New Six-Membered pH-Insensitive Rhodamine Spirocycle in Selective Sensing of Cu2+ through C-C Bond Cleavage and Its Application in Cell Imaging.

A new rhodamine-based chemosensor 1 with a six-membered spirocyclic ring has been synthesized, which exhibits excellent pH stability and shows selective "turn-on" fluorescent detection of Cu2+ ions over a series of other metal ions including Cu+ ions. The expansion of spirocycle improves the stability and selectivity of the chemosensors in sensing of metal ions. Till today only few rhodamine structures R1-R5 with thiourea-, hydrazine amide-, or pyrrole-decorated six-membered spirocyclic rings are known that exhibit metal-ion sensing via C-N bond cleavage of the spiro ring. In this context, rhodamine compound that responds to the metal ion through C-C bond cleavage of the six-membered spiro ring is completely unknown. The present example is a first-time report that demonstrates selective sensing of Cu2+ ions through C-C bond cleavage over the conventional existing systems in the literature. The chemosensor 1 is cell permeable and can detect Cu2+ in live cells using confocal microscopy in the biologically relevant pH range with high photostability.

Introduction

The design and synthesis of fluorogenic and chromogenic probes for selective sensing of biologically relevant metal ions have drawn considerable attention for several years.[1−7] Of the different metal ions, copper, after iron and zinc, is the third most abundant essential trace element in the human body. It plays a crucial role in many fundamental physiological processes in organisms.[8,9] However, under overloading conditions, copper exhibits toxicity that causes several neurodegenerative diseases (e.g., Menkes syndrome, Alzheimer’s disease, Wilson’s disease, and familial amyotropic lateral sclerosis), probably through the production of reactive oxygen species.[10,11] Therefore, it is essential to maintain the balance of copper ion in the body. Owing to such different features of copper ion in the biological system, it is desirable to develop selective fluorescent and colorimetric sensors of Cu2+ ions.

Fluorescent sensors deserve attention due to high sensitivity and spatial resolution in combination with being nondestructive to the samples and less cell damaging in microscopy. In pursuing the sensors of this class, exploitation of rhodamine probes that show excellent photophysical properties[12] and ion-induced facile five-membered lactam ring opening accompanying a color change from colorless to pink color, is worth mentioning.

The literature reveals that numerous rhodamine-based Cu2+ probes contain five-membered spirolactam rings with appropriate binding groups that participate in metal-ion binding involving amide ion through the ring opening and result in color and fluorescence changes.[13−24] In many cases, the probes of this kind are pH sensitive too. In this capacity, metal-ion sensing using six-membered spirolactam-based rhodamine sensors is almost unexplored. The expansion of spirocycle improves the stability and selectivity of the chemosensors in sensing of metal ions. Only four kinds of examples are known till today where either the thiourea (R1 and R2),[25,26] hydrazine amide (R3)[27] or the pyrrole-based (R4 and R5)[28] six-membered spiro rings are involved in sensing of bio-relevant metal ions such as Cu2+ and Hg2+ by ensuing the cleavage of the C–N bond in the spirocycle (Chart ). In this context, the existing five-membered rhodamine chemosensors are also known to interplay via C–N bond cleavage. Therefore, structural modification related to stability, sensitivity, and selectivity is desirable.

Chart 1

Reported and Present Six-Membered Rhodamine Chemosensors for Metal-Ion Sensing

In continuation of our work on rhodamine sensors,[29−35] we wish to report in this full account the design, synthesis, and metal-ion sensing behavior of a new six-membered spirocycle-based rhodamine compound 1. In comparison to the existing six-membered spirocycles[25−28] (Chart ), the present chemosensor 1 contains amide functionality in the spirocycle and has been proven to be photostable and pH-insensitive. Further, it has been established, for the first time, as an excellent chemosensor for selective sensing of Cu2+ ions involving C–C bond cleavage of the spiro ring. Importantly, organic transformation involving C–C bond cleavage followed by its activation through several ways is an attractive issue in organic synthesis.[36] This is a challenging aspect in spite of the inertness of the C–C bond. In many organometallic reactions for organic synthesis, the C–C bond activation (cleavage) is usually thermodynamically less favored than the C–C bond formation due to formation of weak metal–carbon bonds at the expense of a relatively stable C–C bond (90 kcal mol–1).[37] To make the C–C bond cleavage facile, several strategies which are well explained in several reviews[36] are followed. Of the different strategies, formation of a stable metal complex resulting from C–C bond cleavage is unique.

