Rhodamine-Based Dual Chemosensor for Al3+ and Zn2+ Ions with

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Article Cite This: ACS Omega 2019, 4, 6864−6875

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Rhodamine-Based Dual Chemosensor for Al3+ and Zn2+ Ions with Distinctly Separated Excitation and Emission Wavelengths Ankita Roy,† Uday Shee,† Abhishek Mukherjee,‡ Sushil Kumar Mandal,§ and Partha Roy*,† †

Department of Chemistry, Jadavpur University, Jadavpur, Kolkata 700 032, India Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067, India § Department of Ecological Studies and International Centre for Ecological Engineering (ICEE), University of Kalyani, Kalyani, Nadia 741235 West Bengal, India Downloaded via 185.14.194.102 on April 16, 2019 at 22:48:46 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: A rhodamine-based compound, 2-(2-((3-(tert-butyl)-2-hydroxybenzylidene)amino)ethyl)3′-6′-bis(ethylamino)-2′,7′-dimethylspiro[indoline-1,9′-xanthen]-3-one, (HL-t-Bu), is reported here as a dual chemosensor for Zn2+ and Al3+ ions. This compound has been synthesized under mild conditions with high yield and characterized by elemental analysis and different standard spectroscopic methods. Its structure has been confirmed by single-crystal X-ray diffraction analysis. It acts as a fluorescent dual sensor for Zn2+ and Al3+ in 10 mM HEPES buffer in the methanol/water mixture (9:1, pH = 7.4) with wellseparated excitation and emission wavelengths. The fluorescence intensity at 457 nm of HL-t-Bu increases by ∼7 folds in the presence of 1 equiv of Zn2+ when it is excited at 370 nm. With the excitation at 500 nm, the emission intensity of the probe at 550 nm increases massively (∼650 times) in the presence of 1 equiv of Al3+. No other metal has any significant effect on the enhancement of the emission intensity of the probe in the detection process for either of the metal ions. The quantum yield of HL-t-Bu enhances significantly in the presence of Zn2+ and Al3+. Rhodamine derivatives are generally colorless and nonfluorescent when the spirolactam ring is closed, and they are pink in color and highly fluorescent when it exists in the ring-open form. In the presence of Al3+, the probe is pink and highly fluorescent, indicating that this metal ion is able to the open spirolactam ring of the probe. However, Zn2+ is not able to open the spirolactam ring, but it is coordinated through phenolic oxygen and imine nitrogen (salicylaldehyde unit) of the Schiff base restricting PET (photoinduced electron transfer) and imposing CHEF (chelation-enhanced fluorescence) to enhance the emission intensity of the probe. 1H NMR, FT-IR, elemental analysis, and pH-dependent studies support these mechanisms. Limit of detection values are in the nanomolar range for both the metal ions, confirming very high sensitivity of the probe. The probe has been used in cell imaging studies for both of the metal ions.

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INTRODUCTION

In recent times, there are considerable number of reports on the synthesis and applications of fluorescent chemosensor for several cations3 and anions.1c However, most of them are for a single analyte. Less attention has been paid to develop dual or multi-analyte chemosensors. Dual sensors generally have two different emission maxima with the same or separate excitation wavelength(s). Signal magnitude may increase in the presence of one analyte, whereas a second analyte can quench the signal. In another instance, intensities at two emission maxima increase. Chemosensors, which are useful for two or more analytes, have advantages over the chemosensors for single species. They are usually able to produce alterations in the emission intensity and wavelength at the same time, which may offer diverse fluorescence signals or chance to amplify the responding signals. This will give opportunity for simultaneous monitoring of multiple analytes and/or to improve the two most important properties i.e. selectivity and sensitivity of the sensors. These dual chemosensors, on the other hand, can save

There is considerable interest of the researchers from all over the globe on the development of chemosensors for the detection of several analytes including cations and anions.1 Fluorescence spectroscopy is one of the most widely used techniques to design and monitor the progress of chemosensors for the small ions because this technique provides useful advantages such as easy operation, high sensitivity, high selectivity, low instrumental cost, nondestructive sample analysis, etc. The signal of a fluorescent sensor is usually monitored as a change of its emission intensity, fluorescence lifetime, or a shift of emission wavelength. Mostly, fluorescence signal transduction occurs through inhibition of photoinduced electron transfer (PET), chelation-enhanced fluorescence (CHEF), fluorescence resonance energy transfer (FRET), intermolecular charge transfer (ICT), twisted intramolecular/ intermediate charge transfer (TICT), and planar intramolecular charge transfer (PICT).2 Other two recently developed phenomena regarding sensing mechanisms are excited-state intramolecular proton transfer (ESIPT) and aggregation-induced emission (AIE) mechanisms.2 © 2019 American Chemical Society

Received: February 20, 2019 Accepted: March 25, 2019 Published: April 16, 2019 6864

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Scheme 1. Synthesis of the Probe

there is less chance of interference in the detection process. The pH of the medium may influence the sensing procedure as well.5d There is a possibility to improve the properties of the probes. A new probe could offer well-separated excitation and emission wavelengths for both the cations. Aluminum is the most prevalent metal in the earth’s crust. It is not necessary for biological processes, but excess accumulation in the human body could be toxic. It could enter in our body through many human activities. It is used in food, textile, and paper industries. It is used to wrap food items and as a container for packing food. Utensils made of aluminum are widely used for domestic purposes. Its relevance with nerve-related diseases like Alzheimer’s disease and Parkinson’s disease has been established.9 Thus, identification and quantification of this metal ion becomes essential. On the other hand, Zn2+ is the second most abundant metal present in the human body. It is related to many metalloenzymes. Its role is important in various processes such as cellular metabolism, regulation of metalloenzymes, gene transcription, neural signal transmission, apoptosis, etc.10 However, excess zinc in the human body has been related to many health issues such as Alzheimer’s disease, Parkinson’s disease, etc.11 Thus, the development of a chemosensor for these two metal ions is important. In this respect, a rhodamine derivative could be a potential candidate as the dual chemosensor. These compounds have long absorption and emission wavelengths, high absorption coefficient and quantum efficiency, and good tolerance to photobleaching.1d It is being used widely as a dye, biomarker, and fluorophore probe.1d,e It has been established that many derivatives of rhodamine undergo equilibrium between spirocyclic and ring-open amide forms, and the two forms always show completely different fluorescent properties. The commonly existing spirocyclic forms of these dyes are generally nonfluorescent, whereas the ring-open amide compound is a highly fluorescent one. There are few reports on fluorescent chemosensors based on rhodamine 6G.1d,e,12 Rhodamine 6G-based chemosensors have been constructed from either 2-(2-aminoethyl)-3′,6′-bis(ethylamino)-2′,7′-dimethylspiro[isoindoline-1,9′-xanthen]-3-

