities (s = singlet, d = doublet, m = multiplet), coupling constants (Hz), integration, and interpretation. All spectrophotometric titration curves were fitted with gnu plot software 5.0.

3.2. Photo-Physical Studies: Parameters and Conditions

All metal ion-induced color changes, UV–Vis, and fluorescence spectra were obtained in ACN (CH₃CN) or a binary solvent (HEPES (pH 7.4): ACN (1:4)). All absorption and fluorescence scans were saved as ACSII files and further processed in Excel^TM to produce the graphs shown. A stock solution of probe 1 was prepared at 1 mM in ACN and was used for photo-physical studies following appropriate dilution with ACN or a binary solvent (HEPES (pH 7.4): ACN (1:4)). The UV–Vis and fluorescence studies were performed using the probe at 5 and 50 µM, respectively. The titration experiments were carried out by adding Cu²⁺ to the probe 1 solution until the absorption/fluorescence spectra of the solutions reached saturation. The association constants between the probe and Cu²⁺ were determined by fitting the absorption and fluorescence spectral data obtained from the titration experiment of probe 1 with Cu²⁺. The titration data were fitted with the global analysis program gnu plot software.

3.3. Theoretical Calculations

Energy-optimized conformations of probe 1 and its Cu²⁺ complex were obtained via gradient-correlated DFT (density functional theory) calculations using Becke’s three-parameter exchange functional [45] and the Lee–Yang–Parr (B3LYP) exchange–correlation function [46] with 6-31G* basis sets for C, H, N, and O. B3LYP/6-31G* calculations were employed for excited-state optimization. For simplicity, all calculations were carried out in a vacuum to avoid the solvent effect. All stationary points were verified as the minima via calculations from Hessian and harmonic frequency analyses [47,48].

3.4. Synthesis of Probe 1

3.4.1. Tert-butyl 2-(1-amino-2,7-dioxo-2,7-dihydro-3H-cnaphtho[1,2,3-de]quinolin-3-yl)acetate (2)

A solution of 1-amino-3H-naphtho[1,2,3-de]quinoline-2,7-dione (compound 3, 1.13 g, 5 mmol) in DMSO (10 mL) containing K₂CO₃ (1.38 g, 10 mmol) and TBAHSO₄ (phase transfer catalyst; 2%) was stirred at room temperature for 20 min. Next, tert-butyl bromoacetate (0.975 mg, 5.0 mmol) was added to the reaction mixture, and the resulting solution was stirred at room temperature for 48 h. The progress of the reaction was monitored by TLC; upon reaction completion, a brine solution was added. A solid material collected by filtration was identified as unreacted compound 3. The filtrate was concentrated and purified by column chromatography to yield the product (2) (50%) as a yellow solid. ¹H NMR (400 MHz, CDCl₃): δ 1.49 (s, 9H, 3 × CH₃), 5.13 (s, 2H, CH₂), 5.89 (s, 2H, NH₂), 7.35 (d, J = 8.0 Hz, 1H, ArH), 7.51–7.57 (m, 2H, 2 × ArH), 7.76 (t, J = 8.0 Hz, 1H, ArH), 8.33 (d, J = 8.0 Hz, 1H, ArH), 8.51 (d, J = 8.0 Hz, 1H, ArH), and 8.54 (d, J = 8.0 Hz, 1H, ArH); ¹³C NMR (100 MHz, CDCl₃): δ 28.24, 46.02, 83.40, 107.65, 117.91, 120.93, 123.85, 125.96, 126.00, 128.11, 128.91, 131.85, 132.62, 133.54, 135.57, 136.77, 159.03, 166.82, and 182.69. HRMS (M + H⁺) = 377.1499; C₂₂H₂₁N₂O₄ (theoretical (M + H⁺) = 377.1501).