In addition, the compound 1 is useful for cell imaging and the complex of 1 with Cu2+ ion detects S2– ions selectively over a series of other anions. Sulfide ion is known to react with Cu2+ ion to form a stable CuS species, which has a low-solubility product constant ksp = 6.3 × 10–36. Therefore, among various approaches to sensing sulfide anions, sensors exploiting copper sulfide affinity[38,39] attract attention.

Results and Discussion

Synthesis

The six-membered spirocyclic compound 1 was achieved according to Scheme . The Boc-protected benzimidazole 2, obtained from our reported procedure,[35,40,41] was used for methylation of both the ring and aliphatic amino nitrogens using CH3I in dry tetrahydrofuran (THF) to afford the compound 3.[42] Removal of the Boc-group in 3 using trifluoroacetic acid (TFA) gave the amine 4, which on reaction with rhodamine B acid chloride in the presence of Et3N yielded the desired six-membered spirocyclic compound 1 possibly through the simple reaction mechanistic pathway, as shown in Scheme . Compound 1 was fully characterized by usual spectroscopic methods.

Scheme 1

Reaction Conditions: (i) CH3I, NaH, Dry THF, 2 h; (ii) 50% TFA in CH2Cl2, 2 h; (iii) Rhodamine B Acid Chloride, Et3N, Dry CH2Cl2, 8 h

Interaction Study

Metal-ion sensing properties of the new chemosensor 1 toward the perchlorate salts of metal ions, such as Ag+, Na+, Cu+, Ca2+, Co2+, Cd2+, Cu2+, Ni2+, Mg2+, Hg2+, Mn2+, Pb2+, Zn2+, Fe2+, Fe3+, Al3+, and Cr3+ were evaluated in CH3CN/H2O (CH3CN/H2O = 4:1, v/v, 10 mM tris HCl buffer, pH 6.5). In absence of metal ions, the chemosensor 1 is colorless and nonfluorescent, indicating the predominant existence of the spirocyclic form in the compound. Upon excitation at 510 nm, the chemosensor 1 gave strong emission at ∼595 nm [quantum yield[43,44] (Φ): 0.58] in the presence of varying concentrations of Cu2+ ions, accompanying a color change from colorless to pink. Under identical conditions, other metal ions in the study brought insignificant change in emission spectra (Figure S1, Supporting Information). Figure a, in this regard, represents the emission titration spectra of 1 with Cu2+ ions, and upon progression of titration, the colorless solution of 1 became pink. Figure b displays the selective fluorescence enhancement of 1 only in the presence of Cu2+ ions over various other metal ions including Cu+ ion taken in the study, indicating that compound 1 is highly selective to Cu2+ ion.

Figure 1

(a) Fluorescence titration spectra of 1 (c = 2.5 × 10–5 M) in CH3CN/H2O (4/1, v/v; 10 mM tris HCl buffer; pH 6.5) upon successive addition of Cu2+ (c = 1 × 10–3 M) [inset: emission of 1 at 595 nm as a function of Cu2+ concentration and color change of the solution of 1 under illumination of UV light] and (b) fluorescence spectra of 1 (c = 2.5 × 10–5 M) measured in CH3CN/H2O (4/1, v/v; 10 mM tris HCl buffer; pH 6.5) with respective metal cations (6.5 equiv) (λexc = 510 nm, slit = 2/2).

The gradual addition of Cu2+ ions to the solution of 1 brought about a marked change in the absorption spectra (Figure ). Importantly, although the absorbance at 267 nm was increased to a negligible extent, the new absorbance at 565 nm (ε/ε0 = 825, ε = 3.3 × 104 M–1 cm–1) emerged with significant intensity, resulting in pink coloration of the receptor solution. The other metal ions including Cu+ ion used in this study were silent in bringing such changes (Figure S2, Supporting Information).

Figure 2

UV–vis titration spectra of 1 (c = 2.5 × 10–5 M) in CH3CN/H2O (4/1, v/v; 10 mM tris HCl buffer; pH 6.5) upon addition of Cu2+ (c = 1 × 10–3 M) [inset: absorbance of 1 at 565 nm as a function Cu2+ concentration and color change upon addition of Cu2+ ion].