time, cost, and effort to develop an additional chemosensor for the second analyte. With a dual chemosensor, we can monitor the presence of two species simply by changing the excitation wavelengths and/or observing the emission properties. Thus, there is a need for new and effective dual chemosensors. There are few reports on dual chemosensors for some cations such as Fe3+ and Hg2+ ions,4 Zn2+ and Al3+ ions,5 Cu2+ and Zn2+ ions,6 Mg2+ and Zn2+ ions,7 etc.8 Recently, Liu et al. reported a Schiff base fluorescent sensor based on picolinohydrazide and 4-(diethylamino)salicylaldehyde for Zn2+ and Al3+ ions, which provides the same binding pocket for both the ions.5a A diarylethene derivative containing 3-(4-methylphenyl)-1H-pyrazol-5-amine behaves as a fluorescent dual chemosensor for these metal ions with a single excitation wavelength, and it also provides the same binding donor atom for both the cations.5b N-salicylidenehydrazide ligands have been used for the detection of Al3+ with large Stokes shift, whereas these compounds have been used for sensing of Zn2+ and Al3+ ions in the solid state.5c A probe consisting of 2-hydroxynaphthaldehyde and glutamide moieties has been reported for as a dual sensor for these metal ions with the same excitation (385 nm) and emission (445 nm) wavelengths.5d 7-(2,4-Dihydroxybenzylideneimino)-4-methylcoumarin shows high selectivity toward Zn2+ and Al3+ ions. The fluorescence enhancement of the probe occurs in the presence of Al3+ at 427 nm (λex = 357 nm) and Zn2+ at 489 nm (λex = 405 nm).5e 3-Hydroxymethyl-5methylsalicylaldehydenaphthyl-hydrazone behaves as a dual chemosensor for these metal ions with the same excitation (398 nm) and emission (498 nm) wavelengths as it provides a similar coordination environment.5f We have recently reported dual sensors based on quinoline derivatives for these cations with various excitation and emission wavelengths.5g A probe based on biphenyl and rhodamine has been recently reported for multimetal ion recognition including these metal ions.5h,i However, these probes possess some limitations. They show low water solubility, low selectivity, low sensitivity, etc. Some of them show the same excitation and emission wavelength for both the metal ions. This can seriously interfere the detection of either of the cations. If the excitation and emission wavelengths differ significantly for each of the metal ions, then 6865

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one (L1) or 2-amino-3′,6′-bis(ethylamino)-2′,7′dimethylspiro[isoindoline-1,9′-xanthen]-3-one (L2) containing an −NH2 functionality (Scheme 1). Few chemosensors have been prepared from the reaction between L1 and various aldehydes, and they are selective for various cations.13 Depending on the substituent(s) on the aldehyde, the derivative could become selective for a particular cation.13e In other words, the substitution on the aldehyde can influence the metal ion selectivity. We have recently reported two fluorescence chemosensors based on L1 and salicylaldehyde derivatives that show completely different metal ion selectivities. The 5-bromosalicylaldehyde derivative is selective for trivalent cations,13a whereas the 5-methylsalicylaldehyde derivative13e is selective for the Al3+ ion. Different substitutions on the salicylaldehyde unit make it a selective chemosensor for a single analyte but for different metal ions. However, no compound based on rhodamine 6G derivative has been reported as a dual sensor. We report here a chemosensor based on the reaction between L1 and 3-tert-butylsalicylaldehyde, leading to the formation of 2-(2-((3-(tert-butyl)-2-hydroxybenzylidene)amino)ethyl)-3′-6′-bis(ethylamino)-2′,7′-dimethylspiro[indoline-1,9′-xanthen]-3-one (HL-t-Bu) (Scheme 1). We wanted to check the effect of variation of substitutions on the aldehyde ring. So, we use here the tertiary butyl group instead of the bromo or methyl group in the aldehyde ring hoping for different results. Surprisingly, it has been found as a dual fluorescent chemosensor for Zn2+ and Al3+ ions. HL-t-Bu has been synthesized under mild conditions. Its characterization has been done with elemental analysis, different spectroscopic techniques, and single-crystal X-ray analysis. Exploration of the spectral properties of HL-t-Bu has been performed. Cell imaging studies have been performed for both of the ions.

Figure 1. ORTEP of HL-t-Bu with atom numbering scheme (30% thermal ellipsoid probability). Hydrogen atoms were omitted for clarity.

parameters are given in Table S1. Selected bond length and bond angles of the compound are given in Table S2. The presence of the rhodamine platform and salicylaldehyde moiety can be easily observed in the compound. It is also clear that the compound is not planar. The presence of an azomethine moiety has been indicated from the shorter C−N bond distance compared to other C−N bond distances. Absorption Spectral Studies. The spectrophotometric titrations of HL-t-Bu have been carried out with Al3+ and Zn2+ ions separately, and their spectral changes have been recorded. UV−vis spectra of the compound (40 μM) have been recorded in the absence and in the presence of different concentrations of Al3+ (0−48 μM) and Zn2+ (0−48 μM) in 10 mM HEPES buffer in the methanol/water mixture (9:1, pH = 7.4) at room temperature (Figure 2). In the absence of any metal ion, HL-tBu shows a band at 303 nm, and there is no band in the visible region due to the presence of the spirolactam ring. Absorption spectra of the compound show significant differences in the presence of Al3+ ion and Zn2+ ion separately, and these two metal ions behave completely different toward the probe. However, other metal ions could not produce any marked changes in the spectrum (Figure S4). With the addition of Al3+ ion, a new band in the UV−vis spectrum of HL-t-Bu emerges at 528 nm (Figure 2A), while in the presence of Zn2+, a new band originates at 375 nm (Figure 2B). In both cases, the intensities of newly generated bands increase with increasing concentrations of metal ions in the solutions and reach saturation when one equivalent of each of the metal ions are added. The appearance of new peak at 528 nm may be attributed to the opening of the spirolactam ring of HL-t-Bu in the presence of Al3+ ion and formation of the HL-t-Bu−Al3+ complex.2,3 However, in the presence of Zn2+, spirolactam ring opening does not occur, but the probe molecule undergoes chelation with Zn2+ (also supported by other data, vide infra). Thus, the aluminum ion is able to open the spirolactam ring, and the zinc ion could not open the ring but forms a chelate with the probe. The probe shows a very distinct change in color during the interaction with Al3+ and Zn2+ (vide infra). Al3+ changes the yellow color of HL-t-Bu solution into a strong red color. However, Zn2+ shows no vivid change in the color of HL-t-Bu solution under visible light, indicating that the lactam