3.4.2. Probe 1

Compound 2 (190 mg, 0.5 mmol) was stirred vigorously in THF (20 mL) at room temperature, followed by the addition of K₂CO₃ (1.38 g, 10 mmol) and TBAHSO₄ (phase transfer catalyst; 2%) for one hour under an N₂ atmosphere. Then, t-butylbromoacetate (1.95 g, 10 mmol) was added dropwise to the reaction mixture, followed by stirring at room temperature for 72 h. TLC was applied to monitor the progress of the reaction; upon reaction completion, a brine solution was added. This solution was extracted with an equal portion of DCM (x2) to extract the organic compounds completely. The solution was dried with anhydrous Na₂SO₄, concentrated, and subjected to column chromatography to afford the pure product (1) (50%) as a red solid. ¹H NMR (CDCl₃, 400 MHz): δ 1.32 (s, 9H, 3 × CH₃), 1.38 (s, 9H, 3 × CH₃), 1.45 (s, 9H, 3 × CH₃), 3.95 (d, J = 18.0 Hz, 1H (NCH), one H of rigid two NCH′H), 4.93 (d, J = 17.2 Hz, 1H (NCH′), one H of rigid two NCH′H), 5.15 (s, 1H of NCH₂, the other H of this CH₂ could exchange with D due to its H-bonding with –C=O (oxygen)), 5.16 (d, J = 17.2 Hz, 1H (NCH′), one H of rigid two NCH′H), 5.63 (d, J = 18.0 Hz, 1H (NCH), one H of rigid two NCH′H), 7.29 (d, J = 8.0 Hz, 1H ArH), 7.53 (t, J = 8.0 Hz, 1H ArH), 7.55–7.60 (m, 2H, 2 × ArH), 8.32 (d, J = 8.0 Hz, 1H ArH), and 8.37–8.39 (m, 2H, 2 × ArH); ¹H NMR (DMSO-d₆, 400 MHz): δ 1.25 (s, 9H, 3 × CH₃), 1.32 (s, 9H, 3 × CH₃), 1.43 (s, 9H, 3 × CH₃), 4.22 (d, J = 18.0 Hz, 1H (NCH), one H of rigid two NCH′H), 5.12 (q, J = 17.6 Hz, 2H, 2 × (NCH′)), 5.28 (d, J = 18.0 Hz, 1H (NCH′), one H of rigid two NCH′H), 5.67 (s, 1H of NCH₂, the other H of this CH₂ could exchange with D due to its H-bonding with –C=O (oxygen)), 7.64 (t, J = 8.0 Hz, 1H ArH), 7.71–7.75 (m, 2H, 3 × ArH), 8.18 (d, J = 8.0 Hz, 1H ArH), and 8.25–8.27 (m, 2H, 2 × ArH); ¹³C NMR (CDCl₃, 100 MHz): δ 28.03, 28.19, 45.60, 56.48, 68.17, 82.11, 82.95, 83.21, 111.17, 117.55, 118.89, 123.57, 126.43, 127.75, 127.93, 128.27, 128.62, 129.32, 131.40, 134.26, 135.03, 157.67, 166.99, 168.93, 169.22, and 182.65. HRMS (M + H⁺) = 605.2861; C₃₄H₄₀N₂O₈ (theoretical (M + H⁺) = 605.2857).

3.5. Live Cell Imaging

HeLa cells were obtained from the KCLB (Korean Cell Line Bank, Seoul, Republic of Korea) and grown in MEM (Minimum Essential Media) supplemented with 10% fetal bovine serum (FBS), 100 µg/mL streptomycin, and 100 IU/mL penicillin. The cells were maintained in a humidified incubator at 37 °C at 5% CO₂. A total of 3.8 × 10⁴ cells were seeded onto a 13 mm glass bottom of a 35 mm confocal dish and grown for one day/24 h (until 60–70% confluence). The probe stock solution was prepared by dissolving probe 1 in DMSO. Experiments were performed in triplicate in FBS and antibiotic-free medium as follows. HeLa cells were incubated with probe 1 (5 µM) at 37 °C at 5% CO₂ for half an hour. The final DMSO concentration was 0.5% in the sample. After washing the incubated sample twice with 1 x phosphate-buffered saline (PBS) (pH = 7.4), the cells were further incubated with Cu²⁺ (20 µM, H₂O) for half an hour under the same conditions and were washed three times with 1 × PBS. A K1-Fluo confocal laser scanning microscope with a 40× oil immersion objective lens was used to obtain confocal microscopy images. Bright-field images after treatment with probe 1 and Cu²⁺ were obtained to evaluate the cell viability.

4. Conclusions

We prepared a new naphthoquinoline-dione-based probe 1 containing three ester groups starting from compound 3 with no ester groups. While compound 3 was selective for Hg²⁺ sensing, the new probe 1 exhibited high selectivity for Cu²⁺ in ACN, as well as in a 20% aqueous ACN solution. The probe displayed a color change from yellow to colorless upon selective interaction with Cu²⁺. A ratiometric approach using two absorption values (A₄₁₃/A₄₇₆) allowed the detection of Cu²⁺ at as low as 1.0 µM. In addition, the yellow fluorescence emission of the probe in the aqueous solution (HEPES (pH 7.4): ACN = 1:4) was selectively quenched in the presence of Cu²⁺ among various metal ions. Hence, probe 1 provides a dual sensing method for Cu²⁺ detection (absorption and fluorescence emission) and is chromo-fluorogenic. Fluorescence titration of the probe with Cu²⁺ produced a low calculated LOD of 5 nM. Fluorescence, UV–visible, and ¹H-NMR titration studies, in addition to DFT calculations, supported the 1:1 complex formation between Cu²⁺ and probe 1. This probe showed the ability to detect Cu²⁺ in a real sample (sea/drain water) with an observed LOD of 0.5 µM, which signifies its potential for on-site monitoring of the metal ion. In addition, the probe was successfully used for Cu²⁺ detection after adsorption onto solid supports, such as cotton, filter paper, a TLC plate, and silica gels. Importantly, the probe was able to penetrate cell membranes and detect Cu²⁺ in the cytoplasm of HeLa cells. Therefore, this study provides insight into the development of new chromo-fluorogenic heteroaromatic sensors for the detection of different analytes.