The appearance of the peaks at 565 nm in UV–vis absorption and 595 nm in emission spectra are attributed to the six-membered spiro ring opening in 1 to form a metal complex. In this regard, the possible structure of the n class="Chemical">copper complex can be either 1A or 1B, shown in Figure .

Figure 3

Possible structures of 1–Cu2+ complex.

To be acquainted with the actual structure of the metal complex, we tried to grow single crystals of the Cu complex, but we failed. Therefore, we followed density functional theory (DFT)[45] calculations using B3LYP function and 6-31G basis set. The DFT optimization of 1 and all of its possible metal complex structures 1A and 1B was performed in CH3CN (Figure ). In the structure 1, the planes of the spiro ring and xanthene ring are almost perpendicular to each other for facile opening of the ring in the presence of the metal ion to attain either 1A or 1B complex. Although in 1A, the amide oxygen and the benzimidazole nitrogen participate in chelation of Cu2+ ion, in 1B the benzimidazole nitrogen and the amide nitrogen are involved in metal chelation. DFT optimizations of these two possible forms were done in CH3CN and the structure 1B was observed to be higher in energy than 1A by 1.54 kcal mol–1. Time-dependent density functional theory (TDDFT) calculations were performed on each suggested complex. In the case of structure 1A, the theoretically found absorption at 555 nm with oscillator strength f = 1.1757 matched almost with the experimentally observed absorption (Figure ). In comparison, under identical conditions, complex 1B gave absorption theoretically at 603 nm (Figure ) with oscillator strength f = 0.6135, which did not match with the experimental observation. This, therefore, ruled out the possibility of formation of the complex structure 1B.

Figure 4

DFT-optimized structures of 1 and its copper complexes 1A (a = 1.92, b = 1.95, and c = 2.02 in Å) and 1B (a = 2.20 and b = 1.93 in Å). The associated absorption spectra of Cu complexes, determined by TDDFT calculation, are also shown.

DFT-optimized structures of 1 and its copper complexes 1A (a = 1.92, b = 1.95, and c = 2.02 in Å) and 1B (a = 2.20 and b = 1.93 in Å). The associated absorption spectra of n class="Chemical">Cu complexes, determined by TDDFT calculation, are also shown.

Further, to support the proposed binding structure of 1 with Cu2+ ions, we recorded the Fourier transform infrared (FTIR) and 1H NMR of 1 in the presence and absence of Cu2+ ions. In FTIR, the amide carbonyl stretching appeared as doublet at 1654 and 1632 cm–1 and underwent change and merged to the signal 1647 cm–1 upon complexation of Cu2+ (Figure A). Furthermore, the stretching for C=N of benzimidazole[35] at 1614 cm–1 was reduced to 1590 cm–1, which clearly indicated the participation of the imine bond (C=N) in complexation of Cu2+.

Figure 5

(A) Partial FTIR spectra of 1 and 1–Cu2+ complex and (B) 1H NMR (CD3CN, 400 MHz) of (i) 1 (c = 1.17 × 10–2 M), (ii) 1 with 1 equiv amount of Cu(ClO4)2.

(A) Partial FTIR spectra of 1 and 1–Cu2+ complex and (B) n class="Chemical">1H NMR (CD3CN, 400 MHz) of (i) 1 (c = 1.17 × 10–2 M), (ii) 1 with 1 equiv amount of Cu(ClO4)2.

1H NMR spectra of the compound 1 in the absence and presence of Cu2+ ions (although paramagnetic) were recorded in CD3CN (Figure B). In the presence of 1 equiv amount of Cu(ClO4)2, the disappearance of the signal for the assigned proton Ha at 4.84 ppm confirmed the opening of the spirolactam ring through C–C bond cleavage. The exact location of the signal for Ha was difficult to identify due to the broadening nature of the spectra. During complexation, the signals in 1H NMR became broad and some aromatic protons moved to the downfield region. Additionally, the signals for CH3 groups of benzimidazole and spiro rings that appeared at 2.90 and 2.63 ppm also moved to the downfield directions by 0.16 and 0.09 ppm, respectively.