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RESULTS AND DISCUSSION Synthesis and Structure of HL-t-Bu. Synthesis of HL-tBu has been done following Scheme 1. N-(Rhodamine 6G)lactam-ethylenediamine (L1) has been synthesized following a previously published method.16 Condensation between 1 equiv of L1 and 1 equiv of 3-tert-butylsalicylaldehyde in methanol enables the formation of HL-t-Bu with high yield. The compound has been characterized by elemental analysis and different spectroscopic methods including FT-IR, ESI, and NMR. Its structure has been confirmed by single-crystal X-ray diffraction analysis. The FT-IR spectrum of the compound (Figure S1) shows peaks at 3427 cm−1, indicating the presence of phenolic OH group. Peaks in the range of 2970 to 2940 cm−1 indicate the presence of CH and CH2 moieties. Bands in the range of 1652 to 1619 cm−1 show the presence of >CO and >CN moieties. In the ESI mass spectrum of the compound, the m/z peak at 617.56 is attributed to the presence of [HL-t-Bu + H+] species (Figure S2). Discussion on 1H NMR spectra is explained below. The 13C NMR spectrum of the compound is given in Figure S3. Both the spectra support the formation of the compound. All of the spectral analyses of HL-t-Bu have been performed in the presence Al3+ and Zn2+, and discussion about these analyses is explained below. Single crystals of HL-t-Bu suitable for X-ray diffraction analysis have been obtained from slow evaporation of a methanolic reaction mixture. An ORTEP view of HL-t-Bu is given in Figure 1. Data collection and structure refinement 6866

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Figure 3. Fluorescence intensity of HL-t-Bu (40 μM) upon addition of (A) 0, 4, 8, 12, 16,..., 40, 44, 48 μM Al3+ and (B) 0, 4, 8, 12, 16,..., 40, 44 μM Zn2+ in 10 mM HEPES buffer in H2O/methanol (1:9 (v/ v), pH 7.4) at room temperature (λex = 500 nm for Al3+ and 370 nm for Zn2+). Inset: nonlinear plot of fluorescence intensity (at 550 nm) versus [Al3+] in (A) and nonlinear plot of fluorescence intensity (at 370 nm) versus [Zn2+] in (B).

Figure 2. Absorption spectra of HL-t-Bu (40 μM) in the presence of (A) Al3+ and (B) Zn2+ (0, 4, 8, 12, 18,..., 40, 44, 48 μM) in 10 mM HEPES buffer in H2O/MeOH (1:9 (v/v), pH 7.4) at room temperature.

ring of rhodamine is still in the closed state even after HL-t-Bu combined with Zn2+. Emission Spectral Studies. Fluorescence spectra of HL-tBu (40 μM) have been recorded in the absence and in the presence of different concentrations of Al3+ (0−48 μM) and Zn2+ (0−44 μM) in 10 mM HEPES buffer in the methanol/ water mixture (9:1, pH = 7.4) at room temperature (Figure 3). The study reveals that upon excitation at 500 nm, the free HLt-Bu itself is almost nonfluorescent, but with the addition of Al3+, a peak in the fluorescence spectrum emerges at 550 nm (Figure 3A). Upon gradual addition of Al3+, the fluorescence intensity enhances at 550 nm, and this enhancement saturates gradually when 1 equiv of Al3+ has been added. This results in ∼650 times fluorescence intensity enhancement due to an Al3+-induced opening of the spirolactam ring of the probe and subsequent formation of the HL-t-Bu−Al complex. The recognition is completed immediately (within few seconds) after the interaction of Al3+ ion. However, the fluorescence spectral behavior of the probe is different when spectra are recorded in the presence of Zn2+. Different excitation wavelength has been used in the titration with zinc ion. Upon excitation at 370 nm, the probe shows a very weak fluorescence intensity at 454 nm. In the presence of Zn2+, the intensity of the probe shifts slightly to 457 nm (Figure 3B) with increasing intensity. The intensity enhances with increasing concentration of Zn2+ and reaches to a plateau when 1 equiv of Zn2+ is added. This study indicates that complexation of Zn2+ occurs with HL-t-Bu without the opening of the spirolactam ring. The fluorescence intensity of HL-t-Bu, in this case, increases by about 7-fold during complexation with Zn2+. Insets of Figure 3A,B show that the

fluorescence response of the probe in the presence of both cations is linear to some concentration ranges. For Zn2+, linearity is observed up to 3.6 × 10−5 M, while for Al3+, the linearity results are 0−1.2 × 10−5 M and 1.2 × 10−5 to 4.0 × 10−5 M. Fluorescence spectra of HL-t-Bu (40 μM) have been obtained in the presence of five equivalents of different metal ions in 10 mM HEPES buffer in the methanol/water mixture (9:1, pH = 7.4) at room temperature (Figure 4 and Figures S5 and S6). Two different excitation wavelengths such as 370 and 500 nm have been used, and emission has been monitored at 457 and 550 nm, respectively. As the fluorescence intensity of the probe increases at 457 nm in the presence of Zn2+ ion (λex = 370 nm), we monitor emission at 457 nm to check whether other metal ions have any effect on the emission intensity of the probe. For the same reason, we also monitor emission at 550 nm as the intensity of the probe increases in the presence of Al3+. It is clear from Figure 4A that only aluminum is able to increase the fluorescence intensity of the probe in few hundred of folds, whereas other metals including Zn2+ could not make any significant alteration in the fluorescence intensity of the probe. When emission is monitored at 457 nm, it is evident that only the Zn2+ ion can induce considerable enhancement in the emission intensity. Other metal ions could not induce a significant change. These indicate that HL-t-Bu is selective for Al3+ when emission is monitored at 550 nm and for Zn2+ when emission is observed at 457 nm. There is almost no 6867