In the interaction process, the stoichiometry of the Cu2+ complex was determined to be 1:1, as confirmed by Job’s plot[46] (Figure S3, Supporting Information). The binding constant from nonlinear fit[47] of the emission titration data gave a value of (1.25 ± 0.019) × 104 M–1 (Figure S4, Supporting Information). Because of poor change in emission, binding constant values for other ions were difficult to determine. Moreover, the detection limit[48] of Cu2+ for 1 was calculated from the calibration curve using the equation 3σ/S, where σ is the standard deviation of the blank solution and S is the slope of the calibration curve, and the value was noted to be 5.54 × 10–7 M (Figure S5, Supporting Information).

In the selectivity study, the change in emission of 1 was observed in the presence and absence of other metal ions. No metal ion in the study interfered in the binding of Cu2+ (Figure S6, Supporting Information). This is in contrast to the observation of Wang et al.,[26] where the selective detection of Cu2+ by six-membered spirocycle rhodamine R2 was interfered largely by Ag+ and Hg2+ ions. To understand the sensitivity of 1 to Cu2+, we further explored the fluorescence titration experiments of 1 with Cu2+ and other tested metal ions in pure CH3CN and CH3CN containing different proportions of water (e.g., CH3CN/H2O = 1:1 and 1:5, v/v). As can be seen from Figures S7 and S8, the chemosensor 1 reserves its selectivity for Cu2+ ion by showing a slight variation in sensitivity. On increasing the amount of water in CH3CN the sensitivity decreases slightly. The time-dependent fluorescence of the sensor in the presence of Cu2+ ions in Figure represents that the reaction of 1 with Cu2+ ions in aqueous CH3CN is slow compared with the case in CH3CN, evident from the slow change in emission of the sensor. In pure CH3CN, the reaction of 1 with Cu2+ is rapid and completed within 5 min, after which the fluorescence intensity changes scarcely.

Figure 6

Time course for the fluorescence response of 1 upon addition of Cu2+ in different solvent systems.

As rhodamine-based compounds are responsive to H+ also, we evaluated the effect of pH on the absorbance and emission of 1 without or with 6.5 equiv amount of Cu2+ ions (Figure ). The results indicate that the chemosensor 1 has no absorbance as well as emission in a wide range of pH 4–12. Only at pHs 2 and 3, a small change in absorption as well as emission was observed but the solution remained colorless (Figure S9, Supporting Information). This indicates the intactness of the six-membered spirolactam ring of 1 even in strongly acidic condition. On addition of Cu2+ to the solution of 1 at different pHs, dramatic enhancement of absorbance and emission was observed in the pH range 3–7 and the colorless rhodamine solution became pink (Figure S9, Supporting Information). So the chemosensor 1 was fairly applicable in a reasonable pH range.

Figure 7

Responses of 1 and 1–Cu2+ solutions in (a) UV–vis (absorption at 565 nm) and (b) fluorescence (emission at 596 nm) at different pHs in CH3CN/H2O (4/1, v/v; 10 mM tris HCl buffer).

Responses of 1 and 1–Cu2+ solutions in (a) UV–vis (absorption at 565 nm) and (b) fluorescence (emission at 596 nm) at different pHs in n class="Chemical">CH3CN/H2O (4/1, v/v; 10 mM tris HCl buffer).

Next, the usefulness of the colored copper complex of 1 was explored in anion sensing in details. Among various anions, only S2– responded by exhibiting a marked change in absorption as well as in emission. In both cases, the intensity dropped near to zero (Figure ) and the pink solution of 1–Cu2+ complex became light yellow (Figure S10, Supporting Information). This change in color and optical spectra is attributed to the demetallation followed by generation of the spirocyclic ring. Importantly, this spirocyclic ring was not the six-member-based compound 1, instead it was established as five-membered spirocycle 1C(49) (confirmed by 1H NMR and mass analysis, Figure S11, Supporting Information). Further addition of excess Cu2+ ions to the decomplexed solution did not produce any change in absorption and emission as well as in color. To our opinion, after decomplexation of Cu2+, S2–-induced elimination afforded stable amide ion 1C′ that participated in the cyclization reaction to furnish five-membered rhodamine-based compound 1C,[49] which is insensitive to Cu2+ ions (Figure ).

Figure 8

Changes in (a) absorbance and (b) emission of 1–Cu2+ complex in CH3CN/H2O (4/1, v/v; 10 mM tris HCl buffer; pH 6.5) upon addition of different guests (c = 2 × 10–3 M).

Figure 9

S2–-induced cyclization reaction of 1–Cu2+ complex.

Changes in (a) absorbance and (b) emission of 1–Cu2+ complex in n class="Chemical">CH3CN/H2O (4/1, v/v; 10 mM tris HCl buffer; pH 6.5) upon addition of different guests (c = 2 × 10–3 M).

To confirm the insensitivity of 1C toward Cu2+ ions, UV–vis and emission titrations of 1C(49) as isolated from the reaction mixture, were conducted under similar conditions with Cu2+ ions in CH3CN/H2O (CH3CN/H2O = 4:1, v/v, 10 mM tris HCl buffer, pH 6.5). No significant spectral changes were observed (Figure S12, Supporting Information).

In anticipation of anion sensing of the copper complex of 1, as depicted in Figure , the sensing properties of the compound 1 itself toward several common ions and reactive sulfur species were examined (Figure S13, Supporting Information). Importantly, the compound 1 did not show any measurable interaction.

Biological Study

Next, the utility of 1 to detect Cu2+ in live cells was investigated. We labeled HeLa cells with 3 μM of 1 for 30 min (Figure ). Live cell imaging was carried out in a chamber while maintaining temperature (37 °C), humidity (90%), and CO2 concentration (5%). The confocal microscopy image of the 1-labeled HeLa cells showed weak fluorescence, which may reflect the low content of Cu2+ in the basal level (Figure a–c).[50] On the other hand, the fluorescence intensity was dramatically increased by pretreatment of Cu2+ (200 μM) for 30 min (Figure d–f).

Figure 10

Confocal microscope images of HeLa cells labeled with 3.0 μM of 1. Images were captured (a) before and (d) after treatment with 200 μM CuCl2 and (g) after addition of 40 mM Na2S to image (d) for 30 min at 37 °C. (b), (e), (h) are bright-field images. (c), (f), (i) are merged images of (a), (d), (g) and (b), (e), (h), respectively. Cells shown are representative images from replicate experiments (n = 5). Images were collected using 525 nm excitation and emission windows at 550–700 nm. Scale bar: 40 μm.

Moreover, the fluorescence intensity at a given region in the 1-labeled HeLa cells was maintained nearly the same after continuous irradiation of the 525 nm excitation for 1 h. This outcome indicated the high photostability of 1 in the cell imaging condition (Figure ). These data demonstrated that 1 is capable of detecting Cu2+ ions in live cells with good cell permeability and brightness and minimum interference of photoinstability. To confirm that the chemosensor 1 could not affect the viability of HeLa cells in our incubation condition, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay (Cell Titer 96H; Promega, Maidson, Wisconsin) was used according to the manufacture’s protocol (Figure S14, Supporting Information).

Figure 11

(a) Confocal microscope image of HeLa cells labeled with 3.0 μM of 1 for 30 min at 37 °C. (b) Relative intensity measured at the region of interest 1–2 in (a) as a function of time. The signal was collected at 550–700 nm upon excitation at 525 nm. Scale bar: 45 μm.

Conclusions

In conclusion, we have synthesized six-membered spirocyclic fluorescent chemosensor 1 that selectively sensed Cu2+ over a series of other metal ions and discriminated Cu2+ from Cu+ ion effectively. The six-membered spiro ring opening in the presence of Cu2+ proceeded through C–C bond cleavage. This is in comparison to the reported six-membered (R1–R5)[25−28] and other five-membered rhodamine compounds[13−24,29−35,51−57] where complexation of a selective metal ion takes place through C–N bond cleavage (Table S1). The present example is thus a first-time report that demonstrates selective sensing of Cu2+ ions through C–C bond cleavage over the conventional existing systems in the literature (Table S2). The sensor is extremely pH stable and works well in both organic and aqueous organic solvents for Cu2+ with good detection limit without facing any interference of other ions examined.

The chemosensor 1 is cell permeable and can detect Cu2+ in live cells using confocal microscopy in the biologically relevant pH range with high photostability. The complex of 1 with Cu2+ ion additionally detects S2– ions selectively over a series of other anions. Such six-membered spiro ring-based pH-insensitive rhodamine is new addendum to the existing reports, shown in Chart . Structural tuning of this new rhodamine derivative is underway in the laboratory.