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formation of the 1:1 complex is furthered supported by the mass spectral analysis. The free probe shows an m/z peak at 617.38, indicating the formation of [HL-t-Bu + H]+ (Figure S2). However, the probe exhibits m/z peaks at 704.25 and 760.16 in the presence of Al(NO3 )3 and Zn(NO 3)2 , respectively. The peak at 704.25 may be attributed to the formation of [Al(L-t-Bu)NO3] in which the aluminum complex is formed with the ring-opened ligand (Figure S13). The other peak is assigned to the formation of [Zn(HL-tBu)(NO3)(H2O)]+ species (Figure S14). Attempts have been made to get suitable single crystals for X-ray analysis of the probe with both the metal ions; unfortunately, we could not obtain the crystals. However, the products have been isolated, and they have been characterized by FT-IR spectra (Figure S1) and elemental analysis. The peak at 3422 cm−1 for the phenolic OH group is retained for the Zn complex, whereas no such peak is observed for the Al complex. This indicates the retention of phenolic proton in the Zn complex, while deprotonation occurs with Al3+. From this spectral analysis, it has been observed that the characteristic stretching frequency of “CO” bond of the amide of HL-t-Bu−spirolactam ring appears at 1652 cm−1. This peak is completely disappeared in the FT-IR spectrum of the Al complex, in which a new peak is observed at 1642 cm−1. This observation confirms the opening of the spirolactam ring of the probe due to the complexation with Al3+ and also indicates that the oxygen of the spirolactam ring is attached to the Al3+. However, in the case of the Zn complex, the observed stretching frequency at 1657 cm−1 confirms the retention of the basic spirolactam structure of HL-t-Bu in the complex. The band at 1634 cm−1 may be attributed to the presence of the CN moiety in the free probe and is shifted to 1623 and 1605 cm−1 in the Zn and Al complex, respectively, indicating retention of the CN bond in the complex. A sharp peak at 1315 cm−1 indicates the presence of the nitrate group in the zinc complex.14 A corresponding peak in the aluminum complex is observed at 1305 cm−1. The composition of the isolated compounds has been determined by elemental analysis. Results are shown in Table S3. It is clear that the elemental analysis supports the formation of the proposed complexes as indicated in mass and infrared spectral analyses. The formation of the complexes has been further confirmed by NMR spectra (vide infra). The binding constants for the probe with both Al3+ and Zn2+ ions have been determined using the Benesi−Hildebrand equation15

Figure 4. Fluorescence intensity of HL-t-Bu (40 μM) at (A) λem = 550 nm and (B) at λem = 457 nm in the presence of five equivalents of different metal ions in 10 mM HEPES buffer in H2O/MeOH (1:9 (v/ v), pH 7.4) at room temperature. HL in the diagram denotes HL-tBu.

interference for the detection of Al3+ ion in the presence of Zn2+ ion or vice versa. Other metal ions may have an effect on the emission intensity of the probe with equimolar Al3+ and Zn2+ ions. To check the effect of other metal ions, we have monitored the emission intensity at 457 nm (λex = 370 nm) and 550 nm (λex = 550 nm) when the probe is mixed with Zn2+ and Al3+ ions, respectively (Figures S7 and S8). In the case of HL-t-Bu, in the presence of an equivalent amount of Zn2+, it could be seen that most of other metals have little effect. Fe3+, Cu2+, and Co2+ ions reduce the emission intensity to some extent. However, for Al3+, a similar tendency has been observed, that is, Fe3+, Cu2+, and Co2+ ions can decrease the emission intensity of the probe with Al3+ in small magnitude. This experiment shows that HL-t-Bu could be used to detect both the metal ions even in the presence of other relevant metal ions. Color changes of HL-t-Bu are instantaneous in the presence of both Al3+ and Zn2+ ions. Within few seconds, color changes could be marked under visible and UV light, signifying immediate recognition of both the cations. We have recorded fluorescence spectra of HL-t-Bu in the presence of Al3+ and Zn2+ in some time intervals to get information about the stability and time-dependent interaction of HL-t-Bu with these cations (Figures S9 and S10). It could be seen from the figures that these interactions are the same up to 3 h, indicating the stability of the probe and the complexes with these cations. Binding of HL-t-Bu with Zn2+ and Al3+. To determine the binding mode of HL-t-Bu with both metal ions, the stoichiometry of the complex formation has been determined by Job plot analysis (Figures S11 and S12). The analysis shows that both metal ions form a complex (1:1) with the probe. The

1/(F − F0) = 1/(Fmax − F0) + (1/K a[C]){1/(Fmax − F0)}

where F0, F, and Fmax are the emission intensities of HL-t-Bu in the absence of any metal ion, with the intermediate metal ion concentration, and with the saturation concentration of Mn+, respectively, [C] is the concentration of Mn+, and Ka is the binding constant. Plots are given in the Supporting Information (Figures S15 and S16). The binding constant values have been determined to be 9.38 × 103 and 4.75 × 104 M−1 for Al3+ and Zn2+ ions, respectively. Sensitivity of the Probe. Sensitivity of a probe is dependent on the limit of detection (LOD) value. The lower the LOD value, the better the sensitivity of the probe. The LOD of HL-t-Bu toward both the metal ions has been determined following the 3σ method.16 Details are given in the Supporting Information (Figures S17 and S18). The values of LOD have been determined to be 10.98 and 76.92 nM for Al3+ 6868