Experimental Section

tert-Butyl Methyl((1-methyl-1H-benzo[d]imidazol-2-yl)methyl)carbamate (3)[42]

To a stirred solution of 2(35,40,41) (0.7 g, 2.83 mmol) in dry THF (30 mL), sodium hydride (0.27 g, 11.25 mmol) was added. The mixture was stirred for 30 min, and a solution of methyl iodide (1.21 g, 8.49 mmol) in dry THF (5 mL) was added, and the reaction mixture is further stirred for 90 min. After completion of the reaction monitored by thin-layer chromatography (TLC), the solvent was removed under vacuuo and the reaction mixture was then extracted with CH2Cl2 (50 mL). The organic layer was washed with brine, dried over Na2SO4, and evaporated under reduced pressure. The purification of the crude mixture by silica gel column chromatography using petroleum ether/ethyl acetate (9:1, v/v) as eluent yielded the yellow gummy compound 3(42) (0.6 g, yield: 77% ); 1H NMR (400 MHz, CDCl3): δ 7.59 (d, 1H, J = 8 Hz), 7.19 (dd, 1H, J1 = 8 Hz, J2 = 2 Hz), 7.17–7.07 (m, 2H), 4.63 (s, 2H), 3.66 (s, 3H), 2.70 (s, 3H), 1.34 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 155.6, 150.7, 142.1, 136.2, 122.8, 122.1, 119.6, 109.3, 80.3, 44.5, 33.5, 29.9, 28.3; FTIR: ν cm–1 (KBr): 2972, 2933, 1692, 1615, 1480, 1452, 1409, 1388, 1306.

Synthesis of Compound 1

To synthesize compound 1, a solution of rhodamine B (0.5 g, 1.04 mmol) in 1,2-dichloroethane (25 mL) was stirred and phosphorus oxychloride (750 μL) was added dropwise at room temperature. Then, the resulting solution was refluxed for 2 h. The reaction mixture was cooled and evaporated in vacuo to give rhodamine B acid chloride, which was directly used in the next step. The crude acid chloride was dissolved in the dry dichloromethane (20 mL) and was added dropwise for 10 min to a solution of amine 4 (0.23 g, 1.31 mmol), obtained by removal of the Boc-group of 3 using TFA and Et3N (300 μL) in dichloromethane (20 mL) at room temperature. The reaction mixture was stirred for 8 h. After completion of the reaction monitored by TLC, the solvent was removed under pressure and the residue was dissolved in chloroform (60 mL), extracted with water, and dried over anhydrous Na2SO4. The crude mass on purification by silica gel column chromatography using petroleum ether/ethyl acetate (4:1, v/v) as eluent yielded the light pink powdery compound 1 (0.13 g, yield: 20%); mp 136 °C; 1H NMR (400 MHz, CDCl3): δ 8.47 (d, 1H, J = 8 Hz), 7.62–7.60 (m, 1H), 7.43 (t, 1H, J = 8 Hz), 7.35 (t, 1H, J = 8 Hz), 7.15–7.13 (m, 2H), 7.02–7.00 (m, 1H), 6.97 (d, 1H, J = 8 Hz), 6.64 (d, 1H, J = 8 Hz), 6.43 (s, 1H), 6.36 (s, 1 Hz), 6.26 (dd, 1H, J1 = 8 Hz, J2 = 2.4 Hz), 5.76 (d, 1H, J = 8 Hz), 5.65 (d, 1H, J = 8 Hz), 4.76 (s, 1H), 3.36–3.27 (m, 4H), 3.26–3.17 (m, 4H), 2.96 (s, 3H), 2.84 (s, 3H), 1.16 (t, 6H, J = 6.8 Hz), 1.07 (t, 6H, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3): δ 166.3, 154.7, 151.1, 150.3, 148.1, 148.0, 142.0, 141.3, 135.6, 131.8, 131.2, 130.6, 130.5, 128.9, 127.4, 127.1, 122.2, 121.7, 120.3, 113.2, 108.6, 107.3, 106.8, 98.4, 97.6, 67.0, 46.5, 44.4, 44.3, 35.2, 29.7, 12.6, 12.4; FTIR: ν cm–1 (KBr): 2968, 2928, 1654, 1632, 1613, 1577, 1542, 1510, 1466, 1399, 1375; HRMS (TOF MS ES+): calcd for (M + H)+: 600.3339, Found: 600.3344.

Synthesis of Compound 1C(49)

To a stirred solution of 1 (0.01 g, 0.016 mmol) in CH3CN/H2O (4:1, v/v) (10 mL), Cu(NO3)2·3H2O (0.005 g, 0.02 mmol) was added and stirred for 5 min. Then, Na2S (0.008 g, 0.03 mmol) was added to the 1–Cu2+ complex solution and stirred for another 15 min; solvent was then removed under vacuuo, and the reaction mixture was then extracted with ethyl acetate (15 mL). The organic layer was washed with brine, dried over Na2SO4, and evaporated under reduced pressure. The purification of the crude mixture by silica gel column chromatography using petroleum ether/ethyl acetate (4:1, v/v) as eluent yielded the compound 1C(49) (0.004 g, yield: 52%); 1H NMR (400 MHz, CDCl3): δ 7.91–7.89 (m, 1H), 7.41–7.39 (m, 2H), 7.08–7.06 (m, 1H), 6.45 (d, 2H, J = 8 Hz), 6.40 (s, 2H), 6.29 (d, 2H, J = 8 Hz), 3.36–3.31 (m, 8H), 2.66 (s, 3H), 1.17 (t, 12H, J = 6.8 Hz); HRMS (TOF MS ES+): calcd for (M + H)+: 456.2651, Found: 456.2626.

Quantum Yield Determination

Quantum yield of the compound 1 in presence of Cu2+ was determined in CH3CN by the relative comparison procedure using rhodamine B as the standard (φrhB = 0.68 in ethanol).[43] The general equation used in the determination of relative quantum yields is as follows[44]where Φ is the quantum yields, F is the integrated area under the corrected emission spectrum, A is the absorbance at the excitation wavelength, λex is the excitation wavelength, η is the refractive index of the solution, and the subscripts “u” and “s” refer to the unknown and the standard, respectively.

Theoretical Calculation

Structures of compound 1 and its complexes 1A and 1B were optimized in acetonitrile. TDDFT calculations were carried out on 1A and 1B using the same level of theory with the polarizable continuum model in acetonitrile (ε = 35.688). All of the calculations have been performed with Gaussian 09 suite of program.[41]

General Procedure for Fluorescence and UV–Vis Titrations

Stock solution of the compound 1 was prepared in CH3CN/H2O (4/1, v/v; 10 mM tris HCl buffer; pH 6.5) mixture solvent in the concentration of 2.5 × 10–5 M. Stock solutions of guests were also prepared in same solvent in the concentration of 1 × 10–3 M. Solution of compound 1 (2 mL) was taken in the cuvette and to this solution different guests were individually added in different amounts. Upon addition of guests, the change in emission of the compound was recorded. The same stock solutions were used to perform the UV–vis titration experiment in the same way.

Method for Job Plot[46]

The stoichiometry was determined by the continuous variation method (Job plot). In this method, solutions of host (compound 1) and guest (Cu2+) of equal concentrations were prepared in CH3CN/H2O (4/1, v/v; 10 mM tris HCl buffer; pH 6.5) solvent. Then, the solutions of compound 1 and Cu2+ were mixed in different proportions, maintaining a total volume of 3 mL of the mixture. The related compositions for host/guest (v/v) were 3:0, 2.75:0.25; 2.5:0.5, 2.25:0.75, 2:1, 1.75:1.25, 1.5:1.5, 1:2, 0.75:2.25, 0.5:2.5, and 0.25:2.75. All of the prepared solutions were kept for 1 h, with occasional shaking at room temperature. Then, fluorescence and absorbance of the solutions of different compositions were recorded. The concentration of the complex, i.e., [HG] was calculated using the equation [HG] = ΔI/I0 × [H] or [HG] = ΔA/A0 × [H], where ΔI/I0 and ΔA/A0 indicate the relative emission and absorbance intensities, respectively. [H] corresponds to the concentration of the pure host. The mole fraction of the host (XH) was plotted against concentration of the complex [HG]. In the plot, the mole fraction of the host at which the concentration of the host–guest complex concentration [HG] is maximum, gives the stoichiometry of the complex.