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and Zn2+, respectively. This indicates that the probe is highly sensitive toward both metal ions. Determination of Quantum Yield and Lifetime. Quantum yields of HL-t-Bu and its complexes with Al3+ and Zn2+ have been determined using two standards (Table S4). Two standards have been used because increments in absorption and emission maxima of the probe are different in the presence of Al3+ and Zn2+ ions. The quantum yield of the free probe is very small (0.0013, wrt rhodamine 6G; 0.0704, wrt quinine sulfate). The quantum yield increases massively in the presence of both metal ions. The quantum yield of HL-t-Bu is 0.635 in the presence of 1 equiv of Al3+ where rhodamine 6G is used as the standard, whereas the quantum yield of the probe is 0.508 in the presence of 1 equiv of Zn2+ wrt quinine sulfate as the standard. Thus, the quantum yield increases by ∼500 and ∼7 times in the presence of Al3+ and Zn2+ ions, respectively. To study the photophysical properties of HL-t-Bu, timeresolved emission experiments have been performed in the absence and in the presence of Al3+ with an excitation wavelength of 450 nm (Figure S19) and 370 nm in the presence of Zn2+ (Figure S20). The lifetime data are given in Table S4. The lifetime of HL-t-Bu is 2.76 ns, and that in the presence of aluminum is 2.77 ns. Lifetimes of the probe and probe with zinc are 1.20 and 3.68 ns, respectively. HL-t-Bu and both its complexes show a bi-exponential decay, probably due to the formation of different hydrogen-bonded species in polar solvents like water or methanol. Effect of pH on Fluorescence Spectra. The effect of pH in the pH range of 2.0−12.0 on the emission intensity of HL-tBu has been tested both in the absence and in the presence of Al3+ and Zn2+ ions separately. The results are shown in Figure 5. However, the emission intensity of the probe in the presence of metal ions in equimolar ratio has changed significantly with the alteration in pH value. For Al3+ and Zn2+ ions, emission is monitored at 550 and 457 nm, respectively. For the 550 nm band, the probe without any metal shows high emission intensity at low pH. This indicates that the spirolactam ring is opened at low pH in the usual manner. The emission intensity of the free probe is significantly high until pH 4.0. Then, the emission intensity decreases sharply and becomes almost nonfluorescent after pH 6, indicating ring-closed species. However, HL-t-Bu shows high emission intensity at 550 nm until pH 8.0 in the presence of Al3+. This signifies that the Al3+ ion is able to induce ring opening at higher pH medium. However, the response toward pH variation is different for HL-t-Bu when its emission intensity is monitored at 457 nm. Its emission intensity almost does not change in the said pH range. Here, Zn2+ can induce significant changes in its emission spectral behavior (Figure 5). In the presence of Zn2+, the emission intensity does not increase up to pH 6.0. Probably, at low pH, coordination with the metal ion does not initiate. However, its intensity increases after pH 6.0. These pH-dependent studies indicate that the interactions of the metal ions with the probe are different, that is, two metal ions interact with the probe through different mechanisms. These studies also show that both the metal ions can produce high emission intensity at two different wavelengths with the probe in the physiological pH region where the free probe shows low emission intensity, indicating the fact that the probe could be utilized for biological applications. 1 H NMR Spectral Studies. 1H NMR spectra of HL-t-Bu and HL-t-Bu in the presence of one equiv of each of Al3+ and

Figure 5. Fluorescence intensity of HL-t-Bu and HL-t-Bu in the presence of (A) Al3+ and (B) Zn2+ at various pH values. For Al3+, emission is monitored at 550 nm (λex = 500 nm), whereas for Zn2+, emission of the probe is monitored at 457 nm (λex = 370 nm).

Zn2+ have been recorded in DMSO-d6 (Figure 6). The signal for the phenolic proton of the free probe appears at 13.91 ppm. The peak at 8.03 ppm may be attributed to the imine proton (Figure 6A). Various aromatic protons appear in the range of 6.07−7.75 ppm. The peak at 1.32 ppm may be due to the presence of the methyl protons of the tert-butyl group in the salicylaldehyde unit. The peak at 1.81 ppm may be attributed to other methyl protons. Methylene and secondary amine protons appear at 8.03 and 5.05 ppm, respectively. There is an appreciable change in 1H NMR spectra of HL-tBu when its spectra have been recorded in the presence of both the metal ions. In the presence of Al3+, the peak for phenolic OH of HL-t-Bu disappears, indicating deprotonation of the phenolic proton (Figure 6B) and bond formation between deprotonated O and metal center, whereas this peak is retained in the presence of Zn2+ but just a little bit deshielded (14.25 ppm, Figure 6C). This observation indicates that the phenolic proton of the probe is retained in its Zn complex, yet the oxygen atom is coordinated to zinc. All of the other peaks have been shifted and widened. Mechanism Fluorescence Enhancement. Mechanism for the enhancement of the fluorescence intensity of HL-t-Bu in the presence of Al3+ and Zn2+ ions could be proposed based on pH-dependent emission studies, FT-IR, and 1H and 13C NMR spectral analyses. As these two metal ions show emission enhancement at different wavelengths, the mode of interaction may be different for the metal ions (Scheme 2). First, we discuss the interaction with aluminum ion. pHdependent emission spectra studies show that free HL-t-Bu shows characteristic high emission intensity at 550 nm when the pH of the medium is low. At high pH, the emission intensity of the free probe is low, whereas its fluorescence 6869

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intensity is considerably high at 550 nm in the presence of aluminum at low pH and also at high pH. At high pH, the spirolactam ring of the free probe remains in the closed form with low emission intensity. Thus, Al3+ induces the opening of the spirolactam ring, leading to high emission intensity at the high pH region. The interaction of the probe with Zn2+ does not lead to the opening of the spirolactam ring as evident from the emission intensity increment at 457 nm. The enhancement of the emission intensity at 457 nm may be explained based on photoinduced electron transfer (PET) and chelation enhanced emission (CHEF) mechanisms. Transfer of electrons from the imine nitrogen to the aromatic system leads to the quenching of intensity. However, coordination of the imine nitrogen to zinc atom leads to turn-off PET, and thereby emission intensity of the system increases. Coordination of the metal center with HL-t-Bu gives rigidity to the system as revealed from a strong binding constant value. Apart from these, CN isomerization may also be involved in the emission enhancement process.17 When zinc is not bound, in the free probe, CN isomerization is possible as a result of free rotation in the excited state. However, metal ion binding interrupts the free rotation, leading to the significant fluorescence enhancement. However, at low pH, the emission intensity of the probe at 457 nm is not high because coordination with zinc ion, probably, does not initiate. The proposed mechanism is well supported by FT-IR spectra. The FT-IR spectrum of the probe in the presence of aluminum indicates that the spirolactam ring is opened and the oxygen atom of the amide group is bonded to this metal ion. However, in the case of Zn2+, there is no formation of the ringopened compound, but imine nitrogen is linked to zinc. Reversibility Test. Reversibility of a probe is an important character for judging its chances for its application. This test also indicates the stability of the probe in vitro. We have done the reversibility test of HL-t-Bu with both Al3+ and Zn2+ ions (Figures S21 and S22). For both the metal ions, this test has been performed in a similar process. Fluorescence spectra of the probe have been recorded with 1 equiv of Al3+ and Zn2+ ions at 550 and 457 nm, respectively. High emission intensity is observed for both cases. Next, EDTANa2 has been added, and the emission spectrum has been measured. The emission intensity has been quenched severely in the presence of EDTANa2 as it snatches the metal ion from the probe−metal complex and liberating the probe free. Again, addition of the metal ion causes emission enhancement, and further addition of EDTA forces the decrease of emission intensity. This cycle repeats for several times. This indicates that the probe is reversible for metal ion binding of both ions. Naked Eye Detection. HL-t-Bu has been used for naked eye detection of Al3+ and Zn2+ ions. Naked eye detection is always handy as it needs no sophisticated tool. Photographs of the probe and probe with different metal ions have been recorded under UV and visible light (Figure 7). Other than Al3+ and Zn2+, no other metal ions could produce any change in color under both UV irradiation and visible light. All of them are colorless under visible light, but they are indigo under UV light. Zn2+ could not produce a color change under visible light, but it becomes blue under UV irradiation. Al3+ produces different coloration from the rest of the metal ions under both UV irradiation and visible light. A solution of HL-t-Bu becomes pink in the presence of this metal ion due to the opening of the spirolactam ring of HL-t-Bu. It turns bright yellow when observed under UV light. Thus, the presence of

Figure 6. 1H NMR spectra of (A) HL-t-Bu and HL-t-Bu in the presence of 1 equiv of (B) Al3+ and (C) Zn2+ in DMSO-d6.

Scheme 2. Mechanism for Emission Enhancement of HL-tBu in the Presence of (A) Al3+ and (B) Zn2+

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Figure 7. Colors of HL-t-Bu in the presence of different metal ions under visible light (upper row) and UV light (lower row) in 10 mM HEPES buffer in H2O/MeOH (1:9 (v/v), pH 7.4) at room temperature. HL in the figure denotes HL-t-Bu. (Photograph courtesy of Ankita Roy.)

Al3+ and Zn2+ ions could be marked under UV light, whereas the former ion could be ascertained under visible light. Cell Imaging Studies. MTT assay reveals that HL-t-Bu shows no significant cytotoxicity in BV2 cells even at 100 μM concentration (Figure S23). BV2 cells show almost 83% viability when treated with 100 μM HL-t-Bu. In fluorescence imaging studies, HL-t-Bu does not show any fluorescence itself (Figure 8). The addition of Zn2+ ions to the cells pre-incubated with 10 μM HL-t-Bu shows bright fluorescence, the intensity of which is a function of Zn2+ concentration (Figure 8, third panel). The same probe could also produce considerable fluorescence after the addition of Al3+ ions, and the intensity of which increases with an increase in Al3+ concentration. The addition of TPEN (N,N,N′,N′tetrakis(2-pyridylmethyl)ethylenediamine) quenches the fluorescence arising due to the presence of Zn2+ ions (Figure 8, fourth panel); however, the fluorescence due to Al3+ was still recognizable (Figure 8, fifth panel). The intracellular Zn2+ and Al3+ imaging behavior of HL-t-Bu has been studied in BV2 cells by fluorescence microscopy. The cells do not exhibit any fluorescence when it has been incubated with HL-t-Bu alone. The addition of Zn2+ and Al3+ results in a gradual increase in the fluorescence in a concentration-dependent manner. The fluorescence arising due to Zn2+ has been suppressed in the presence of TPEN since TPEN is a strong scavenger of Zn2+ ions. The chemosensor molecules have been inhibited to bind with Zn2+ ions, thus resulting in the disappearance of fluorescence. This observation leads to the fact that the probe is selective for sensing Zn2+. A different fluorescence signal detectable after TPEN addition may further prove that the probe is also able to sense Al3+. The considerable difference in the emission wavelengths for Zn and Al detection may help to exclude spectral overlap while detecting these metals simultaneously. The cytotoxicity study (MTT assay) shows that HL-t-Bu has no significant toxic effects on BV2 cell viability for at least up to 12 h. Discussion on Recently Published Dual Sensors. Few aspects of this chemosensor for Al3+ and Zn2+ may be compared with recently published related dual sensors (Table S5).5a,b,d−i,18 Most of the chemosensors possess the same excitation and emission wavelengths. Subsequently, emission enhancement occurs following the same mechanism for both the cations. However, 7-(2,4-dihydroxybenzylideneimino)-4methylcoumarin shows two different mechanisms. Enhancement of the fluorescence intensity is explained by hydrolysis of the probe for Al3+ and based on PET for Zn2+ (Table S5, Sl. No. 4). Thus, it exhibits two different excitations and two

different emission wavelengths for both the cations. In our case, HL-t-Bu shows emission enhancement via the opening of the spirolactam ring by Al3+ and PET, inhibition of CN isomerization, and CHEF mechanisms with Zn2+. This is a clear advantage over the other probes. Same or close excitation and emission wavelengths for both the cations may interfere the process of identification of one cation in the presence of other cations. For example, a rhodamine hydrazide-based probe shows dual sensing properties, but there is FRET mechanism, which may interfere the sensing process.5i Distinctly separated excitation and emission wavelengths for both the cations show less interference chance for this process. Hydrolysis of the probe is a disadvantage as recombination of the hydrolyzed product may become difficult. Questions about reversibility of the chemosensor may be aroused. HL-t-Bu does not undergo hydrolysis to detect either of the cations showing its stability. Most of the probes show a strong binding constant than that with the present probe. Our probe exhibits very good sensitivity toward both the cations (in the nanomolar range). Some of the recently published probes have LOD values in the micromolar range. Many of the previously published chemosensors have not been applied for cell imaging studies. HL-t-Bu along with other sensors have been used for cell imaging studies. This finds the utility of the chemosensor.

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CONCLUSIONS In summary, 2-(2-((3-(tert-butyl)-2-hydroxybenzylidene)amino)ethyl)-3′-6′-bis(ethylamino)-2′,7′-dimethylspiro[indoline-1,9′-xanthen]-3-one (HL-t-Bu) has been successfully synthesized and characterized by standard techniques including single-crystal X-ray analysis. The fluorescent probe is able to differentially detect Al3+and Zn2+ even in the presence of either cation. Detection of Al3+ (excitation at 500 nm and emission at 550 nm) and Zn2+ (excitation at 370 nm and emission at 457 nm) is realized through two distinct fluorescence changes. No other cations are capable of increasing the emission intensity of the probe as is obvious from the study. The change of color of HL-t-Bu in the presence of Al3+ can also be marked by the naked eye. HL-t-Bu has a high sensitivity toward both the cations with LOD values in the nanomolar range. This probe has been used for cell imaging studies. The study suggests that HL-t-Bu can be readily used as an efficient, selective, and sensitive tool for biological imaging without cytotoxic effects. Such a fluorophore can be a promising and potential candidate to probe Zn2+ and Al3+ simultaneously, which may be helpful in understanding the alterations in Zn and Al dynamics in cells. 6871

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However, it always surprised us that different substitutions offer different metal ion sensors. For example, the tert-butyl group containing the probe in this presentation is a dual sensor for Al3+ and Zn2+. A methyl-substituted compound13e is selective for Al3+, whereas a corresponding bromo compound13a is selective chemosensor for trivalent cations. However, metal ion selectivity could not be ascertained based on the nature of substituents. Probably, more examples are needed to draw a conclusion.

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EXPERIMENTAL SECTION Materials and Physical Measurements. Rhodamine 6G, ethylenediamine, and 3-tert-butylsalicylaldehyde were purchased from Sigma-Aldrich, and metal salts such as nitrates of Al3+, Zn2+, Pb2+, Ni2+, NH4+, Na+, Mn2+, Mg2+, K+, Hg2+, Fe3+, Cu2+, Cr3+, Cd2+, and Ca2+ were purchased from MERCK and used as received. Other chemicals were received from commercial sources and used as received. Solvents used during spectroscopic studies were purified and dried by the standard procedures.19 Elemental analysis was carried out on a 2400 Series-II CHN analyzer (Perkin Elmer, USA). FT-IR spectra were recorded on a Perkin Elmer spectrometer (Spectrum Two) with the samples using the attenuated total reflectance (ATR) technique. Absorption spectra were studied using a Shimadzu UV 2100 spectrophotometer. Emission spectra were recorded on a HORIBA Fluoromax-4C spectrofluorometer. The ESI-MS+ spectra were recorded on a QTOF Waters’ HRMS spectrometer (model XEVO G2QTof). 1H and 13C NMR spectra were recorded in D2O or DMSO-d6 or CDCl3 on a Bruker 300 MHz spectrometer using tetramethylsilane (δ = 0) as an internal standard. Luminescence lifetime measurements were performed using a TCSPC (time-correlated single photon counting) setup from Horiba Jobin Yvon. The luminescence decay data were recorded on a Hamamatsu MCP photomultiplier (R3809) and analyzed using the IBH DAS6 software. Emission quantum yields (Φ) of HL-t-Bu and its complexes with Al3+ and Zn2+ were measured using the formula Φsample = {(ODstandard × A sample × η2 sample) Figure 8. Upper panel (first panel) represents the bright-field images (C1) of BV2 mouse microglial cell line. The second panel represents the fluorescence images of BV2 cells treated with 10 μM HL-t-Bu alone separately. The third panel indicates the blue fluorescence images (C2) of BV2 cells pre-incubated with 10 μM HL-t-Bu and different concentrations of Zn2+ ions (10, 20, and 100 μM separately). The fourth panel represents the disappearance of blue fluorescence after treatment with 100 μM TPEN separately. The fifth panel indicates the appearance of the green fluorescence images (C3) of BV2 cells pre-incubated with 10 μM HL-t-Bu and different concentrations of Al3+ ions (10, 20, and 100 μM respectively). The sixth panel represents the merged images of C1 and C2. The last panel indicates the merged images of C1 and C3.

/(ODsample × A standard × η2 standard )} × Φstandard

where A is the area under the emission spectral curve, OD is the optical density of the compound at the excitation wavelength, and η is the refractive index of the solvent. The quantum yield of the zinc compound has been determined by using quinine sulfate (0.546) as the standard, and that of the aluminum complex is determined using rhodamine 6G (0.94) as the standard.20 N-(Rhodamine 6G)lactam-ethylenediamine (L1) was synthesized following a published procedure.21 Synthesis of 2-(2-((3-(tert-Butyl)-2hydroxybenzylidene)amino)ethyl)-3′-6′-bis(ethylamino)-2′,7′-dimethylspiro[indoline-1,9′-xanthen]-3-one (HL-t-Bu). To a solution of L1 (0.125 g, 0.5 mmol) in 20 mL of methanol, 3-tert-butylsalicylaldehyde (0.085 mL, 0.5 mmol) was constantly added drop by drop, and the mixture was stirred until a clear solution was obtained. Then, it was refluxed for 5 h, and the solution became yellow. After that, the mixture was cooled to room temperature and filtered to remove any suspended material or precipitate. It was kept in the dark for slow evaporation. Finally, block-shaped

Thus, the easy-to-synthesize rhodamine derivative HL-t-Bu can serve as a very good dual chemosensor for Al3+ and Zn2+ ions, and it may find application in biomedical fields. It is pertinent to discuss the effect of substitution(s) of the salicylaldehyde unit of the probe on the metal ion selectivity. In our previous report, we have summarized different fluorescent probes based on rhodamine 6G and salicylaldehyde derivatives. It has been seen that substitution(s) on the salicylaldehyde part can control the metal ion selectivity. 6872

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concentrations (10−100 μM) of zinc nitrate and aluminum nitrate at a 10 min interval. Also, fluorescence images were taken after further addition of TPEN (100 μM). Cell Cytotoxicity Assay. To test the cytotoxicity of HL-tBu, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed with BV2 cells according to the standard procedure.28 Briefly, after treatment of BV2 cells (103 cells in each well of a 96-well plate) with HL-t-Bu (1, 10, 50, and 100 μM) for 12 h, 10 μL of MTT solution (1 mg/mL in PBS) was added to each well and incubated at 37 °C for 3 h. The media were then removed, and 100 μL of acidic isopropyl alcohol was added into each well. The intracellular formazan crystals (blue-violet) formed were solubilized with 0.04 N acidic isopropyl alcohol, and the absorbance of the solution was measured at 595 nm with a microplate reader (model THERMO Multiskan EX). The cell viability was expressed as the optical density ratio of the treatment to control. Values were expressed as mean ± standard error of three independent experiments.

yellow crystals of HL-t-Bu suitable for single-crystal X-ray diffraction analysis were obtained after a few days. The product was collected by filtration and dried in air. Yield: 0.127 g, 75%. Anal. calc. (%) for C39H44N4O3: C, 75.94; H, 7.19; N, 9.08. Found: C, 75.80; H, 7.11; N, 9.22; 1H NMR (DMSO-d6, 300 MHz) (δ, ppm): 13.91 (1H, s), 8.02 (1H, s), 7.75 (1H, d), 7.49 (1H, dd), 7.48 (1H, d, J = 3.3 Hz), 7.22 (1H, d, J = 6.84 Hz), 7.09 (1H, dd), 6.99 (1H, d), 6.76 (1H, dd), 6.26 (2H, s), 6.07 (2H, s), 5.04 (2H, s), 3.18−3.08 (broad), 1.81 (6H, s), 1.31 (9H, s), 1.21 (6H, t, J = 6.96 Hz); 13C NMR (CDCl3, 75 MHz) (δ, ppm): 167.60, 167.53, 160.36, 153.89, 151.52, 148.20, 136.66, 133.24, 130.82, 130.46, 129.52, 128.76, 128.08, 124.10, 122.82, 118.81, 118.69, 118.09, 105.15, 96.11, 64.62, 56.34, 49.07, 37.95, 34.82, 29.62, 17.48, 14.62; ESI-MS+ (m/ z): 617.56 [(HL-t-Bu + H+)]. Crystallographic Data Collection and Refinement. A suitable single crystal of HL-t-Bu was mounted on the tip of a glass fiber with the help of commercially available glue. X-ray data collection of the single crystal was performed at room temperature using a Bruker APEX II diffractometer, equipped with a normal focus, sealed tube X-ray source with graphite monochromated Mo Kα radiation (λ = 0.71073 Å). The data integration was performed using the SAINT program,22 and the absorption correction was made with SADABS. The structure was solved by SHELXS 9723 using the Patterson method followed by successive Fourier and difference Fourier syntheses. Full-matrix least-squares refinements were performed on F2 using SHELXL-97 with anisotropic displacement parameters for all the nonhydrogen atoms.24 All the hydrogen atoms were fixed geometrically by the HFIX command and placed in ideal positions. Calculations were carried out using SHELXL 97, SHELXS 97, PLATON v1.15,25 ORTEP-3v2,26 and WinGX system Ver-1.80.27 Fluorescence and UV−Vis Spectral Experiments. Absorption and fluorescence spectra of HL-t-Bu were recorded in the absence and in the presence of different metal cations including Al3+ in 10 mM HEPES buffer in H2O/MeOH (1:9 (v/v), pH 7.4) at room temperature. Nitrate salts of different cations (Al3+, Zn2+, Pb2+, Ni2+, NH4+, Na+, Mn2+, Mg2+, K+, Hg2+, Fe3+, Cu2+, Cr3+, Cd2+, and Ca2+) were used for spectroscopic measurements. Typically, for the titration, the probe and different metal salts were mixed in such a way to get the final concentration of the probe to be 40 μM with the preferred concentration of the metal ion. Cell Culture. BV2 mouse microglial cell line was obtained from the National Centre for Cell Science (Pune, India) and used throughout the experiment. Cells were grown in RPMI (HiMedia) supplemented with 10% FBS (HiMedia) and an antibiotic mixture (1%) containing penicillin and streptomycin (HiMedia) at 37 °C in a humidified incubator with 5% CO2. Cell Imaging Study. The stock solution (1.0 mM) was prepared by dissolving HL-t-Bu in DMSO/water. BV2 cells at approximately 50% confluence were used for the experiment. Cells were rinsed with phosphate-buffered saline (PBS) and incubated with RPMI containing 10 μM HL-t-Bu for 30 min at 37 °C. After incubation, DIC (differential interference contrast) and fluorescence images of BV2 cells were captured with the help of a Zeiss Axio Imager wide-field fluorescence microscope using a 63× oil immersion objective. For each experimental condition, three coverslips were prepared. At least 10 different fields were examined in each coverslip. Similarly, fluorescence images of BV2 cells (pre-incubated with 10 μM HL-t-Bu) were taken with addition of different

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00475. FT-IR spectra of HL-t-Bu and its complexes with Al3+ and Zn2+, ESI mass spectra of HL-t-Bu and HL-t-Bu in the presence of Al(NO3)3 and Zn(NO3)2., 13C NMR spectrum of HL-t-Bu, UV−vis and fluorescence spectra of HL-t-Bu in the presence of different cations, fluorescence intensity of HL-t-Bu and Zn2+ along with other metal ions, fluorescence intensity of HL-t-Bu and Al3+ along with other metal ions, Job plot analysis with Al3+ and Zn2+, fluorescence intensity of HL-t-Bu in the presence of Zn2+ and Al3+ at different time intervals, plot of (Fmax − F0)/(F − F0) against 1/[C] for HL-t-Bu to determine the binding constants for Al3+ and Zn2+, details for the determination of LOD of HL-t-Bu for Al3+ and Zn2+, excited-state fluorescence decay behavior of HL-t-Bu and its complexes with Al3+ and Zn2+, reversibility test of HL-t-Bu for Al3+ and Zn2+, % cell viability of BV2 cells, crystal data of HL-t-Bu, selected bond lengths and selected bond angles of HL-t-Bu, elemental analysis of HL-t-Bu and its complexes with Al3+ and Zn2+, lifetime of HL-t-Bu and its complexes Al3+ and Zn2+ ions, and comparison of a few aspects of recently published dual chemosensors (PDF) Crystallographic data of HL-t-Bu (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; proy@chemistry. jdvu.ac.in. ORCID

Partha Roy: 0000-0001-5387-5626 Notes

The authors declare no competing financial interest. 6873

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ACKNOWLEDGMENTS A.R. gratefully acknowledges DST-INSPIRE for her fellowship. A.M. was supported by UGC DS Kothari Postdoctoral Fellowship.

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DOI: 10.1021/acsomega.9b00475 ACS Omega 2019, 4, 6864−6875